sRNA-mediated regulation of gal mRNA in E. coli: Involvement of transcript cleavage by RNase E together with Rho-dependent transcription termination

Heung Jin Jeon; Yonho Lee; Monford Paul Abishek N; Xun Wang; Dhruba K Chattoraj; Heon M Lim

doi:10.1371/journal.pgen.1009878

. 2021 Oct 28;17(10):e1009878. doi: 10.1371/journal.pgen.1009878

sRNA-mediated regulation of gal mRNA in E. coli: Involvement of transcript cleavage by RNase E together with Rho-dependent transcription termination

Heung Jin Jeon ^1,^2,^#, Yonho Lee ^1,^#, Monford Paul Abishek N ^1,^#, Xun Wang ³, Dhruba K Chattoraj ⁴, Heon M Lim ^1,^*

Editor: Jue D Wang⁵

PMCID: PMC8577784 PMID: 34710092

Abstract

In bacteria, small non-coding RNAs (sRNAs) bind to target mRNAs and regulate their translation and/or stability. In the polycistronic galETKM operon of Escherichia coli, binding of the Spot 42 sRNA to the operon transcript leads to the generation of galET mRNA. The mechanism of this regulation has remained unclear. We show that sRNA-mRNA base pairing at the beginning of the galK gene leads to both transcription termination and transcript cleavage within galK, and generates galET mRNAs with two different 3’-OH ends. Transcription termination requires Rho, and transcript cleavage requires the endonuclease RNase E. The sRNA-mRNA base-paired segments required for generating the two galET species are different, indicating different sequence requirements for the two events. The use of two targets in an mRNA, each of which causes a different outcome, appears to be a novel mode of action for a sRNA. Considering the prevalence of potential sRNA targets at cistron junctions, the generation of new mRNA species by the mechanisms reported here might be a widespread mode of bacterial gene regulation.

Author summary

sRNAs are regulators of gene expression in all forms of life. In bacteria such as E. coli, sRNAs base-pair with mRNA, which can have many consequences such as premature transcription termination by Rho and degradation of mRNA by the endoribonuclease RNase E. Here we show that the two processes can occur on the same sRNA-mRNA pair and produce shorter but stable mRNA that can be of functional significance. Thus, in sRNA regulation of mRNA, transcript cleavage can be an additional mechanism to well-known transcription termination to generate new mRNA species. The choice between the mechanisms is dictated by where on the mRNA the base pairing with sRNA takes place.

Introduction

The galactose (gal) operon of Escherichia coli has been the subject of many seminal studies on transcription regulation in bacteria [1]. Four structural genes comprise this operon: galE, galT, galK, and galM (Fig 1A). In addition to the full-length gal mRNA, galETKM (formerly called mM1), four other mRNA products are also found: galE, galE1, galET and galETK [2]. These mRNAs have the same 5’ end at the transcription initiation site, but different 3’ ends immediately after the galE, galT, or galK genes (yielding galE, galET, or galETK mRNA, respectively). The 3’ end of the galE1 mRNA lies in the middle of the galT gene.

Fig 1 — A) Schematics of the *galETKM* operon showing the position of the last nucleotide of the stop codon of each gene from the transcription initiation site of the P1 promoter (+1) (top line). The Spot 42 (red) binding site spans about 75 nucleotides and is located at the *galT-galK* junction [20]. The region of *galE* used to probe northern blots is marked as E-probe. The next two lines show the nucleotide sequence of the 3’ ends of *galET-short* and *galET-long* mRNA. The letters in bold are a putative Shine-Dalgarno sequence (GGAG, red letters) for *galK*, the stop codon (UAA, blue letters) of *galT*, and the initiator codon (AUG, green letters) of *galK*. The sequence of the entire Spot 42 RNA is shown in red and its regions that are complementary to *gal* mRNA are indicated by black dots and are marked as Region I, II and III. B) Northern blot with the E probe of *gal* mRNA from MG1655 (WT, lane 1) and MG1655Δ*spf* (Δ*spf*, lane 2) cells. Note that the *galET* mRNA band is missing in lane 2. The identity of the two faint bands in MG1655Δ*spf* cells where the *galET* band resides has not been determined. Shown at the bottom is 16S rRNA, which was used as a loading control. C) 3’ RACE assay of 3’ ends of *galET* mRNA from MG1655 (lane 1) and MG1655Δ*spf* (lane 2) cells. DNA sequencing ladders that serve as length markers are in lanes marked T and C. The data presented in these blots are representative of three independent experiments.

The 3’ end of the full-length galETKM mRNA is located at gal operon nucleotide coordinate 4,313, where 1 is the transcription initiation site of the galP1 promoter (Fig 1A) [2,3]. The 3’ end is generated due to Rho-dependent transcription termination (RDT) followed by exo-nucleolytic processing up to 4,313, the currently accepted mature 3’ end of galETKM mRNA [4]. Another type of transcription termination, known as intrinsic or Rho-independent transcription termination, generates the 3’ end at 4,315, which is immediately processed by RNase II to 4,313 [4]. Rho also causes natural polarity in the gal operon, which results in greater levels of promoter-proximal transcripts [5]. The Rho-dependent termination that occurs at the end of each gene has been suggested as the key molecular event underlying the natural polarity in gal gene expression [1,2].

Bacteria cope with a rapidly changing environment by regulating gene expression. One such adaptive response is mediated by small RNAs (sRNAs), a group of regulatory RNAs that are encoded in trans to the target mRNAs to which they base-pair [6]. Most sRNAs bind near the 5’ end of target mRNA and mediate negative control [7]. sRNA binding near the translation initiation site of target mRNA blocks translation initiation. This may also allow recruitment of RNase E, which cleaves in the ribosome-free zones of mRNA that are created as a result of sRNA binding; this augments negative control due to degradation of the target mRNA [7–12].

Another consequence of sRNA binding at the 5’ end of target mRNA is Rho-dependent termination (RDT). Transcription without accompanying translation makes the ribosome-free transcript available for Rho binding and subsequent transcription termination [1,13–16]. For example, the sRNA ChiX binds to the 5’ end of its target mRNA chiPQ, which causes decoupling of translation from transcription, resulting in RDT downstream from the point of decoupling [17–19]. At the intercistronic region between galT and galK, the stop codon of galT (blue colored UAA) and the initiator codon of galK (green colored AUG) are separated by 3 nucleotides (Fig 1A). A putative ribosome-binding site for galK (red colored GGAG) exists two nucleotides upstream of the galT stop codon. The sRNA, Spot 42, is known to be involved in the regulation of this galT-galK region [20]. Spot 42 is a 109-nucleotide RNA found in E. coli and some 196 other species of Gammaproteobacteria [21]. Spot 42 binds near the galK start codon and inhibits the translation of galK (Fig 1A) [20,22]. This binding also results in the decrease of galK transcription [23]. In a previous study, we reported that Spot 42 decreases galK-specific mRNA by accelerating the degradation of an internal product of the gal operon, the galKM mRNA (formerly called mK2) [24]. However, we also found that Spot 42 binding causes transcription termination at the end of galT, generating a new mRNA species, galET (formerly called mT1). Thus, by generating the promoter-proximal galET mRNA and degrading the promoter-distal galKM mRNA, Spot 42 enhances natural polarity in the gal operon [24].

In this study, we further explore the role of Spot 42 binding at the galT-galK junction in the generation of galET mRNA species. We find that the galET mRNA has two alternate 3’ ends located at positions 30 and 60 nucleotides downstream of the galT stop codon (Fig 1A) [24]. We show that these two different 3’ends are generated as a result of base-pairing of two different regions of Spot 42 with the corresponding complementary regions of mRNA and involvement of two different mechanisms: RDT (as was already known) [24] and a previously unreported RNase E-mediated transcript cleavage. We demonstrate that RNase E generates exclusively the longer product and Rho primarily the shorter product after processing by RNase III. Spot 42 binding thus causes generation of different galET species by two different 3’ end-generating mechanisms. sRNA-control is known to be primarily at the 5’ end of messages. gal operon is the first example where sRNA regulation is seen internal to the message. Finally, we discuss the possible physiological relevance of generating variously truncated stable versions of a polycistronic message.

Results

Spot 42 promotes processing of gal operon mRNA to galET mRNA

We performed a northern blot of total RNA prepared from WT (MG1655) cells grown in LB supplemented with 0.5% galactose to an OD₆₀₀ of 0.6, the growth phase up to which Spot 42 production remains maximal [23]. We analyzed the blot with an E-probe that hybridizes to the first half of the first gene of the operon, galE (Fig 1A). The results show that the following gal specific mRNAs are present in WT cells: galE, galE1, galET, galETK, and the full-length galETKM (Fig 1B, lane 1). In our previous study, we determined the 3’ ends of gal mRNAs across the entire operon and found that the 3’ ends mentioned in Fig 1B are the major 3’ ends produced from the operon [2].

In this study, we measured the location of the 3’ ends of the galET mRNA more precisely using the 3’ RACE assay. The 3’ends of galET clustered at two different locations in the gal operon between nucleotide coordinates 2,125–29 and 2,161–63 (Fig 1C, lane 1). The two 3’ end clusters were located approximately 30 and 60 nucleotides downstream from the stop codon of galT (Fig 1A). Since the 5’ end of galET resides at the transcription initiation site [2], these results indicate that the single mRNA band observed in the northern blot was actually composed of two clusters of mRNA species that differ in their 3’ ends.

We found that the galET mRNA was specifically reduced to 30 (±3) % of WT in MG1655Δspf cells in which the gene for the Spot 42 sRNA is deleted (Fig 1B, lane 2). All the other mRNA bands in MG1655Δspf showed more or less the same amount of WT (Fig 1B). In such cells, both the 3’ end clusters of the galET mRNA were missing (Fig 1C, lane 2) (S1 Fig). These results indicate that Spot 42 is required to generate both the clusters. In this study, we referred to the galET mRNA that has 3’ ends about 60 nucleotides downstream from the galT stop codon as galET-long, and that has 3’ ends about 30 nucleotides downstream from the galT stop codon as galET-short (Fig 1C).

Specific base-pairing of Spot 42 and gal mRNA is critical for the generation of galET 3’ ends

i) Mutations in region I of Spot 42 abrogate the generation of galET-long but not galET-short. sRNAs commonly function through base pairing to target mRNAs. Nucleotide sequence analysis indicated that regions I, II and III of Spot 42, identified by Beisel and Storz [23], could each make eight to ten perfect base pairings with three different regions of the gal mRNA (Fig 1A). We investigated the role of each of the base pairing regions of Spot 42 on the generation of the 3’ ends of galET.

Region I is located at the 5’ end of Spot 42 that has a guanine complementary to the cytosine at coordinate 2,158 of gal mRNA (Fig 2A). It is likely that region I basepairs with 8 consecutive nucleotide sequences of gal mRNA, a few nucleotides upstream from what would be the 3’ ends of galET-long. We deleted either one or three nucleotides from the 5’ end of region I. Using 3’RACE, we assayed the 3’ ends in MG1655Δspf cells that harbor a pBR322-derived expression plasmid pSpot42, in which different mutations in the Spot 42 gene (spf) were introduced [23]. The 3’RACE results revealed that a single nucleotide deletion in Spot 42 (as in Spot42DMI-1, Fig 2A) resulted in production of the 3’ end of galET-long about two nucleotides shorter than WT galET-long (Fig 2B, lane 2). The deletion of three nucleotides (as in Spot42DMI-2, Fig 2A) resulted in a drastic reduction in the level of galET-long (Fig 2B, lane 3). The level of galET-short in the two mutants was essentially the same as it was when Spot 42 was WT (Fig 2B, lane 1). These results indicate that the 5’ end of region I of Spot 42 plays an important role in the formation of galET-long specifically.

We next introduced substitution mutations in region I of Spot 42. The three nucleotides at the 5’ end of region I (5’ GUA) were changed to their complementary nucleotides to yield Spot42MMI-1, and the next three nucleotides (5’ GGG) were changed to their complementary sequence to generate Spot42MMI-2 (Fig 2A). Results from 3’RACE revealed that the formation of galET-long was completely eliminated when Spot 42 had either of the substitution mutations (Fig 2B, lanes 4 and 5). Again, these region I changes had only a marginal effect on the level of galET-short (Fig 2B, lanes 1–5). Thus, the results of both the deletion and substitution mutations in region I of Spot42 suggest that pairing of region I with its complementary sequences in gal mRNA plays a critical role in the generation of galET-long specifically.

ii) Mutations in region II of Spot 42 abrogate the generation of galET-short but not galET-long. Spot 42 region II has perfect complementarity to a stretch of 10 contiguous nucleotides of gal mRNA (Fig 2A). The 3’ end of region II of Spot 42 is complementary to the second nucleotide following the initiator codon of galK (the guanine at 2,107, Fig 2A). We made a series of substitution mutations in region II and assayed the effect of these mutations on the formation of galET 3’ ends. One, two or three nucleotides from the 3’ end of region II were changed to their complementary sequences, generating Spot 42 mutants MMII-1, -2 and -3, respectively (Fig 2A). Results of 3’RACE showed that substituting a single-nucleotide in Spot 42, as in Spot42MMII-1, caused a drastic reduction in the level of galET-short (Fig 2C, lane 2). The results were the same when two- and three-nucleotide substitutions were made (Fig 2C, lanes 3 and 4). Generation of galET-long was not affected by the presence of these region II mutants (Fig 2C, lanes 1–4), suggesting that the pairing event that generates galET-long was not affected by un-pairing of the three nucleotides of Spot 42 region II.

iii) Mutations in region III of Spot 42 do not affect the generation of either galET-long or galET-short. Region III of Spot 42 has perfect complementarity to 9 consecutive nucleotides partially covering the Shine-Dalgarno sequence of galK mRNA (GGAG, Fig 2A). Unlike mutations in regions I and II, none of the substitution mutations in region III of Spot 42 affected generation of either of the 3’ ends of galET mRNA significantly (Fig 2D, lanes 1–7). We note that when Spot 42 mutants caused drastic changes in the level of galET mRNAs, they were not due to drastic changes in Spot 42 mutant RNA levels (Fig 2B–2D, bottom row).

To further examine the specific base-pairing requirements of Spot 42 and gal mRNA, we took a complementary approach: Instead of changing Spot 42, we made substitution mutations in some of the gal sequences implicated in base-pairing with Spot 42 and assayed the formation of galET 3’ ends. Mutations were generated in a pBAC-derived low copy number plasmid, pGal that carries the entire gal operon [25]. The effect of gal mutations was assayed in the MG1655Δgal strain [25].

We substituted 3 nucleotides of pGal, generating galMMI-1 that would change gal mRNA from 5’ UGC to 5’ ACG, and thus break the complementarity with the AUG at the 5’ end of Spot 42 (Fig 2A, lower set of sequence lines). Results of 3’ RACE showed that the 3’ end of galET-long formation was severely impaired (Fig 2E, lane 2). The results were same when we substituted the next 3 nucleotides of gal, from CCC to GGG, generating galMMI-2 (Fig 2A, also see Fig 2E, lane 3). In contrast, neither of the gal mutants caused any impairment in the generation of galET-short. These results, in conjunction with the results from the Spot 42 region I mutants Spot42MMI-1 and -2 (which have changes in the same base-pairing region as the gal mutants galMMI-1 and -2), led us to conclude that specific base-pairing between region I of Spot 42 and gal mRNA is critical for the formation of the 3’ ends of galET-long.

We took a similar approach to test the base-pairing requirements for the generation of galET-short. The guanine at 2,107 in gal mRNA, which pairs with the 3’ end of region II of Spot 42, was changed to cytosine, generating the gal mutant galMMII-1 (Fig 2A, last but one of the sequence lines). Results of 3’ RACE showed that the level of the 3’ end of galET-short decreased to 40 (±3) % of the level observed in the absence of the mutation (Fig 2E, lane 4). When the next base was changed additionally, generating galMMII-2, the level of the 3’ end of galET-short decreased to less than 20 (±4) % of the level observed in the absence of the mutations (Fig 2E, lane 5). Generation of galET-long was not affected by these mutations (Fig 2E, lanes 1, 4 and 5). Together with the results of Spot 42 region II mutants Spot42MMII-1 and -2 (which have changes in the same base-pairing regions as the gal mutants galMMII-1 and -2), these results led us to conclude that base-pairing between region II of Spot 42 and gal mRNA is critical for the formation of 3’ ends of galET-short.

Next, we tested if these gal mutants could restore galET production to its WT level in the presence of Spot 42 mutants with complementary changes (Fig 2F). When MG1655ΔgalΔspf cells harboring both pGal and pSpot 42 mutant plasmids with complementary changes were assayed for the presence of the 3’ ends of the galET mRNA, the levels of both of the 3’ ends were restored to WT levels in all cases (Fig 2F). These results confirm that base pairing is required to generate both galET-long and galET-short, and the regions of base-pairing involved in their generation are different.

RNase E is involved in the generation of galET-long 3’ ends

In our previous study, we suggested that RDT is the molecular event that generates the 3’ end of galET mRNA [24]. Because we found that there are two different 3’ ends of galET mRNA, we tested the possibility that endoribonuclease-mediated transcript cleavage could be another mechanism to generate one of the 3’ ends. We assayed the 3’ ends of galET mRNA in strains defective in endoribonucleases RNase G (rng^-), RNase P (rnp^ts) and RNase E (rne^ts). In rng^- [26], both galET-long and galET-short were produced in amounts comparable to that in WT, indicating that RNase G is not involved in the generation of the two galET species (Fig 3A, lanes 1 and 2).

Fig 3 — 3’RACE assay of *galET* RNA 3’ ends in three different endoribonuclease mutant strains. Strains used are GW10 (*rng+ rne+*; WT for RNase G and RNase E, lanes 1, 7 and 8), GW11 (*rng*::*cat*; [*rng-*] RNase G down mutant, lane 2), NHY312 (*rnpA+*; [*rnp+*] WT for RNase P, lanes 3 and 4), NHY322 (*rnpA49*; [*rnp*^ts] RNase P temperature-sensitive mutant, lanes 5 and 6), GW20 (*ams1*^ts; [*rne*^ts] RNase E temperature-sensitive mutant, lanes 9 and 10). The data presented in these blots are representative of three independent experiments.

To test the requirement for the other two endoribonucleases, rnp^ts and rne^ts strains [26,27] were grown to an OD₆₀₀ of 0.3 at a permissive temperature (30°C), together with their respective WT strains. The cultures were then incubated at the non-permissive temperature (44°C) for an additional 25 min before harvesting the cells for analysis. By this time, the culture OD₆₀₀ reached about 0.6 in most cases. In rnp^ts, no difference in the levels of the two 3’ ends were observed in cells harvested at the permissive and non-permissive growth temperatures, indicating non-involvement of RNase P in the generation of either of the galET species (Fig 3A, lanes 3–6). In contrast, in rne^ts at the non-permissive temperature, the level of the 3’ end of galET-long decreased to less than 10% of the level observed in WT cells (Fig 3A, lanes 8 and 10). In comparison, the difference in the level of the 3’ ends of galET-short was negligible. These results indicate that RNase E plays a major role in the generation of galET-long specifically.

Rho-dependent transcription termination generates galET-short 3’ ends

To test the involvement of Rho in the generation of galET-short, we assayed transcription by adding pGal in a cell-free system prepared from the E. coli strain BL21 (λDE3) [28]. The cell-free system contains all necessary factors for transcription and translation but not template DNA. Reactions were run for 30 min at 30°C after adding the template DNA, pGal. Results of 3’ RACE after adding pGal produced no noticeable 3’ ends (Fig 4A, lane 3). However, in the presence of galactose (to a final concentration of 0.5%) produced noticeable but faint 3’ ends (Fig 4A, lane 4), indicating that the bands are from transcription of the gal operon. Interestingly, the band at 2,184 increased 5 times in the presence of plasmid pRho, where rho is under the T7 promoter control (Fig 4A, lane 5). These results show that the 2,184-band is the product of RDT.

Fig 4 — A) 3’RACE assay of the *galET* mRNA 3’ ends from MG1655 *in vivo* (lane 1) and from the cell-free system with no DNA template (lane 2), with DNA template pGal (lane 3), with pGal and 0.5% galactose (lane 4), and with pGal, pRho and 0.5% galactose (lane 5). T, C: DNA sequencing ladders. B) 3’RACE assay of the *galET* mRNA 3’ ends from the cell-free system as in (A) except for the absence of pRho plasmid and presence of Spot 42 RNA. Lane 1 is same as in lane 1 of (A); Lane 2 is same as in lane 4 of (A); Lanes 3–5: the same reaction as in lane 2 but with 1.5 nM (lane 3), 15 nM (lane 4) and 150 nM Spot 42 RNA (lane 5). T, C: DNA sequencing ladders. (C) The nucleotide sequence of a Rho-utilization site *rut*, known as the ‘C-rich region’, which lies downstream of the *galT* gene and spans *gal* coordinates 2,075 to 2,175 (shown in orange). The binding sites in *gal* mRNA for Regions I—III of Spot 42 are boxed. The +1, -1 and -10 of the consensus sequence for the RNA polymerase pause site are presented in bold. Below is shown the cytosine and guanine contents of the region in the green and the blue lines, respectively. The C-rich region (boxed with dotted line) is shown over the abscissa with *gal* coordinates. A sliding window of 70 nt was used to plot the GC content. The data presented in these blots are representative of three independent experiments.

To confirm if the band at 2,184 is the result of RDT, we added increasing concentration of the Rho inhibitor Bicyclomycin (BCM) to the cell-free reaction in the presence of pGal, 0.5% galactose and pRho. The band at 2,184 decreased with increasing concentrations of BCM from 0.5 to 10 μg/ml (S2 Fig, lanes 2–6). The BCM at 10 μg/ml inhibited formation of the band at 2,184 by ~90% of the level seen without the drug (S2 Fig, lane 1). These results indicate that the band at 2184 is the result of RDT.

Furthermore, the cytosine at 2,184 is nine nucleotides downstream from a Rho-utilization site (rut), known as the “C-rich region”, present downstream of the galT gene spanning coordinates 2,075 to 2,175 (Fig 4C) [24]. We found a perfect consensus sequence for the RNA polymerase pause site [29,30], in which the cytosine at 2,184 is located at -1 position of the consensus sequence. These results indicate that Rho terminates gal transcription paused at 2,184 (Fig 4A).

Next, we tested the effect of Spot 42 RNA in RDT. We prepared Spot 42 RNA using an in vitro transcription reaction (See Materials and Methods). We added increasing amounts of Spot 42 RNA to the cell-free reaction together with pGal and 0.5% galactose. Results showed that the band at 2,184 increased with increasing concentration of Spot 42 RNA. The band increased 5, 9, and 12 times when Spot 42 was 1.5, 15, and 150 nM, respectively (Fig 4B). These results do show that Spot 42 directly controls RDT in gal.

We observed a cluster of 3’ ends at 2,125–2,129 that also increased with increasing Spot 42 concentration. Interestingly, these 3’ ends are formed exactly where the 3’ ends of galET-short are found (Fig 4B). These results indicate that RDT generates galET-short 3’ ends. These 3’ ends are not formed without the added Spot 42 (Fig 4A, lane 5). It appears that the galET-short 3’ ends are produced by Spot 42-mediated processing of the RDT product at 2,184 by unknown exoribonucleases in the cell-free system.

RNase III-mediated transcript cleavage results in generation of galET-short 3’ ends

To identify exoribonucleases that process the 3’ ends of galET-long and -short to their observed locations, we performed 3’RACE on RNA prepared from MG1655Δrnb (RNase II deleted), MG1655Δrnr (RNase R deleted) and W3110Δpnp (PNPase deleted). The results show that generation of neither 3’ end was compromised in the exoribonuclease deficient strains tested (Fig 5A). This prompted us to test endoribonucleases, specifically the RNA double strand-specific endonuclease RNase III.

Fig 5 — A) 3’RACE assay of *galET* 3’ends in E. *coli* mutants that are deficient in exoribonucleases. Strains used are MG1655Δ*rnb* (where the gene for RNase II is deleted from the chromosome) (lane 2), MG1655Δ*rnr* (where the gene for RNase R is deleted from the chromosome) (lane 3), W3110Δ*pnp* (where the gene for PNPase is deleted from the chromosome) (lane 5). The results of the WT for strains Δ*rnb* and Δ*rnr* are shown in lane 1, and for Δ*pnp* strain is shown in lane 4. B) 3’RACE assay of the *galET* mRNA from the cell-free system in presence of pRNIII plasmid, where the gene for RNase III is under control of the T7 promoter. The data presented in these blots are representative of three independent experiments.

We assayed transcription using the cell-free system in the presence of plasmid pRNIII, where the RNase III gene (rnc) is under the T7 promoter control. In the presence of RNase III, the 3’ ends of galET-short at 2,125–2,129 increased 2 times and the 2,184 band decreased by 50 (±5) % (Fig 5B, lane 4). These results further support that the generation of the 3’ ends of galET-short at 2,125–2,129 require Spot 42 and are processed from the RDT product at 2,184, and unexpectedly, reveal the involvement of an endoribonuclease in this processing.

Spot 42 tunes production of enzymes for competing pathways of galactose anabolism and catabolism to optimize growth

Spot 42 is known to downregulate galK transcriptionally [23] and translationally [20]. Spot 42 is also known to expedite degradation of one of the gal mRNA species, galKM, which harbors the last two genes of the operon [24]. Here we also found and confirmed that Spot 42 binding leads to the generation of the galET mRNA with the two promoter-proximal genes. The expectation from these results is that in the presence of Spot 42 the production of Gal proteins from the promoter-distal genes, galK and galM, would decrease, while production from promoter-proximal genes, galE and galT, would increase.

To test this prediction, we measured Gal proteins from MG1655 and MG1655Δspf using western blots. Cells were grown in M9 medium with 0.5% galactose (M9-galactose). The results showed that at OD₆₀₀ of 0.6, MG1655 produced about 83 (±16) % and 68 (±25) % more of GalE and GalT proteins, respectively, but 17 (±5) % and 12 (±5) % less of GalK and GalM, respectively, than what were produced from MG1655Δspf (Fig 6A). These results indicate that as expected, Spot 42 increases the production of GalE and GalT, and decreases production of GalK and GalM, although the decrease was relatively small. GalE is an epimerase involved in the biosynthesis of the UDP-glucose/galactose required for producing LPS and other polysaccharides, and GalK is a kinase involved in galactose catabolism [31,32]. These results suggest that Spot 42 regulates gal expression facilitating the anabolic role of the pathway.

Fig 6 — A) Western blot analysis of the protein products of *gal* operon (GalE, GalT, GalK and GalM) from WT and *Δspf* cells. B) Physiological effect of Spot 42 deletion. This was measured by comparing growth rate of MG1655 and MG1655Δ*spf* cells in various media: LB with 0.5% galactose (light blue and green), M9 with 0.2% glucose (grey and yellow) and M9 with 0.5% galactose (blue and red). The growth of MG1655Δ*spf cells* was identical to that of MG1655 in LB-galactose and M9-glucose media. In M9-galactose medium, MG1655Δ*spf* cells (red) showed slower growth than that of MG1655 cells (blue). Error bars represents the mean fold-change ± standard deviation of three replicates from three independent experiments (n = 3).

Based on the above results, we reasoned that MG1655Δspf cells would grow slower than MG1655 cells in a medium, like M9-galactose, where both anabolism and catabolism of galactose are required. To test this, we measured the growth of MG1655 and MG1655 Δspf cells in various media. In LB-galactose or M9-glucose media, where galactose is not required or relevant, respectively, there was no growth difference between the two bacteria (Fig 6B). However, in M9-galactose, MG1655Δspf grew slower than MG1655. In exponential growth phase (OD₆₀₀ between 0.05–0.4), the doubling time of MG1655Δspf was 23% longer than that of MG1655 (S3A Fig and Table 1).

Table 1. Doubling time of various gal and spf mutants in M9-galactose medium.

Strain / plasmid	Doubling time (min) in exponential phase (OD₆₀₀ 0.05–0.4)
MG1655	71±3
MG1655 Δspf	88±5
MG1655 Δgal /pgal	65±5
MG1655 Δgal /pgalMMI-1	79±3
MG1655 Δgal /pgalMMII-1	82±6
MG1655 Δgal Δspf /pgal /pSpot42	108±11
MG1655 Δgal Δspf /pgalMMI-1 /pSpot42	136±8
MG1655 Δgal Δspf /pgalMMI-1 /pSpot42MMI-1	107±2
MG1655 Δgal Δspf /pgalMMII-1 /pSpot42	144±26
MG1655 Δgal Δspf /pgalMMII-1 /pSpot42MMII-1	112±5

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By the same token, we reasoned that gal mutants defective in the generation of galET mRNA would grow slower in M9-galactose medium. To test this, we measured the growth of MG1655Δgal cells encoding galMMI-1 (defective in generating galET-long), or galMMII-1 (defective in generating galET-short) in M9-galactose medium. In exponential growth phase, the doubling times of cells encoding galMMI-1 or galMMII-1 (S3B Fig) were 22 and 26% longer than those encoding the WT gal sequence, respectively (Table 1). The growth defect was restored when the gal mutants were complemented with Spot 42 mutants that would restore base pairing of Spot 42 with gal mRNA (Fig 2F) (S3C Fig). The doubling time of galMMI-1 and galMMII-1 in the presence of the Spot 42 mutants with complementary changes became 21 and 22% shorter, respectively, than when Spot 42 was WT (Table 1). These results reveal the physiological relevance of differential expression of the gal operon genes by Spot 42, which is to optimize growth by directing gal expression towards anabolic pathway.

Discussion

Transcript cleavage and transcription termination are both involved in the generation of galET mRNA

In this study, we explored mechanisms by which a noncoding sRNA, Spot 42, regulates expression of the gal operon of E. coli at post transcription-initiation level. Our results indicate that in the presence of Spot 42, two galET species are generated. These mRNAs have the same 5’ end as the full-length galETKM mRNA [33], but have two different 3’ ends, herein referred to as galET-short and galET-long. These RNAs are generated by base pairing of two different regions of Spot 42 (regions I and II) with their complementary sequences near the beginning of galK ORF. Region II base pairing leads to the formation of galET-short and requires the transcription termination factor Rho, and region I base pairing leads to the formation of galET-long and requires the endoribonuclease RNase E. The involvement of Rho in galET generation was known and that it is also generated by RNase E is reported here. Below, we discuss how base pairing in one region brings about transcription termination and, in another region, transcript cleavage.

Transcription is coupled to translation in E. coli [34–36]. When coupled, RNA polymerase is physically connected to the leading ribosome that translates the nascent transcript [37–39]. This coupling is a stochastic event, such that RNA polymerase often transcribes an ORF without a linked ribosome, generating regions of ribosome-free mRNA [40,41].

Based on the experimental results of Moller et al [20], which show that Spot 42 binding inhibits translation initiation of galK, we propose that when transcription is stochastically uncoupled from translation, Spot 42 initiates base-pairing with gal mRNA and particularly the region II base-pairing immediately downstream of the galK start codon blocks the initiation of galK translation (Fig 7A) [42]. The resultant generation of ribosome-free RNA, which includes an Rho utilization site (rut), known as a ‘C-rich region’ [24], allows Rho binding to the rut site and subsequent termination of transcription that is paused at 2,184 (Fig 7B) [1,15,17,43,44]. RNase III is involved either alone or in conjunction with an unknown exoribonuclease in the processing of Rho-terminated transcript with 3’ end at 2,184 to galET-short products with 3’ends at 2,125–2,129 (Fig 7C).

Fig 7 — A) Generation of *galET-short* by Rho: The model shows the (leading) ribosome at the stop codon of *galT* on a nascent chain of *gal* mRNA at the *galT-galK* intercistronic region uncoupled from a transcribing RNA polymerase. In this situation, the mRNA sequences that pair with Spot 42 region II are not covered by the ribosome. B) The Region II binding is assumed to maintain downstream mRNA ribosome-free. This provides the platform for Rho loading and generation of *galET-short* by transcription termination by Rho at 2184. The 3’end of *galET-short* is underlined. C) The subsequent processing of the Rho-terminated transcript by RNase III cleavage is shown at the 3’end of *galET-short* (downward green arrow). D) Production of *galET-long* by RNase E: This process requires hybridization with Spot 42 Region I. We assume that translation termination at *galT* does not always lead to translation initiation of *galK* (due to stochastic uncoupling of *galK* translation from *galT* translation), which would keep *galK* RNA ribosome-free and promote hybridization with Region I. (E, F) The hybridization is assumed to ensure that the downstream RNA remains ribosome-free in the nascent transcript (E) or in non-nascent transcripts (F) to allow RNase E cleavage. The hybrid Region I could also block 3’-end processing by 3’→ 5’ exonucleases after RNase E cleavage. Note that Spot 42 Region III cannot base pair with the corresponding *gal* sequences in this situation due to the presence of the ribosome at *galT* stop codon, which might explain why mutations in that region are inconsequential to *galET* mRNA production. “RNase (?)” represents currently unidentified RNases(s) that may be involved in the further processing events of the *galET* transcripts.

Unlike Rho loading, which depends on uncoupling of transcription from translation, cleavage by RNase E does not have to be so restricted. It is likely that both nascent and non-nascent transcripts could serve as substrates for Spot 42-mediated galET-long production. The probability of cleavage by RNase E would thus depend on the probability of uncoupling of translation from transcription in the case of nascent transcripts, and the uncoupling of galK translation from galT translation in non-nascent transcripts (Fig 7D–7F). Spot 42 binding to transcripts is expected to increase the probability of downstream RNA staying ribosome-free, thus facilitating RNase E action (Fig 7E). Spot 42 could also recruit RNase E by allowing RNase E to bind to the 5’ mono-phosphorylated end of Spot 42 [45]; however, this is not supported by our data (S4 Fig). It appears that RNase E is accessing ribosome-free mRNA by the direct entry pathway [45].

In summary, our results demonstrate that like transcription termination, transcript cleavage by RNase E is another mechanism to generate galET mRNA species. We believe that this is the first example of a bacterial sRNA generating essentially a single mRNA product using at least two different mechanisms. Simultaneous regulation by both RNase cleavage and RDT, however, has been described for riboswitches [46–48].

Our data also suggest that the two events that generate galET happen independently, i.e., the frequency with which one event occurs does not affect the frequency with which the other occurs (Fig 2B and 2C). Although these events could influence each other by depleting substrates, we note that a significant fraction of the substrates (galETKM and galETK) remain intact (Fig 1B), suggesting that the substrate availability may not be a limiting factor. We believe both events are probabilistic and occur on different transcripts with comparable efficiencies. These results can also be understood if Spot 42 exists in two forms that are not in equilibrium. In that case, loss of one will not affect the outcome from the other.

Processing of the 3’-OH ends generated by transcription termination or transcript cleavage

The Spot 42 region I mutant Spot42DMI-1 (which has a single nucleotide deletion at the 5’ end) generates galET-long mRNA that is two nucleotides shorter at the 3’ end (Fig 2B, lane 2). One possible explanation of these results is that the base-paired double stranded RNA between region I and gal mRNA blocks 3’→ 5’ exoribonuclease digestion, because it is well known that a hairpin (stem-loop) structure at the 3’ end of E. coli mRNA can protect the free 3’-OH end from such digestion [49–52].

The 3’ ends of RDT-generated transcript are known to be immediately subjected to 3’→ 5’ exoribonuclease digestion [4,50]. For the first time, however, we report in this study that RNase III, which is a double-stranded RNA specific endoribonuclease, can process the 3’ end of a transcript that is released from RDT (Fig 5). It is not clear, however, whether the RNase III cleavage sites are on the double stranded RNA region formed between Spot 42 and gal RNA or that formed intramolecularly in gal RNA sequences.

sRNA binding at intercistronic region and at 5’ UTR may have different outcomes

sRNA binding to the 5’untranslated region (5’UTR) generally causes inhibition of translation initiation. This results in generation of ribosome-free region downstream from the sRNA binding region, which could allow either transcription termination by Rho [17,24,53,54] or transcript cleavage by RNase E [7–12]. Usually, a new mRNA 3’ end generated by either of these events becomes immediately subjected to 3’→5’ exoribonuclease digestion, which results in the degradation of target mRNA.

However, if sRNA binds to intercistronic regions of a multi-cistronic mRNA, this sRNA could inhibit the initiation of translation of the downstream gene, making recruitment of Rho or RNase E possible. In these cases, 3’→5’ exoribonuclease digestion (beginning at the newly generated 3’ end) is likely to stop at the sRNA-mRNA hybrid and not likely to reach the 5’ end of the target mRNA and degrade the message to completion. Thus, sRNA binding at the intercistronic region in multi-cistronic mRNA could protect upstream cistrons, resulting in the accumulation of relatively stable new mRNA species. We observed an example of this in our study: Spot 42 binding at the intercistronic region of galT-galK led to the generation of a new mRNA species, galET. Considering the prevalence of potential sRNA targets at cistron junctions [55], the generation of new mRNA species by the mechanisms described here may be a general function of sRNA in polycistronic gene regulation.

Relationship of cAMP, Spot 42 and polarity in gal operon

Polarity in gal operon is conspicuous in glucose medium in WT cells (where cAMP level is low) or in cAMP mutant cells [2,56–58]. These two observations indicate that cAMP is an inhibitor of polarity. Spot 42 synthesis is repressed by cAMP and is induced in glucose medium or in cAMP mutant cells. In other words, polarity in gal operon seen in the absence of cAMP can be attributed to the induction of Spot 42.

Materials and methods

Bacterial strains and primers

E. coli strains used were GW10 (rng+, rne+; WT for RNase G and RNase E), GW11 (rng::cat; RNase G down mutant), GW20 (ams1^ts; RNase E temperature-sensitive mutant), NHY312 (rnpA+; WT for RNase P), and NHY322 (rnpA49; RNase P temperature-sensitive mutant). Chromosomal deletion strains of E. coli MG1655 were generated using phage Lambda Red-mediated recombineering [59]. Bicyclomycin (BCM) was a generous gift from M. Gottesman (Columbia University, USA). Primers used in this study are listed in S1 Table. Bacterial strains used in this study are listed in S2 Table.

Bacterial growth conditions

All cells were grown at 37°C in Lysogeny Broth (10 g tryptone, 5 g yeast extract, and 10 g NaCl per liter of water) supplemented with 0.5% (w/v) galactose and chloramphenicol (15 μg/ml) or ampicillin (100 μg/ml) to an OD₆₀₀ of 0.6 before RNA isolation. To determine generation time during exponential growth, cultures in M9-galactose grown to OD₆₀₀ of 1.0 were diluted to a fresh medium to OD₆₀₀ of 0.01, and growth was monitored by taking OD₆₀₀ periodically up to ~0.4.

RNA preparation

Total RNA was purified from clarified cell lysates using the Direct-zol RNA MiniPrep kit (Zymo Research, USA). To generate cell lysates, 2 × 10⁸ cells were resuspended in 50 μl of Protoplasting buffer (15 mM Tris-HCl [pH 8.0], 0.45 M sucrose and 8 mM EDTA). Five μl of lysozyme (50 mg/ml) was added and the sample was incubated for 5 min at 25°C. A phenolic detergent (1 ml TRI Reagent; Molecular Research Center, USA) was added and vortexed for 10 sec before incubating for 5 min at 25°C. The RNA was isolated according to the manufacturer’s recommendations and dissolved in 30 μl of RNA storage buffer (Thermo Fisher Scientific, USA). RNA concentrations were determined by measuring the absorbance at 260 nm using a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA).

Northern blot analysis

Total RNA (10 μg in 10 μg/ml ethidium bromide) was resolved by 1.2% (w/v) formaldehyde-agarose gel electrophoresis at 8 V/cm for 2 h. RNA integrity was assessed under a UV light and was transferred overnight to a positively charged nylon membrane (Thermo Fisher Scientific, USA) using a downward transfer system (Whatman TurboBlotter, USA). The RNA was fixed to the nylon membrane by baking at 80°C for 1 h. The northern blot probes were prepared as follows. First, a 500-bp DNA fragment from the galE region (from +27 to +527) was prepared by PCR, followed by labeling with ³²P. The template DNA (0.15 pmol) was mixed with random hexamers (4 μl of 1 mM) (Takara, Japan) in a total volume of 28 μl and then heated at 95°C for 3 min. The reaction was then rapidly cooled for 5 min on ice and a mixture containing 5 μl of 10× Klenow buffer, 5 μl deoxynucleoside triphosphate (dNTP) mix (0.2 mM dATP, dGTP, and dTTP), 10 μl α³²P-dCTP, and 2 μl Klenow fragment (2 U/μl) (Takara, Japan) was added. The reaction was incubated at 37°C for 1 h, followed by inactivation of the Klenow fragment at 65°C for 5 min. The product was purified by passage through a G-50 column. The blot was pre-hybridized in 7 ml ULTRAhyb Ultrasensitive Hybridization buffer (Invitrogen, USA) at 65°C for 30 min. The DNA probe was denatured at 95°C for 5 min and 5 μl of the probe was added to the hybridization buffer. The hybridization was performed at 42°C overnight, and the blot was then washed twice in low-stringency wash buffer (2× SSC, 0.1% SDS) for 5 min each at room temperature and then twice in high-stringency wash buffer (0.2×SSC, 0.1% SDS) for 15 min each at 42°C. Finally, the radioactive bands were visualized after exposure to X-ray film. The films were scanned and RNA bands were quantified using ImageJ software (NIH, USA).

Rapid amplification of cDNA ends (RACE)

Contaminating DNA in the RNA preparation was removed using Turbo DNase I (Thermo Fisher Scientific, USA). For the amplification of the 3’ RNA end (3’ RACE) or the 5’ RNA end (5’ RACE), we performed an RNA ligation reaction in a 25 μl reaction volume containing 1 μl of a 100 nM synthetic RNA oligo (27 mer for 3’RACE (S1 Table) and E. coli 5S rRNA for 5’ RACE), 2.5 μg or 5 μg total RNA for 3’RACE or 5’RACE, respectively, 2.5 μl 10x reaction buffer (Thermo Fisher Scientific-USA), 10 U T4 RNA ligase (Thermo Fisher Scientific-USA), and 10 U of rRNasin Ribonuclease Inhibitor (Promega, USA). The ligation reaction was incubated at 37°C for 3 h, and the resulting RNA was purified using a G-50 column (GE Healthcare, USA). Four micrograms of the ligated RNA was reverse transcribed at 37°C for 2 h in a 20 μl reaction mixture containing 4 U of Omniscript reverse transcriptase (Qiagen, Germany), 0.5 mM of each dNTP, 10 μM random hexamers and 10 U of rRNasin. For 3’ RACE, the specific 3RP primer (S1 Table), which hybridizes to the ligated RNA, was used. Either 2.5 or 10 μl was used as the template for PCR amplification of the target RNA for 3’RACE or 5’RACE, respectively, using a target RNA specific primer set and HotStarTaq DNA polymerase (Qiagen, Germany) in a 50 μl reaction volume. The amplified cDNA was purified and used as a template for the primer extension reaction, which was performed in a 20 μl volume with a ³²P-labeled specific primer and one unit of Taq polymerase (Qiagen, Germany) as previously described [2]. The reaction products were resolved on an 8% (w/v) polyacrylamide-urea sequencing gel, and the radioactive bands were visualized using X-ray films.

Reverse Transcription-quantitative PCR (RT-qPCR)

Reverse transcription was performed after removal of genomic or plasmid DNA from the reaction mixtures using Turbo DNase I according to the manufacturer’s recommendations (Thermo Fisher Scientific, USA). One microgram of total RNA was incubated at 37°C for 2 h in a 20-μl reaction volume containing 4 U of Omniscript reverse transcriptase, 0.5 mM of each dNTP, 8 μM random hexamer primer, and 10 U of rRNasin. Using a CFX96 PCR instrument (Bio-Rad, USA), RT-qPCR was performed in a 10 μl reaction volume containing 5 μl of iQ SYBR Green Supermix (Bio-Rad, USA), 3 μl of RNase-free water, 0.5 μl each of 10 μM forward and reverse primers, and 1 μl of the cDNA template. The following conditions were applied: an initial denaturation step at 95°C for 3 min and 40 cycles of 10 s of denaturation at 95°C, 20 s of hybridization at 60°C, and 10 s of elongation at 72°C. The results from each sample were normalized using rrsB, which encodes 16S rRNA.

Mutagenesis and quantification of Spot 42

Spot 42 mutations were generated in the pSpot42 plasmid [23] using the mega-primer method [60]. All mutations were confirmed by DNA sequencing. The 5’RACE method was used to quantify Spot 42 production.

In vitro synthesis and purification of Spot42 RNA

In vitro synthesis of Spot42 RNA was performed with T7 RNA Polymerase (New England BioLabs, USA) according to the manufacturer’s instructions. The DNA template containing the T7 promotor was generated by PCR using the T7-spot42-for and T7-spot42-rev primers using pSpot42 plasmid [20] as the template. Briefly, DNA template (1μg) was incubated at 37°C for 1 h in reaction buffer (40 mM Tris-HCl, pH 7.9; 6 mM MgCl2; 2 mM spermidine; 1 mM dithiothreitol [DTT]) containing 2 U RNA polymerase, 1 U RNase inhibitor and 10 μl rNTP mix (final concentration, 2 mM each NTP) in a 50 μl reaction. Post-incubation at 37°C for 1 h, contaminating DNA in the reaction was removed by adding Turbo DNase I and incubation at 37°C for 15 min. The reaction was stopped by adding 5 μl of DNase I Inactivation reagent (Thermo Fisher Scientific, USA). After vortexing and centrifugation at 2,650 g for 5 min, the aqueous phase (30 μl) was passed through a G-50 column. The purified RNA was quantified and stored at -70°C.

Assay for Rho dependent transcription termination using the cell-free expression system (in vitro transcription translation reaction)

We used the pGal plasmid as DNA template for in vitro transcription-translation reaction. The reaction was performed using E. coli extract (S30) containing E. coli RNA polymerase holoenzyme/T7 RNA polymerase (Bioneer, Korea) for transcription and all essential components for translation [28], according to the manufacturer’s instructions. In short, DNA template (10 nM) and pRho plasmid (where rho is under the T7 promotor/terminator and RBS [Ribosomal Binding Site] control) was incubated at 30°C for 30 min containing the E. coli extract in reaction buffer (Bioneer, Korea) (that includes including amino acids, rNTPs, tRNAs, an ATP generating system, IPTG and appropriate salts) with 0.5% (w/v) galactose in a 60 μl reaction. Post-incubation at 30°C for 30 min, the reaction was stopped by adding equal volume of phenol: chloroform: isoamyl alcohol 25:24:1 (Merck, USA). After vortexing and centrifugation at 2,650 g for 10 min, the aqueous phase (30 μl) was passed through a G-50 column. The purified nucleic acids were used for 3’RACE reactions.

Western blot analysis

For the western blot analysis of the Gal proteins, we inserted in frame a ‘His-tag’ (a string of histidine residues) at the end of the open reading frame of each gal gene of the gal operon DNA in the plasmid pGal, generating plasmids; pGalE-histag, pGalT-histag, pGalK-histag, and pGalM-histag. MG1655 or MG1655Δspf strain harboring each one of the plasmids was grown in M9-galactose to OD600 of 0.6. Proteins in crude cell lysate from each culture (5 μg) were separated on a SDS-acrylamide slab gel electrophoresis and blotted on a nitrocellulose membrane using a protein blotter (Bio-Rad, USA). The blots were probed with the His-tag antibody (Merck, Germany) according to manufacturer’s instructions.

Supporting information

S1 Fig. Correspondence of intensities of galET bands between northern blots and 3’RACE analysis results.

In the absence of Spot 42, particularly the galET band in (A) disappears from the northern blot (lane marked Δspf), as was seen in Fig 1B. Similarly, in Δspf cells, generation of the 3’ ends of galET-short and -long is significantly (~ 95%) inhibited. When Spot 42 was overproduced (spf*), the galET production increased 150% of WT (A), and the production of the 3’ ends of galET-short and -long also increased correspondingly (B).

(TIF)

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S2 Fig. Rho-dependent transcription termination in vitro assayed in a cell-free system.

3’RACE assay of galET mRNA 3’ ends from the cell-free system with pGal, pRho and 0.5% galactose, and either with no BCM (lane 1) or increasing concentrations of BCM (0.1, 0.5, 1, 5 and 10 μg/ml) (lanes 2–6).

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S3 Fig. Growth rate of various cells in the exponential phase in the M9-galactose medium.

(A) MG1655 (blue) and MG1655 Δspf (red). (B) MG1655Δgal /pGal (blue), MG1655Δgal /pgalMMI-1 (green) and MG1655Δgal /pgalMMII-1(red). (C) MG1655ΔgalΔspf /pGal/pSpot42 (blue), MG1655Δgal Δspf /pgalMMI-1/pSpot42 (brown), MG1655ΔgalΔspf /pgalMMI-1/pSpot42MMI-1 (gray), MG1655ΔgalΔspf/pgalMMII-1/pSpot42 (green) and MG1655ΔgalΔspf /pgalMMII-1/pSpot42MMII-1(red).

(TIF)

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S4 Fig. 3’ RACE assay of galET 3’ends in MG1655ΔrppH, a strain deficient in RNA pyrophosphohydrolase.

RNase E binds to mono-phosphorylated 5’ end of sRNA. This ability enables RNase E to be recruited to the site of cleavage on target mRNA (1). To see if RNase E cleavage that generates galET-long depends on mono-phosphorylated 5’ end of Spot 42, we assayed the 3’ ends of galET in MG1655ΔrppH strain where the gene for the RNA pyrophosphohydrolase is deleted from the chromosome. Without the RNA pyrophosphohydrolase, most of the 5’ end of RNA remains in di-phosphorylated state [33]. Results showed no difference in generation of the 3’ end of galET-long and -short between WT (lane 1) and MG1655ΔrppH (lane 2) strains. These results demonstrate that the mono-phosphorylated 5’ end of Spot 42 is not a requirement for the generation of galET-long.

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S1 Table. Primers used in this study.

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S2 Table. Bacterial strains used in this study.

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Click here for additional data file.^{(19.8KB, docx)}

Acknowledgments

Authors are grateful to Prof. Gerhart Wagner (Uppsala U, Sweden) and Dr. Nadim Majdalani (NIH, USA) for their thoughtful comments. Authors thank Jeongok Park for cell growth measurements.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This research was funded by the Basic Science Research Program of the National Research Foundation of Korea: 2017R1D1A3B03027965, 2018R1D1A3B07044032, 2020R1A2C200633611 to H.M.L., and 2017R1A5A2015385 to H.J.J. This research was also funded by the Intramural Research Program of the Center for Cancer Research, NCI, NIH, of USA to D.K.C. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS Genet. doi: 10.1371/journal.pgen.1009878.r001

Decision Letter 0

Josep Casadesús, Jue D Wang

14 Jun 2021

Dear Dr Lim,

Thank you very much for submitting your Research Article entitled 'sRNA‑mediated regulation of gal mRNA in E. coli: Involvement of transcript cleavage by RNase E together with Rho‑dependent transcription termination' to PLOS Genetics.

The manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important problem, but raised some substantial concerns about the current manuscript. Based on the reviews, we will not be able to accept this version of the manuscript, but we would be willing to review a much-revised version. We cannot, of course, promise publication at that time.

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Josep Casadesús

Section Editor: Prokaryotic Genetics

PLOS Genetics

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: This is a beautifully written manuscript dealing with the regulation of the E. coli gal operon by Spot 42, an sRNA. The findings are surprising and quite novel. Spot 42 promotes Rho-dependent termination at galK as well as degradation of the galK transcript. Amazingly, these effects are performed by different SpoT 42 sequences, both complementary to galK mRNA.

Critique:

1. There are several typos that need correcting.

2. Eliminating one of the two SpoT activities does not increase the activity of the other, suggesting that the substrate mRNA is in excess. It may suggest that SpoT has two structures that are not in equilibrium. Is anything known about the structure of this sRNA?

3. The overall efficiency of SpoT processing is very low. I wonder how a SpoT could affect cell growth. And whether it plays a significant role in Gal regulation.

4. If would be useful to indicate the extent of processing under the gel lanes (Fig 1).

5. Fig 3C needs significance calculations - what is Fig3D looking at? Also, what does the time course mean – it the values are quite variable.

6. Fig 4 Is the lettering correct? The band at 2184 doesn’t correspond to anything in vivo. What does this figure add to understanding the mechanism of action of SpoT? Unless you can explain why it isn’t seen in vivo.

7. Fig 5 – statistical significance not given.

Reviewer #2: I have reviewed the manuscript by Jeon et al. submitted to Plos Genetics. The manuscript basically reports investigation of mRNA metabolism originating at the gal operon in E. coli. Specifically, the authors deal with post-initiation processing of RNA and transcription termination, and the regulation of these events. More than 50 years ago, a phenomenon was discovered, called Natural Polarity, which is decreased expression of distal cistrons compared to the expression of promoter proximal cistrons in a polycistronic operon. Regulations both at the transcriptional and translational level have been independently suggested to be responsible for natural polarity but the exact mechanism(s) of natural polarity remained unproven for decades. For the first time, by in depth analysis of the gal operon in E. coli the current authors have demonstrated that the phenomenon is complex and occurs at different levels: transcription termination, RNA processing and translation. A forerunner paper analyzing natural polarity was published in 1980s in the his operon of Salmonella although determining the precise mechanisms was not possible at the time because of lack of modern technologies. In the current manuscript, Jeon et al. showed how a small RNA participates in creating natural polarity in all three steps – transcription termination, RNA processing and translation. It is also noteworthy that involvement of small RNA in Rho-mediated transcription termination is novel.

In gal, the natural polarity is condition dependent – only shown in the absence of cAMP and has huge physiological implications.

The experiments are done in meticulous details with clear and convincing results. Importantly, the authors also nicely correlate the implications of the mechanisms with physiological experiments. I strongly recommend that the manuscript should be published as soon as possible. However, I suggest that several minor editorial corrections before publication. They are:

Line 63. Mention the 3’ endpoint if known.

Line 101. Delete the phrase “rather than by new synthesis”

Line 170. It appears that region III of the small RNA does not have a role. The authors may speculate why not.

Lines 241-3. Bicyclomycin does not inhibit Rho activity completely. It is suffice to see A partial effect of the drug is expected. It is not necessary to invoke another factor.

Line 248. Use the phrase ‘in the presence of’ of ‘adding’.

Line 251. Same. ‘In the presence’ of plasmid pRho instead of ‘adding’.

Lines 260 and 267. Same. ‘In the presence’ of pGal, etc.

Line 270. ‘in the galK gene sequence’.

Line 300. …. at ‘the’ galT…

Line 313. Here ‘we’ also found….

Line 317. “Increased”?

Line 327. It is better to say ‘…. Expression facilitating the anabolic role of the pathway’.

Starting line 328. Great experiments showing the physiological relevance of spot 42 in gal operon expression.

Line 352. Extend the sentence ‘E. coli .. at post transcription initiation level.

Line 373. The unknown enzyme could be RNase II as shown by the authors (ref. 4).

Lines 374-377. See comments on lines 241-243.

Line 410. I agree. PNPase acts the same way as RNAse II does.

Line 433. …in ‘polycistronic’ gene regulation.

Discussion. It is bit long, and may be somewhat condensed by removing a few of the ‘remains to be investigated’ topics.

Discussion. I suggest that the authors mention the relationship of cAMP and polarity in gal that I discuss above simply because the relationship has been unraveled in the paper.

Reviewer #3: The manuscript by Jeon et al investigates regulation of the E. coli gal operon by Rho, RNaseE, and a small RNA Spot 42. This work builds on previous observations published in 2014 and 2015. The authors show that binding of Spot 42 to the gal mRNA leads to Rho-dependent termination (that was already shown by the authors) and RNase E cleavage at two closely-spaced sites.

In my opinion, the insights provided by this work fall short of the PLOS Genetics standards.

Binding of Spot 42 to the TIR of galK is expected to inhibit translation, making RNA susceptible to Rho and RNA degradation. In their previous PNAS paper, the authors proposed just that. The new data presented in this work are (i) effects of mutations in mRNA, sRNA, and compensatory mutations on the formation of two mRNA species and (ii) the involvement of RNaseE, but not RNaseP or G, in mRNA processing. With respect to Rho, the authors add a lot of small new pieces, including in vitro coupled assay in which Rho is expressed. However, nothing principally new is shown and their previous conclusions do not change. It is well established how Rho works. On that note, the authors should brush up on Rho literature – papers they cite on the mechanism of action of BCM and Rho itself do not reflect current knowledge in the field, and their discussion does not incorporate recent findings. Similarly, the collection of transcription-translation references is really odd.

By contrast, data on RNAseE are limited to one gel with a ts mutant. If the authors want to make their study impactful, they should investigate this aspect of the regulatory mechanism.

With respect to Spot 42, the authors should inhibit translation of galK by other means (many possibilities) to distinguish different aspects of its contribution to gal control. The authors conclude that Spot 42 “directly” controls termination based on data in Fig. 4 – this is impossible to tell in the assay used, as all RNA synthesis goes up upon the addition of Spot 42 RNA, not only the species authors mention. In this and other assays, a normalization control is essential.

Some aspects of other assays are puzzling. For example, in Fig. 3, the authors argue that a small effect of Rho inhibitor BCM after extended incubation is supportive of their conclusions. Rho terminates 20% genes in E. coli, and BCM leads to a lot of secondary effects; particularly after 1 hour. In this case, error bars would be critical but are absent.

The authors mention the use of strain with a non-functional Rho – they must elaborate as rho is an essential gene in E. coli unless RNase H/UvsW is overexpressed.

Comparing reporter fusions to tRNA-Arg gene +/-rut makes no sense. One should do RT PCR probing regions before and after terminator. And the effects presumably due to termination are tiny! There are tens of well-characterized mutants in Rho and NusG that could be used to probe the role of Rho.

Making physiological arguments based on plasmids that express regulatory factors is not acceptable. MG1655 can be genetically modified as needed.

Most importantly, the authors discuss possible effects of uncoupling of transcription and translation and the roles of small RNAs. There is no doubt that sRNAs have a lot of targets and control expression of many genes. But the effects reported here are small, not necessarily direct, and need to be investigated in more detail.

Reviewer #4: In this work, Jeon and Lee et al. build upon previous analyses of regulation in the well-studied E. coli gal operon. The authors present the interesting finding that both RNase E and Rho, in conjugation with the sRNA, Spot 42, control formation of alternative forms of the galET sub-transcript (long and short forms, respectively). In particular, base-pairing between Spot 42 region I and the gal transcript favors generation of galET-long, and base-pairing between Spot 42 region II and the gal transcript favors generation of the galET-short transcript. galET-short generation is dependent upon Rho factor activity, whereas galET-long generation is dependent on RNase E activity. Finally, through specific base-pairing interactions, Spot 42 is predicted to help direct metabolites to anabolic pathways during growth in minimal medium. Overall, this work should be of interest to the field, but the manuscript requires both major (including one experiment) and minor revisions as outlined below.

The major revisions needed for this work center around three points. The first point is that information about biological replicates and numbers of blots represented by those presented should be included in the manuscript. The second point centers around Spot 42-mediated, Rho-dependent termination in vivo and whether Fig 5 is sufficient to establish this mechanism, including a need for information about statistical tests used. It is unclear if Spot 42 region I also contributes to Rho-dependent termination from the data presented (i.e., whether RDT may still be promoted via Spot 42 region I, but processing of the gal transcript to galET-short fails without region II base-pairing). Finally, the data presented in Fig 6 seem to test a prediction that was already known to be true rather than directly testing the contributions made by regions I and II to Gal protein level tuning to add biological relevance to the differential processing mechanism described in this work.

Major revisions:

1. The authors do not provide clear statements about the number of biological replicates used to obtain data presented in the figures (e.g., describing the number of replicate blots representative of blots shown represent). This information is especially important for instances where differences are modest and, as such, should be included. In the same vein, it is also not clear where the data for Fig 3D were derived. In particular, were the mean and SD determined from the intensities in Fig 3C? It may be more informative to show comparisons at each time point (e.g., as a line graph), using p-value adjustments for multiple comparisons (error bars should also be included on each point in Fig 3C—the comparisons would, thus, be between galET-short values at each time point or galET-long values at each time point only, rather than between the two isoforms at each time point). As shown in the same figure, the decrease of galET-short over time seems unclear (in fact, from 10 to 20 minutes the level seems to be increasing; the level at 30 minutes is similarly higher than at 10 minutes, and the levels at 40 and 50 minutes seem to be similar to that at 10 minutes). The authors should also mention that the concentration of BCM used is sub-inhibitory and thus is unlikely to completely abrogate RDT. Finally, the authors should provide an explanation for this ambiguous galET-short depletion over time in the text.

2. The authors should revise figure captions to clearly define the data shown in the figures (i.e, with the exception of Fig 3, it is unclear that bars or points represent a mean with error bars representing SD [how many replicates] or some other metric of variability). In Fig 5, are error bars shown for pHL2000? In the same figure, statistical tests should be performed and reported for the comparison of pHL2000 and pHL2184 (all panels). Are these differences significant? If these differences are not (B and E) or are (C and D), it is unclear what this figure adds to the manuscript. Depending on results of the statistical analysis (or if a statistical analysis cannot be performed), the authors should consider removing the figure (and editing in the text for this modification accordingly).

3. Nothing presented in Fig 2, 3, 4, and 5 suggests that some level of RDT cannot be accomplished through Spot 42 region I hybridization (or that region II hybridization alone is sufficient for RDT), just that the short isoform does not accumulate in region II variants. Unless an experiment that determines whether region I/II variants disrupt RDT can be performed, region I hybridization should be depicted in Figure 7A; the possibility of region I contributions to RDT should also be acknowledged in the text.

3. The authors seem to make an assumption (given the data presented in Fig 6) that defective Spot 42 base-pairing (Table 1) leads to an altered (reduced) metabolite flux toward an anabolic pathway (possibly reduced metabolite moving toward UDP-glucose/galactose) through Gal protein fine-tuning. However, such specific data is lacking in the manuscript and would make a substantial contribution to Fig 6 (i.e., western blots of Gal protein levels with ectopic expression of Spot 42 [and region I and II variants, along with associated suppressors]).

Minor revisions:

Ln 31: Use “Rho” in place of “the Rho factor”.

Ln 83: Put commas between Spot 42: “The sRNA, Spot 42,…”

Ln 98: Delete the comma after reference 24.

Lns 100-2: It is unclear what the authors mean by new synthesis not being required for the two different 3’ ends (new synthesis would be required [galET-long cannot be generated from galET-short, and it was not concluded that galET-short can be generated from galET-long]).

Lns 107-10: Please add a reference for Spot 42 remaining maximal in LB (especially w/o glucose) at 0.6.

Lns 134-6: This sentence (“Using 3’ RACE…”) seems like it would be better with the following paragraph (beginning at Ln 137).

Ln 212: This section caption is especially uninformative. Consider changing to: “Both RNase E and Rho serve a function in the mechanisms generating unique galET 3’ ends”

Ln 231: Consider using: “To test whether…” vs. “To test if…” here and elsewhere.

Ln 235: Perhaps it would be better to say “We found that…” vs. “The results showed that…” (less passive).

Ln 237: This can be part of the previous paragraph; the line space is not necessary.

Ln 238: This may be changed to: "In particular, as a functional Rho is present in the rnets mutant grown at a non-permissive temperature, the drastic reduction in galET-long levels indicates Rho does not serve a significant function during the generation of this transcript isoform."

Ln 241: Consider changing "...but this requires RNase E" to "... in an RNase E-dependent manner"

Ln 246: Consider using “The cell-free system contains all…” instead of “The cell-free system is supposed to contain all…”

Ln 273: It seems appropriate to speculate that RNase E may serve some function generating the 2,121 and 2,166 nucleotide RNAs in the text; the BL21(DE3) cells still produce a functional RNase E (vs. the less functional truncated RNase E variant from BL21 Star [DE3]).

Ln 276: Double-check reference formatting (other references are in brackets)

Ln 298: The authors should explain the properties of the rho::AmpR allele. Is the rho nonfunctional? Rho is essential; therefore, this mutation is unlikely to produce a nonfunctional variant of Rho. In what way do the properties of rho::AmpR differ from rho15(Ts)?

Ln 308: This would be a good spot to introduce the supplemental data on exoribonucleases (vs. introducing this particular new data in the discussion). Although, the results are negative, as noted in the manuscript discussion (should also be acknowledged at this line in the text if the figure is added here), these RNases have redundant function (double mutants are not viable). It may be worthwhile to speculate here on the function of RNases toward dsRNA (such as RNase III) for generation of galET-short. The point could be made that some RNase is necessary to generate galET-short (the transcript from RDT alone would produce a transcript that is too long to be this isoform). This point would be parallel to the section beginning at Ln 399 in the discussion.

Ln 313: I believe the line should read: "Here we also found...."

Ln 330: change to “…where both anabolism and catabolism of galactose are required.”

Lns 339-41: Consider editing to: “In exponential growth phase, the doubling times of cells encoding galMMI-1 or galMMII-1 (SI Appendix, S1 Fig B) were 22 and 26 % longer than those encoding the WT gal sequence, respectively (Table 1).”

Ln 386: Although the authors present data suggesting the 5’ phosphorylated state of Spot 42 state does not significantly contribute to RNase E-mediated gal transcript processing, it does not rule out the potential internal entry (5’ bypass) pathway also noted in reference 43 (Bandyra et al. 2012. Mol Cell 47:943-53). As the 5’-dependent pathways seem to have unclear importance (or possibly be redundant to the internal entry pathway) for Spot 42-promoted RNA processing, it seems relevant to note the possibility that this alternative pathway makes a contribution to gal transcript processing in the text.

Ln 775: Delete the comma at the end of the line.

Lns 799-801: The mutation names should match what is shown in the figure (i.e., these can be: “GW20 (amsts [rnets]…”

Ln 806: Colors of the bars should be noted with respective transcript isoforms.

Ln 809: Change the figure panel lettering (and corresponding caption and spots in the text) such that the blots are A-C (look at Ln 813; the ladders are in all three blots, but not B).

Ln 817: I believe this is a sliding window GC-content plot; it is customary to say this is what is being depicted and the window size being used to determine GC-content (in the figure caption and text).

Ln 823: The word “the” should be lowercased. Also, please decide on a consistent choice for capitalization state of “lane” in the caption.

Ln 830: “were taken…” instead of “was taken…”

Ln 834: Technically, the figure does not assay gal expression. The caption should reflect tuning of protein levels toward an anabolic pathway.

Ln 865: The color gray should be lowercased. Also, depending on formatting, these text colors may not be included in the final manuscript version of the manuscript.

Ln 886: Figure S4 is not referenced anywhere in the text. Please make reference to this figure where appropriate.

Ln 935: The font is difficult to read on Fig 6B. It would be more informative to show the individual growth curves (vs. mean and SDs). Do the authors have data points for LB+0.5% galactose (Δspf) beyond 500 minutes?

Ln 958: It would be useful for the authors to show a representation of the assumed cleavage events leading to galET-short in the model (perhaps just by noting “RNase?” in figure panel 7B). It would also be helpful if “RNase E/RNase?” (instead of RNase E alone) cleavages were shown closer at the relevant ends of galET-long and -short (it is unknown precisely where the cleavage/processing events happen, so drawing the cleavage at relevant sites would not be any less inaccurate).

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: None

Reviewer #4: Yes

**********

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Reviewer #1: No

Reviewer #2: Yes: Sankar Adhya

Reviewer #3: No

Reviewer #4: No

PLoS Genet. 2021 Oct 28;17(10):e1009878. doi: 10.1371/journal.pgen.1009878.r002

Author response to Decision Letter 0

7 Sep 2021

Attachment

Submitted filename: Response to reviewers.docx

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PLoS Genet. doi: 10.1371/journal.pgen.1009878.r003

Decision Letter 1

Josep Casadesús, Jue D Wang

29 Sep 2021

Dear Dr Lim,

The manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important topic but identified some concerns that we ask you address in a revised manuscript

We therefore ask you to modify the manuscript according to the review recommendations. Your revisions should address the specific points made by each reviewer.

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1) Provide a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript.

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We hope to receive your revised manuscript within the next 30 days. If you anticipate any delay in its return, we would ask you to let us know the expected resubmission date by email to plosgenetics@plos.org.

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Jue D. Wang

Associate Editor

PLOS Genetics

Josep Casadesús

Section Editor: Prokaryotic Genetics

PLOS Genetics

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: The revised manuscript is much improved and answers the criticisms to this reviewer's satisfaction.

Reviewer #2: The authors have satisfactorily revised the manuscript adequately responded to the criticisms of the reviews. I recommend its publication in Plos Genetics.

Reviewer #3: A revised manuscript by Jeon et al. presents some additional data, most notably on the involvement of RNAses in the gal operon regulation, and remove other pieces of data that were questioned by the reviewers. The manuscript is easier to follow and provides some interesting insights into gal control. It is customary, however, to ask if the authors have addressed all the reviewers’ concerns. This is not the case with mine.

I am still not convinced that the data on Rho have any novelty. It is encouraging that Rho terminates transcription when RNAP is paused at a consensus site, since this is consistent with what we know. But there does not seem to be any direct interplay between Spot 42 and Rho. The authors mention a possibility that Spot 42 RNA binding to region 1 could have an effect on termination (incl. in Figure 7) but nothing in their data suggests this. In the first round or review, I wrote: “the authors should inhibit translation of galK by other means to distinguish different aspects of its contribution to gal control”. This would have been a very simple way to test if Spot 42 does anything in addition to inhibiting translation initiation. Since the plasmids are used in the main “extract” assay, a substitution of the galK start codon would take very little effort. In the absence of this, or any other similar evidence, the null hypothesis is that spot42 inhibits translation, just as expected, and this promotes Rho-dependent termination.

Another request that was made by me and other reviewers but was ignored is a need to report error analysis that would indicate that the results are reproducible. I understand that not every reviewer’s comment has to be addressed, as long as a valid reason is offered. I do not, however, think that the lack of evidence in support of reproducibility is acceptable. The figure legends have to state the number of replicates used. If any fold effect is stated (2-fold, 50% etc), it must be accompanied by error analysis. Otherwise, the numbers should be substituted by some qualitative terms.

With respect to 2 different mechanisms of control, what is really happening here is that the inhibition of translation by spot 42 leads to generation of two shorter RNA species, one via premature termination of the nascent RNA (possibly every transcript, if the pause site in question is strong), the other – by ribonuclease processing of RNAs that have been completely made. In both cases, spot 42 job is to make sure that the proximal RNA, and thus proteins, are present in excess. But from the physiological point of view, these two RNAs should be identical, as their ends are closely spaced. So the outcome is actually the same, just the means differ.

I suggest making the following changes to Figure 7:

Panel A: remove region I base pairing from the picture and draw ribosome at a real scale = it protects ~23 nt and depicting this correctly would help to show why base pairing in region II does its job

Panel B: in late 2020, papers showing that Rho travels with RNAP and terminates transcription when the enzyme pauses and RNA is captured were published. They should be cited, and the cartoon should reflect these new data.

Reviewer #4: The authors have done a good job addressing most the reviewer’s comments and have added new results describing how another well-characterized RNase (RNase III) serves a function generating galET-short. This new finding adds to the authors’ story of how different binding events and parts of Spot 42 RNA potentiate different galET processing events. The authors are suitably careful to leave open the possibility that RNase III processing may be the result of dsRNA that forms intramolecularly vs. between gal and Spot 42—this possibility is acknowledged in the model figure. However, two major, related points raised in the initial review remain unaddressed after revision. These unadressed points concern reproducibility of the results in the form of enumerating the number of replicates performed or their statistical analysis. Addressing these points will greatly strengthen the conclusions presented in this manuscript.

Major points

1. The authors need to include information regarding the number of replicates performed for representative blot presented in the manuscript. This need could be met simply by stating in the figure captions: “The data presented in these blots are representative of X independent experiments.”

2. Fig 6B. Error bars are depicted, but it remains unclear from the text and figure legend what these error bars represent. Presumably, these are the same errors that are propagated to determine the errors in the calculated doubling times presented in Table 1. The error analysis must be fully explained. Are they standard deviations of replicate data? Standard errors of means? The authors should also specify “n=x”, where x is the number of replicates.

Minor point

1. In the Figure 7 caption, it would be better to state directly that “RNase(?)” represents currently unidentified RNases(s) that may be involved in the further processing events of the galET transcripts.

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: No: see review comments.

**********

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Reviewer #1: Yes: max gottesman

Reviewer #2: Yes: Sankar Adhya

Reviewer #3: No

Reviewer #4: No

PLoS Genet. 2021 Oct 28;17(10):e1009878. doi: 10.1371/journal.pgen.1009878.r004

Author response to Decision Letter 1

4 Oct 2021

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PLoS Genet. doi: 10.1371/journal.pgen.1009878.r005

Decision Letter 2

Josep Casadesús, Jue D Wang

14 Oct 2021

Dear Dr Lim,

We are pleased to inform you that your manuscript entitled "sRNA‑mediated regulation of gal mRNA in E. coli: Involvement of transcript cleavage by RNase E together with Rho‑dependent transcription termination" has been editorially accepted for publication in PLOS Genetics. Congratulations!

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Yours sincerely,

Jue D. Wang

Associate Editor

PLOS Genetics

Josep Casadesús

Section Editor: Prokaryotic Genetics

PLOS Genetics

www.plosgenetics.org

Twitter: @PLOSGenetics

----------------------------------------------------

Comments from the reviewers (if applicable):

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #3: The revised manuscript addresses most of my concerns and is suitable for publication.

Reviewer #4: The authors have fixed the remaining small issues with the manuscript.

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Reviewer #3: None

Reviewer #4: Yes

**********

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Reviewer #3: No

Reviewer #4: No

----------------------------------------------------

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PLoS Genet. doi: 10.1371/journal.pgen.1009878.r006

Acceptance letter

Josep Casadesús, Jue D Wang

25 Oct 2021

PGENETICS-D-21-00643R2

sRNA‑mediated regulation of gal mRNA in E. coli: Involvement of transcript cleavage by RNase E together with Rho‑dependent transcription termination

Dear Dr Lim,

We are pleased to inform you that your manuscript entitled "sRNA‑mediated regulation of gal mRNA in E. coli: Involvement of transcript cleavage by RNase E together with Rho‑dependent transcription termination" has been formally accepted for publication in PLOS Genetics! Your manuscript is now with our production department and you will be notified of the publication date in due course.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Correspondence of intensities of galET bands between northern blots and 3’RACE analysis results.

(TIF)

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S2 Fig. Rho-dependent transcription termination in vitro assayed in a cell-free system.

(TIF)

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S3 Fig. Growth rate of various cells in the exponential phase in the M9-galactose medium.

(TIF)

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S4 Fig. 3’ RACE assay of galET 3’ends in MG1655ΔrppH, a strain deficient in RNA pyrophosphohydrolase.

(TIF)

Click here for additional data file.^{(375KB, TIF)}

S1 Table. Primers used in this study.

(DOCX)

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S2 Table. Bacterial strains used in this study.

(DOCX)

Click here for additional data file.^{(19.8KB, docx)}

Attachment

Submitted filename: Response to reviewers.docx

Click here for additional data file.^{(41.8KB, docx)}

Attachment

Submitted filename: Response to Reviewers 2.docx

Click here for additional data file.^{(17.7KB, docx)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

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PERMALINK

sRNA-mediated regulation of gal mRNA in E. coli: Involvement of transcript cleavage by RNase E together with Rho-dependent transcription termination

Heung Jin Jeon

Yonho Lee

Monford Paul Abishek N

Xun Wang

Dhruba K Chattoraj

Heon M Lim

Roles

Abstract

Author summary

Introduction

Fig 1. Spot 42 promotes generation of galET mRNA form galETKM operon.

Results

Spot 42 promotes processing of gal operon mRNA to galET mRNA

Specific base-pairing of Spot 42 and gal mRNA is critical for the generation of galET 3’ ends

Fig 2. Specific base-pairing of Spot 42 and gal mRNA is critical for the generation of galET 3’ ends.

RNase E is involved in the generation of galET-long 3’ ends

Fig 3. RNase E is involved in the generation of galET-long 3’ ends.

Rho-dependent transcription termination generates galET-short 3’ ends

Fig 4. Rho-dependent transcription termination in vitro assayed in a cell-free system.

RNase III-mediated transcript cleavage results in generation of galET-short 3’ ends

Fig 5. Generation of galET-short 3’ ends by RNase III-mediated transcript cleavage.

Spot 42 tunes production of enzymes for competing pathways of galactose anabolism and catabolism to optimize growth

Fig 6. Spot 42 regulates the tuning of gal protein levels towards an anabolic pathway.

Table 1. Doubling time of various gal and spf mutants in M9-galactose medium.

Discussion

Transcript cleavage and transcription termination are both involved in the generation of galET mRNA

Fig 7. Model of Spot 42 sRNA-mediated generation of galET RNA species by Rho and RNase E.

Processing of the 3’-OH ends generated by transcription termination or transcript cleavage

sRNA binding at intercistronic region and at 5’ UTR may have different outcomes

Relationship of cAMP, Spot 42 and polarity in gal operon

Materials and methods

Bacterial strains and primers

Bacterial growth conditions

RNA preparation

Northern blot analysis

Rapid amplification of cDNA ends (RACE)

Reverse Transcription-quantitative PCR (RT-qPCR)

Mutagenesis and quantification of Spot 42

In vitro synthesis and purification of Spot42 RNA

Assay for Rho dependent transcription termination using the cell-free expression system (in vitro transcription translation reaction)

Western blot analysis

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Josep Casadesús

Jue D Wang

Roles

Author response to Decision Letter 0

Decision Letter 1

Josep Casadesús

Jue D Wang

Roles

Author response to Decision Letter 1

Decision Letter 2

Josep Casadesús

Jue D Wang

Roles

Acceptance letter

Josep Casadesús

Jue D Wang

Roles

Associated Data

Supplementary Materials

Data Availability Statement

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