Regulation of ribonuclease E activity by the L4 ribosomal protein of Escherichia coli

Abstract

Whereas ribosomal proteins (r-proteins) are known primarily as components of the translational machinery, certain of these r-proteins have been found to also have extraribosomal functions. Here we report the novel ability of an r-protein, L4, to regulate RNA degradation in Escherichia coli. We show by affinity purification, immunoprecipitation analysis, and E. coli two-hybrid screening that L4 interacts with a site outside of the catalytic domain of RNase E to regulate the endoribonucleolytic functions of the enzyme, thus inhibiting RNase E-specific cleavage in vitro, stabilizing mRNAs targeted by RNase E in vivo, and controlling plasmid DNA replication by stabilizing an antisense regulatory RNA normally attacked by RNase E. Broader effects of the L4-RNase E interaction on E. coli transcripts were shown by DNA microarray analysis, which revealed changes in the abundance of 65 mRNAs encoding the stress response proteins HslO, Lon, CstA, YjiY, and YaeL, as well as proteins involved in carbohydrate and amino acid metabolism and transport, transcription/translation, and DNA/RNA synthesis. Analysis of mRNA stability showed that the half lives of stress-responsive transcripts were increased by ectopic expression of L4, which normally increases along with other r-proteins in E. coli under stress conditions, and also by inactivation of RNase E. Our finding that L4 can inhibit RNase E-dependent decay may account at least in part for the elevated production of stress-induced proteins during bacterial adaptation to adverse environments.

Over the past two decades, an understanding of mRNA decay pathways in Escherichia coli has advanced significantly (for reviews, see ref. 1–3), and RNase E has emerged as a key player in mRNA turnover as well as in the processing and decay of noncoding RNAs (e.g., rRNAs [4, 5], tRNAs [6, 7], M1 RNA [8], and 6S RNA [9]). RNase E is a multifunctional endoribonuclease (10) known to preferentially cleave RNA within AU-rich single-stranded regions (11, 12) enriched in specific sequence determinants (13). The level of this enzyme in vivo is controlled via autoregulation of its own synthesis (14–16).

In addition to its N-terminal catalytic domain (N-RNase E), RNase E contains a C-terminal region (C-RNase E) that serves as a scaffold (17, 18) for association with polynucleotide phosphorylase (PNPase), RhlB RNA helicase, and the glycolytic enzyme enolase to form the RNA-degrading complex known as the “degradosome” (19, 20). C-terminal truncation of RNase E, which prevents degradosome assembly, leads to accumulation of RNase E-targeted mRNAs (21, 22), suggesting that degradosome assembly and functional interactions of degradosome components are necessary for normal mRNA turnover in E. coli.

Although ribosomal proteins (r-proteins) function primarily as components of the translation machinery, some prokaryotic and eukaryotic r-proteins also have extraribosomal functions (23). For example, L4, an essential r-protein encoded by the S10 operon in E. coli is a regulator of both transcription and translation of its own operon (24, 25). The regions within L4 required for these distinct functions differ (26). Here we show that the E. coli L4 protein interacts with RNase E and that this interaction modulates RNase E activity, altering the steady-state level and decay of affected regulatory and messenger RNAs. As the abundance of proteins encoded by some of these mRNAs is known to increase along with free r-proteins in response to environmental stresses, our findings reveal a mechanism by which L4 may regulate the production of stress-induced proteins to enhance the survival of bacteria under adverse conditions.

Results

L4 Directly Interacts with the C-Terminal Region of RNase E in Vivo and in Vitro.

To identify low-molecular-weight (≤ 30 kDa) proteins that bind to RNase E, FLAG-tagged RNase E was overexpressed in E. coli and purified by affinity-chromatography as described previously (19). After electrophoretic analysis on 12% SDS gels followed by Coomassie Blue staining, the polypeptides co-purifying with RNase E were identified by mass spectroscopy. Several r-proteins, including L2, L3, L4, S3, and S4, were co-purified with the RNase E complex (the degradosome) (supporting information (SI) Table S1). We then used an E. coli two-hybrid system (27) to further investigate a possible interaction of each of these r-proteins with the major components of the degradosome: RNase E, PNPase, RhlB helicase, or enolase (Fig. S1). We observed that only L4 directly interacted with degradosome proteins binding to the C-terminal half of RNase E and also to PNPase (Fig. 1 A and B). Co-immunoprecipitation experiments confirmed that L4 bound to RNase E and this interaction of L4 and RNase E was likewise dependent on the C-terminal half of the enzyme (Fig. 1C, compare lane 2 with lane 6), in particular on two regions (684–784 aa and 985-1061 aa, Fig. 1B). Furthermore, by using micrococcal nuclease to digest RNA co-purifying with the RNase E complex (19, 28), we found that the association of L4 with RNase E is independent of RNA (Fig. 1D). In contrast, interaction between L4 and PNPase was weak (data not shown); we thus chose not to characterize it further.

Fig. 1.

L4 interacts with the E. coli degradosome in vivo and in vitro by binding to the C-terminal “scaffold” region of RNase E. (A) E. coli two-hybrid assays demonstrating L4 interactions with RNase E and other major components of the degradosome on MacConkey/maltose agar plates. The T25- and T18-based chimeric protein construct pairs (see SI Materials and Methods) screened for protein-protein interactions as well as positive [C(+), red colonies] and negative [C(−), white colonies] controls (see supporting ref. 3) are indicated for each sector of the plates shown. (B) The schematic representation of expressed polypeptides and results [positive (+) and negative (−)] of two-hybrid screening. (C) Analysis of the RNase E-L4 interaction by immunoprecipitation of cell lysates prepared from the wild-type BL21 (DE3) strain (rne⁺) and its isogenic mutant (rne131) encoding C-terminally truncated RNase E. Protein samples obtained by immunoprecipitation with L4-specific antibodies (IP), preimmune serum (PI) as well as total cell lysate (T), and proteins that were bound to beads nonspecifically (B) were separated by electrophoresis on a 10% SDS gel and analyzed by Western blotting by using antibodies specific for L4, full-length RNase E (FL-RNase E), N-terminal half of RNase E (N-RNase E), and PNPase. The polypeptide nonspecifically interacting with anti-L4 antibody (in PI and IP) is indicated by an asterisk. (D) Co-immunopurification of L4 and RNase E is not dependent on E. coli RNA. FLAG-RNase E (FR) was affinity-purified on an M2 column (Sigma) from E. coli cell extract (T) containing co-expressed FLAG-RNase E and HA-L4 before (−) and after (+) micrococcal nuclease treatment. The resulting samples of affinity-purified FLAG-RNase E were either analyzed by Western blotting (on the left) by using antibodies specific for RNase E or HA-tag or were extracted with phenol and analyzed by electrophoresis on a 2.0% agarose gel followed by ethidium bromide staining (on the right). M, 1 kb DNA ladder (Fermentas).

L4 is a structural protein of the 50S ribosomal subunit and also a regulator of both transcription and translation of its own operon (24, 25). These functions require two independent domains of L4 (26). To examine whether these domains are required also for interaction with RNase E, we separately co-expressed FLAG-tagged RNase E with HA-tagged L4 (control) or L4 mutants lacking either of these functional domains (Fig. S2A). Subsequently, by using affinity purification, we found that both mutant L4 proteins interacted with RNase E (Fig. S2A), thus suggesting that neither domain is essential for L4 binding to RNase E.

The L4-RNase E Interaction Inhibits RNase E Activity in Vitro and in Vivo.

To determine whether binding of L4 to RNase E affects RNase E endonucleolytic activity, we performed cleavage assays in vitro by using full-length RNase E (FLAG-RNase E) or its N-terminal catalytic domain (N-RNase E) in the presence of increasing amounts of FLAG-tagged L4 (FLAG-L4). Before testing cleavage ability, we used an in vitro translation system to confirm that FLAG-L4 was able to inhibit its own translation (Fig. 2A) and therefore functionally resembled the nontagged chromosomally encoded L4. Cleavage assays were carried out with oligonucleotide BR13 derived from RNAI, an antisense RNA, whose cleavage by RNase E controls replication of ColEI-type plasmids in vivo (29). As anticipated from previous studies (30), FLAG-RNase E efficiently cleaved this substrate; however, cleavage was reduced in the presence of increasing amounts of L4 (Fig. 2B, lanes 2–4). In parallel control experiments, no inhibition of the ribonucleolytic activity of RNase A was observed (data not shown). In addition, no inhibition was observed when FLAG-RNase E was replaced with N-RNase E containing the catalytic domain alone (Fig. 2B, lanes 7–9). This result suggests that L4 inhibits RNase E activity by interacting with the enzyme's C-terminal region.

Fig. 2.

Testing the functionality of FLAG-L4 and its ability to inhibit RNase E-mediated cleavage of BR13 in vitro. (A) Testing the ability of FLAG-tagged L4 to inhibit its own synthesis by using an in vitro translation system. The plasmid pET29-ΔS10 (depicted on the left) encoding the S10 leader sequence and the first three genes (rpsJ, rplC, rplD) of the S10 operon was used as a template for in vitro transcription-translation assays. Reactions were performed in the presence of ³⁵S-Met and increasing concentrations of FLAG-L4 and decreasing concentrations of BSA as indicated. The products of translation were analyzed by electrophoresis on a 12% SDS polyacrylamide gel and detected by autoradiography. (B) In vitro cleavage of oligonucleotide BR13 by the full-length RNase E (FL-RNase E) and its C-terminally truncated polypeptide (N-RNase E). Reactions were performed at 30 °C for 15 min in 20 μl with equimolar amounts of RNase E and N-RNase E (500 ng of each protein) and increasing amounts of FLAG-L4 (25 to 100 ng). The bands corresponding to BR13 and its cleavage product BR13₋₅ are indicated by arrows.

To learn whether L4 also can impair RNase E activity in vivo, we tested the effect of L4 ectopic expression on RNase E-mediated decay of RNAI and its consequences on the copy number of a ColE1-type plasmid in E. coli strains N3433 and BZ453 (31) expressing the full-length and C-terminally truncated RNase E polypeptides, respectively. Northern blot analysis revealed that elevation of L4 resulted in a prolongation of the RNAI half-life from 3.4 min to 5.7 min (Fig. 3A) and a reduction in the copy number of a ColE1-type plasmid in strain N3433 strain (Fig. 3C), but that no detectable effect on RNAI half-life or plasmid copy number was seen in the BZ453 strain (Fig. 3C). These results suggest that L4 binding to the C-terminal region of RNase E inhibits RNase E activity and leads to prolongation of the RNAI half-life, decreasing copy number of ColE1-type plasmids. Consistent with the previous data, we also observed that in vivo production of L4 leads to an increased level of endogenous full-length RNase E (Fig. S3) and its mRNA (see microarray data in Fig. 4B) in N3433 but not the BZ453 strain. As RNase E is known to autoregulate its own level (16) by cleaving its cognate mRNA, the observed increase in the full-length RNase E and rne mRNA levels is consistent with the observed inhibition of RNase E activity by L4.

Fig. 3.

Effects of L4 ectopic expression on the RNase E-mediated decay (A–C) and processing (D–F) of noncoding RNAs. (A and B) Northern blot data demonstrating that the half-life of RNAI increases after L4 ectopic expression from pPWflagL4 (L4) in a strain encoding the full-length RNase E (N3433) but not in a strain (BZ453) expressing only the N-terminal domain (1–602 aa) of this protein. The half-life of RNAI in the absence of L4 ectopic expression was also determined in the same strains harboring the plasmid pPW500flag (control) lacking the L4-coding sequence. (C) The observed increase in the half-life of RNAI upon L4 ectopic expression (A and B) correlates with a decrease in the copy number of ColE1-type plasmids in N3433 (lanes 1 and 2) when compared to that in BZ453 (lanes 3 and 4). Chromosomal (Chr) and plasmid (P) DNA are indicated. The larger size of plasmid DNA (P) indicated in lane 2 and 4 is due to the presence of an extra DNA fragment encoding L4. (D–F) Equal amounts of total RNA extracted from the E. coli wild-type N3433 (wt) strain carrying control and L4 expressing plasmids after induction with IPTG (0.5 mM) for 30 min as well as those prepared from the temperature sensitive N3431 (rne^ts) strain at permissive (32 °C) and nonpermissive (44 °C) temperatures were analyzed by Northern blotting. The ³²P -labeled oligonucleotide probes were complementary to M1 RNA (D), 6S RNA (D), 5S rRNA (E), and tRNA^Asn (F). None of the processing intermediates normally accumulating upon inactivation of RNase E in N3431 (lane 4) were detected in the presence of increasing levels of L4 in N3433 at 32 °C (compare lane 2 versus lane 1).

Fig. 4.

Microarray identification of specific mRNA targets dependent on the L4-RNase E interaction. (A) Shown is the experimental strategy used to perform microarray analysis. The E. coli strains N3433 and BZ453 encoding full-length (Rne) and C-terminally truncated RNase E (N-Rne), respectively, and carrying the control plasmids pPW500flag (vector only) and plasmid pPWflagL4 (vector + L4) encoding L4 were individually grown in LB medium at 32 °C to an OD₆₀₀ of ≈0.3 and then induced with IPTG (0.5 mM) for 30 min. Aliquots of total RNA extracted from each cell culture were used to synthesize cDNAs labeled with Alexa Fluor 647 or Alexa Fluor 555 as indicated on the scheme. After hybridization with a DNA microarray, the resulting patterns were analyzed by using GeneSpring software GX 7.3.1 (Silicon Genetics) and the results of this analysis were summarized in Table S3. (B) Microarray data (shown in duplicate) are for selected transcripts known to be controlled either by L4 or RNase E. As anticipated, several 5′-proximal species transcribed from the S10 operon including rpsJ, rplC, rplW, rplB, and rpsS were down-regulated upon L4 (rplD) ectopic expression. In contrast, the level of rne mRNA (rne) was up-regulated, whereas the abundance of other well known RNase E substrates (rpsT, ompA, and rpsO) was not affected. A reference color bar showing correlation between the observed color patterns and quantitative changes in transcript levels is provided in panel C. (C) Microarray data (shown in duplicate) are for 23 transcripts whose relative mRNA levels were significantly increased in N3433 (i.e., in the presence of L4-RNase E interaction) but not in BZ453 (in which L4-RNase E interaction is impaired). The complete list of 65 transcripts that were reproducibly up-regulated upon L4 ectopic expression in N3433 is shown in Dataset S1. The gradient color bar on the left indicates the relative decrease or increase in the level of individual transcripts. The differently colored circles (on the right) indicate functional classification of gene products (red, metabolism; green, transcriptional/translational regulation; blue, stress response; and black, hypothetical/uncharacterized).

As the C-terminal half of RNase E is required for interaction of this endoribonuclease with L4 (present study) but is dispensable for processing of stable RNAs (22), we hypothesized that the L4-RNase E interaction would most likely not affect stable RNA processing. Consistent with this notion, we found that the RNase E-mediated processing of 5S rRNA (32), tRNA (6, 7), 6S RNA (9), and M1 RNA (8), the catalytic RNA subunit of RNase P, in vivo was similar in the presence (L4) or absence (control) of L4 ectopic expression (Fig. 3 D–F, compare lane 2 with lane 1).

This finding enables the use of stable RNAs as internal standards for normalizing the amount of individual transcripts during L4 ectopic expression.

Microarray Identification of Transcripts That Are Affected by L4 in an RNase E-Dependent manner.

To determine the breadth of the effect of L4 inhibition of RNase E activity on the cellular abundance of E. coli transcripts in vivo, we used microarray analysis to compare the steady-state levels of 4290 E. coli transcripts in the presence (L4) or absence (control) of L4 ectopic expression by using the wild-type (N3433) and rne mutant BZ453 strains (Fig. 4A). As the RNase E polypeptide produced in strain BZ453 is C-terminally truncated, and therefore lacks the ability to interact with L4, transcripts whose steady-state level is specifically dependent on the L4-RNase E interaction were expected to be detected as RNAs that are up-regulated during L4 ectopic expression in N3433, but not in BZ453. We used these criteria to identify 65 transcripts whose relative levels were increased at least 1.5 fold in N3433 (see Table S2). Interestingly, these transcripts included products of five genes (cstA, yjiY, lon, hslO, and yaeL) found previously to be involved in bacterial stress responses, which are known to elevate the level of free r-proteins in vivo (33). Genes that encode remaining transcripts have been implicated in carbohydrate and amino acid metabolism and transport (membrane-associated proteins) (e.g., sdhA, sdhA, sdhC, and sdhD), transcription/translation (e.g., rimK, rpoC, rpoS, etc.), and DNA/RNA modification (rmuC, topA, and rne).

In addition, we found that among the 15% to 20% of genomic transcripts that were affected by the ectopic expression of L4 (Table S3), ≈3.6% (154) of transcripts were down-regulated and ≈2.3% (99) of transcripts were up-regulated in both the N3433 and BZ453 strains (i.e., their abundance was decreased or increased by ectopic L4 expression even in the absence of the RNase E segment required for interaction with L4), indicating the RNase E independence of this regulation. The down-regulated species include the polycistronic transcript detected with probes complementary to several ORFs (rpsJ, rplC, rplW, rplB, and rpsS, Fig. 4B) within the 5′-proximal portion of the S10 operon (Fig. 4B and Dataset S1), consistent with the previous finding that L4 is capable of down-regulating transcription and translation of its own operon (24, 25). Unlike its chromosomally encoded counterpart, FLAG-L4 was ectopically expressed from a high copy number plasmid and therefore the level of its own mRNA was among the transcripts up-regulated in both strains (Fig. 4B, and Dataset S1).

To corroborate the microarray data, perturbed expression of several of the genes most prominently affected by the L4-RNase E interaction (see Fig. 4C) was verified by quantitative real-time PCR (qRT-PCR). The qRT-PCR data obtained were in good agreement with the results of the microarray analysis (Table S3).

L4 Can Stabilize E. coli mRNA Species Encoding Stress-Induced Proteins.

Previous findings that the intracellular concentration of free r-proteins is elevated under stress conditions (33) raised the possibility that the observed L4-stimulated RNase E-dependent increase in abundance of stress-responsive transcripts (in particular, cstA, yjiY, lon, hslO, and yaeL mRNA) (Fig. 4C, Table S2), results from inhibition of mRNA decay by the L4/RNase E interaction. To test this notion, we first determined whether the half-lives of these transcripts are increased upon L4 ectopic expression by Northern blot analysis or ribonuclease protection assay. We found (Fig. 5A–D and Fig. S4A) that an increase in the level of L4 prolonged the half-life of each transcript, consistent with the hypothesized role of this r-protein in inhibiting mRNA turnover.

Fig. 5.

Northern blot analysis of transcripts whose rate of decay is controlled by RNase E in a L4-dependent manner. (A–D) Total RNA was isolated from the wild-type N3433 (wt) strain carrying plasmid pPW500flag (control) or plasmid pPWflagL4 encoding L4 (L4) after induction with IPTG (0.5 mM) for 30 min prior rifampicin treatment. Equal amounts of total RNA were analyzed by Northern blotting by using radioactively labeled RNA probes specific for the cstA, yjiY, lon, or hslO mRNAs (A–D, respectively) as described in Materials and Methods. The same procedure and probes were used to analyze RNA extracted from the rne wild-type N3433 (wt) and its isogenic mutant N3431 (rne^ts) at permissive (32 °C) and nonpermissive (44 °C) temperatures (see panel E–H). The half-life (t_1/2) of each transcript and standard deviations were calculated based on the intensity of radioactive signals normalized to the amount 16S rRNA (internal loading control) shown at the bottom of each panel. Due to the low intensity of signals presumably caused by rapid turnover of these transcripts at 44 °C, it was not possible to calculate their half-lives in N3433 (ND).

The above result (Fig. 5 and Fig. S4A) together with microarray data (Fig. 4C) argue that the observed stabilization of cstA, yjiY, lon, hslO, and yaeL mRNAs upon L4 ectopic expression results from inhibition of RNase E-mediated decay. Indeed, further analysis revealed that all five transcripts were stabilized upon inactivation of RNase E at nonpermissive temperature (44 °C) in an RNase E temperature-sensitive mutant (rne^ts) but not in the wild-type strain (Fig. 5 E–H and Fig. S4B; compare rne^ts with wt), demonstrating a role for RNase E in controlling the degradation of these mRNA species (cstA, yjiY, lon, hslO, and yaeL).

Discussion

Previous work has shown that RNase E along with PNPase, RhlB helicase, and enolase form a multienzyme complex termed as the “RNA degradosome.” Although a number of proteins are known to be present in this complex as minor components, very little is known about their specific roles in the regulation of degradosome activity, assembly, or composition (for review, see ref. 3). Here, we affinity purified RNase E and determined the nature of the co-purified proteins with molecular weights below 30 kDa. Although several r-proteins were found to co-purify with FLAG-RNase E (see Table S1), further analysis revealed that only one of them, the r-protein L4, could directly interact with the degradosome by binding to the C-terminal half of RNase E. L4, an integral component of the 50S ribosomal subunit, is known to regulate its own operon (i.e., the S10 operon) by repressing its transcription and translation (24, 25), which are actions involving two distinct nonoverlapping domains (26). In the present study, L4 mutant variants lacking either of these functional domains were still able to interact with RNase E (Fig. S2A), suggesting that L4 includes multiple domains that carry out extraribosomal functions.

Cleavage assays revealed that RNase E cleavage of oligonucleotide BR13 representing the 5′-end single-stranded segment of RNAI, an antisense RNA controlling the copy number of ColE1-type plasmids (29), decreased on L4 binding. Moreover, the L4-dependent inhibition of RNase E activity in vitro was not observed for an RNase E variant protein containing only the catalytic domain of the enzyme (Fig. 2). Consistent with the ability of L4 to inhibit RNase E cleavages in vitro, we found that ectopic expression of L4 in vivo increased the level and stability of RNAI in vivo, in turn resulting in a lower copy number of the resident plasmid in cells synthesizing full length RNase E but not in cells producing an RNase E variant that lacks the C-terminal region. Similarly, the L4-dependent decrease in RNase E activity also increased the level of rne mRNA (see Fig. 4B), which is known to be cleaved by this endoribonuclease during autoregulation of its own synthesis in vivo (15). Microarray analysis revealed that L4-mediated inhibition of RNase E activity was associated with an increase in the steady-state level of numerous mRNAs, including multiple stress-responsive transcripts (cstA, yjiY, lon, hslO, and yaeL). Moreover, Northern blot analysis revealed that the increased abundance of cstA, yjiY, lon, hslO, and yaeL mRNAs upon L4 ectopic expression resulted from inhibition of RNase E-dependent decay of these mRNAs.

The level of free r-proteins is known to increase with E. coli in response to stresses (e.g., temperature upshifts or amino acid starvation, a carbon source, nitrogen, or phosphate [33]) that trigger the nucleolytic attack of ribosomal RNA, leading to ribosome disassembly (reviewed in ref. 34). The stress-induced disassembly of ribosome particles and accumulation of free r-proteins, including L4, can potentially inhibit the RNase E-mediated decay of the above stress-responsive transcripts, thereby facilitating bacterial adaptation to adverse environments. Our finding that L4 can potentially increase the level of Lon by inhibiting the degradation of lon mRNA suggests that the L4-mediated inhibition of RNase E activity might contribute to the Lon-dependent degradation of r-proteins in response to amino acid starvation (stringent response) to provide the extra amino acids necessary for the synthesis of stress-related proteins (35).

Interestingly, our data also suggest that L4 does not act as a general inhibitor of RNase E to stabilize every mRNA whose decay is mediated by this enzyme (e.g., ompA mRNA, see Fig. 4C). This mode of L4 inhibitory action is similar to RraA and RraB, which are also inhibitors of RNase E and affect only selective groups of transcripts (36, 37). However, unlike RraA and RraB, proposed to impede RNA decay by remodeling the degradosome (36), L4 inhibits the RNase E-dependent RNA decay without altering the degradosome composition, which was found to be nearly the same in the absence or presence of L4 ectopic expression (Fig. S2B).

Our microarray data not only suggest the existence of multiple mRNA targets controlled by L4 in an RNase E-dependent manner but also disclose a large number of transcripts that are up- or down-regulated by L4 independently of the L4/RNase E interaction (Dataset S1). The group of down-regulated transcripts includes species that are transcribed from the S10 operon known to be controlled by L4 at the transcriptional and translational levels (24, 25). It seems possible that, similar to its control of the S10-derived transcripts, L4 might down-regulate the level of these mRNAs by impairing their translation, thereby increasing the level of ribosome-free mRNAs that are intrinsically more susceptible to degradation by the RNA decay machinery.

Materials and Methods

Bacterial Strains and Plasmids.

The E. coli strain BL21(DE3) (rne131) (22) encoding a truncated version of RNase E (amino acid 1–585), and its parental strain BL21(DE3) were used to prepare protein samples for immunoprecipitation, copurification of RNase E and L4 variants, and to overexpress FLAG-tagged proteins. The strain DHP1, an adenylate cyclase-deficient derivative of DH1 (38), was used in E. coli two-hybrid assays to study protein-protein interactions in vivo (27) as described in the SI Materials and Methods. The E. coli K-12 strains N3433 (39) and BZ453 (31) encoding the full-length RNase E and its truncated version (amino acid 1–602), respectively, were used for determining RNA stability, plasmid copy number, detection of endogenous RNase E, and microarray analysis. The pnp⁻ strain YHC012 (40) was used to purify FLAG-tagged RNase E, N-RNase E (amino acid 1–597), C-RNase E (amino acid 499–106), and FLAG-L4 proteins.

Basic Biochemical and Molecular Biology Techniques.

Protein purification, RNase E cleavage assays, Northern blot analysis, and plasmid copy number determination were performed as previously described (12, 19, 29) (see SI).

Testing the Functionality of Affinity-Purified FLAG-L4.

To confirm that, similar to its chromosomally encoded counterpart, the recombinant FLAG-L4 can autoregulate its own synthesis by binding to the S10 leader (41), we carried out in vitro translation by using pET29-ΔS10, a pET29-based plasmid encoding the S10 leader-rpsJ-rplC-rplD region, as a template and an in vitro translation kit (Rapid Translation System RTS 100, E. coli HY kit, Roche). The resulting ³⁵S-labeled products of translation were further analyzed on 12% SDS polyacrylamide gels followed by exposure to x-ray films.

Microarray and Quantitative RT-PCR.

The relative mRNA abundance of 4290 E. coli transcripts was analyzed in the presence (L4) or absence (control) of L4 ectopic expression by using the wild-type (N3433) and rne mutant BZ453 strains carrying the plasmids pPW500flag (vector only) and pPWflagL4 (vector plus L4). RNA was isolated from cells grown at 32 °C and induced with IPTG at an OD₆₀₀ ≈ 0.3 for 30 min. RNA isolation, synthesis of fluorescently labeled cDNA, hybridization to microarrays, and data analysis were performed as described in SI Materials and Methods to generate Dataset S1. For quantitative RT-PCR, gene-specific primers (see Table S4) were designed with the Beacon Designer 5 software package (Premier Biosoft). Quantitative PCR and data collection were performed in the real time PCR system (MiniOpticon, Bio-Rad).