Negative Regulation of the EcoRI Restriction Enzyme Gene Is Associated with Intragenic Reverse Promoters

Yaoping Liu; Ichizo Kobayashi

doi:10.1128/JB.00127-07

. 2007 Jul 6;189(19):6928–6935. doi: 10.1128/JB.00127-07

Negative Regulation of the EcoRI Restriction Enzyme Gene Is Associated with Intragenic Reverse Promoters^▿^†

Yaoping Liu ^1,2, Ichizo Kobayashi ^1,2,3,^*

PMCID: PMC2045195 PMID: 17616602

Abstract

Type II restriction-modification systems are expected to possess mechanisms for tight regulation of their expression to suppress the potential of lethal attack on their host bacteria when they establish and maintain themselves within them. Although the EcoRI restriction enzyme has been well characterized, regulation of its expression is still poorly understood. In this study, mutational analysis with lacZ gene fusion and primer extension assay identified a promoter for the transcription of the ecoRIR gene. Further analyses revealed that an intragenic region containing two overlapping reverse promoter-like elements acted as a negative regulator for ecoRIR gene expression. The activity of these putative reverse promoters was verified by transcriptional gene fusion, primer extension and in vitro transcription. Mutations in these reverse promoters resulted in increased gene expression in both translational and transcriptional gene fusions. An RNase protection assay revealed that the transcript level of the wild type relative to that of the reverse promoter mutant at the downstream regions was much lower than the level at the upstream regions. This suggests that these reverse promoter-like elements affect their downstream transcript level. The possible mechanisms of this kind of negative regulation, in addition to their possible biological roles, are discussed.

A type II restriction-modification (R-M) system is typically composed of two enzymatic components: a restriction enzyme and a modification methyltransferase (30). The restriction enzyme cleaves a double-strand DNA within or near its specific recognition sequence, while the cognate modification enzyme methylates the same recognition sequence to protect it from cleavage. On the one hand, a type II R-M system can protect itself and its host cells through direct attack on invading foreign DNA. On the other hand, it can kill the host cells through attack on the chromosome, resulting from the loss of the R-M gene complex in a process termed postsegregational host cell killing or genetic addiction (13, 16, 25). These and other lines of evidence indicate that type II R-M systems behave as selfish mobile elements (16).

For any genetic addiction system, including the R-M systems, fine regulation of the toxin activity, which kills the host, and the antitoxin activity should be crucial. It should keep the two activities in balance and prevents the toxin from attacking the host until it senses loss of its genes. Among the three major types of genetic addiction, the classical proteic genetic addiction systems are autoregulated at the transcription level by the antitoxin protein or by the coordinated action of both the toxin protein and the antitoxin protein (17, 22, 39). In another type of genetic addiction system, translation of a toxin protein from a mRNA is inhibited by an antisense RNA (antitoxin) (11). Loss of these two classes of genetic addiction gene pairs will lead to faster decay of the unstable antitoxin, which allows lethal action by the stable toxin to occur. Such differential stability has not been observed for an R-M system (14).

The regulation of R-M gene complexes has been mostly studied at the transcriptional level. One regulatory mechanism uses a small, so-called “C protein” (40). The C proteins specifically bind a DNA operator sequence through a helix-turn-helix motif to regulate expression of the restriction enzyme, modification enzyme, or both (40, 41). This group of proteins can delay the expression of the restriction enzyme during establishment in a new host cell (26). The coordinated expression of R-M genes in SsoII (15) and EcoRII (37) systems relies on the ability of the modification enzymes to repress the transcription of their own genes. In CfrBI and LIaJI R-M systems, transcription regulation depends on the methylation status of cognate recognition site(s) within the promoter region (3, 28).

For the EcoRI R-M system, whose restriction gene (ecoRIR) lies upstream of its modification gene (ecoRIM) in the same orientation, the transcription pattern and regulation mechanism are not well understood, except for the proposal of a promoter sequence for ecoRIM at the end of the ecoRIR (27). SalI and LlaDII R-M systems have a structural organization similar to that of EcoRI. The transcription pattern of the SalI R-M system has been investigated (1). For the LlaDII R-M system, expression of the modification enzyme was autoregulated by the llaDIIM gene, whereas no regulation was found for the llaDIIR gene (9).

In the present study, we identified a promoter for transcription of the ecoRIR gene and two intragenic reverse promoters associated with negative regulation of the ecoRIR gene.

MATERIALS AND METHODS

Bacterial strains.

Escherichia coli MC1061 [araD139 Δ(ara-leu)7696 galE15 galK16 Δ(lac)X74 rpsL hsdR2 mcrA mcrB1] (7) was used as the host strain for the assay of β-galactosidase (β-Gal) activity. E. coli DH10B [F⁻ mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 recA1 deoR araD139 Δ(ara-leu)7697 galU galK rpsL (Str^r) endA1 nupG] (Invitrogen) was used as the host strain for construction of all of the plasmids.

Plasmid construction.

The plasmids used are listed in Table 1. The promoterless reporter pLY2 was constructed by cloning an XbaI-SalI lacZ gene fragment (amplified by PCR with primers lacF and lacR [see Table S1 in the supplemental material]) from pAB2001 into pACYC184, which was also digested with XbaI and SalI. In the present study, all of the lacZ transcriptional fusions were constructed in a similar way: the corresponding parts of the ecoRIRM operon were amplified by PCR with KOD Plus (Toyobo) and then digested with XbaI and cloned into pLY2, which was also digested with XbaI. All of the lacZ translational fusions were constructed in the same way, except that PCR fragments were digested with both XbaI and HindIII. The pGEM-T plasmid was used to construct the probe-generating plasmids, pGEM-T1, -T2, and -T3. The PCR products from ecoRIR, +133 to +275 and +431 to +612, were cloned into pGEM-T in the forward orientation to construct pGEM-T1 and -T2, respectively. The pGEM-T3 was similarly constructed by cloning the PCR product from ecoRIR (+586 to + 669, together with the 53-bp lacZ sequence) into pGEM-T.

TABLE 1.

Plasmids

Plasmid	Relevant characteristics^a	Source or reference
pACYC184	E. coli cloning vector (Cm^r)	Laboratory collection
pBR322	E. coli cloning vector (Ap^r)	Laboratory collection
pIK163	pBR322 carrying ecoRIRM operon	20
pGEM-T	E. coli TA cloning vector (Ap^r)	Promega
pGEM-T1	pGEM-T carrying a fragment (+133 to +275) of the ecoRIRM operon	This study
pGEM-T2	pGEM-T carrying a fragment (+431 to +612) of the ecoRIRM operon	This study
pGEM-T3	pGEM-T carrying a fragment (+586 to +669) of the ecoRIRM operon and 53-bp 5′ terminus of the lacZ reporter sequence of pLY2	This study
pAB2001	Promoterless lacZ reporter	2
pLY2	pACYC184 carrying promoterless lacZ gene	This study
pLY11	Translational fusion of a fragment (−67 to +45) of ecoRIRM operon to lacZ into pLY2	This study
pLY11mut1	pLY11 derivative with TAT to GCC mutation in −10 box of P_R	This study
pLY11mut2	pLY11 derivative with TA to CC mutation in −10 box of P_R	This study
pLY11mut3	pLY11 derivative with tgttAatcT to tgttCatcC mutation in putative −10-extended box of P_R	This study
pLY58	Translational fusion of a fragment (−67 to +669) of ecoRIRM operon to lacZ into pLY2	This study
pLY59	pLY58 derivative with a 82-bp deletion from +390 to +471	This study
pLY60	pLY58 derivative with TATAAT to CTGCAG and TATATT to CCCGGG mutation in the −10 box of both P_REV1 and P_REV2 reverse promoters	This study
pLY77	pLY58 derivative with TATAAT to CTGCAG mutation in −10 box of P_REV1	This study
pLY79	pLY58 derivative with TATATT to GATATC mutation in −10 box of P_REV2	This study
pLY85	pLY58 derivative with CTAACA to CGAGGA and TTCCCA to GGCCGC mutation in the −35 box of both P_REV1 and P_REV2 reverse promoters	This study
pLY91	Transcriptional fusion of a fragment (+455 to +381) of the ecoRIRM operon to lacZ into pLY2	This study
pLY92	Transcriptional fusion of a fragment (+381 to +455) of the ecoRIRM operon to lacZ into pLY2	This study
pLY66	Transcriptional fusion of a fragment (−67 to +400) of the ecoRIRM operon to lacZ into pLY2	This study
pLY116	Transcriptional fusion of a fragment (−67 to +431) of the ecoRIRM operon containing the minimal promoter P_REV1 to lacZ into pLY2	This study
pLY116mut	pLY116 derivative with TATAAT to CTGCAG mutation in the −10 box of P_REV1	This study
pLY117	Transcriptional fusion of a fragment (−67 to +445, with CTAACA to TTGACA upmutation in −35 box of P_REV1) of the ecoRIRM operon to lacZ into pLY2	This study

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Cm^r, chloramphenicol resistance; Ap^r, ampicillin resistance.

β-Gal assays.

β-Gal activity was determined by using exponentially growing cells. The assay was carried out by the procedure published by Miller (23), except that rich LB broth was used instead of 1xA minimal medium. Duplicate measurements were performed with two clones of each strain. A bar indicates a standard deviation in these four measurements.

Preparation of RNA probes with Sp6 RNA polymerase.

Plasmid DNAs were purified by a plasmid purification kit (QIAGEN). The pGEM-T1, -T2, and -T3 plasmid molecules were linearized with NcoI and purified for in vitro transcription with Sp6 RNA polymerase. In vitro transcription was carried out as recommended by the manufacturer (Promega). One hour after the start of reaction, 10 U of RQ DNase I (Promega) was added to the reaction mixture to digest the DNA templates, and the unincorporated ribonucleotides were removed by passage through a Sephadex G-50 column; the transcribed RNAs of expected size were then purified by 8% polyacrylamide-8 M urea gel electrophoresis.

In vitro transcription by E. coli RNA polymerase.

The region from positions +333 to +458 of the ecoRIR gene was amplified by PCR and used as a template (wild type [WT]). The template containing double mutations of −10 boxes of P_REV1 and P_REV2 (TATAAT and TATATT were substituted by CTGCAG and CCCGGG, respectively) was obtained by two-step PCR (36) using different mutant oligonucleotides. Single-round transcription with E. coli RNA polymerase (Epicenter) was performed using these two templates. RNA polymerase (20 nM) and promoter DNA (10 nM) were mixed and, after incubation at 37°C for 30 min, [³²P]CTP (50 μM), nucleoside triphosphates (500 μM ATP, 10 μM CTP, 200 μM GTP, and 200 μM UTP), and rifampin (300 μg/ml) were simultaneously added to the 50-μl reaction volume. The reactions were stopped after 5, 10, and 30 min, respectively, with sequencing gel loading buffer (Promega), and the resulting mixture was run through a 10% polyacrylamide gel.

Total RNA extraction.

E. coli MC1061 cells harboring corresponding plasmids were harvested in the exponential phase. Total RNA was extracted by using an RNeasy Protect Bacteria Minikit (QIAGEN) according to the manufacturer's instructions for primer extension and an RNase protection assay, respectively, along with a DNase I digestion step. RNA concentration and purity were checked by measuring the absorbance at 260 and 280 nm and by electrophoresis through a 1% formaldehyde-agarose gel.

Primer extension.

For the primer extension reaction, 20 μg of the total RNA was reverse transcribed with 1 U of avian myeloblastosis virus reverse transcriptase from the Primer Extension System (Promega) in the presence of 1 pmol of [γ-³³P] end-labeled primer according to the manufacturer's protocol. The sequencing reaction with Taq DNA polymerase (Promega) was performed by PCR with the same end-labeled primer and template used for the primer extension. The reaction products were resolved through an 8% sequencing gel, dried, and visualized by exposure to a PhosphorImager screen.

RNase protection assay.

Total RNA annealed with the corresponding cRNA probes was digested with E. coli RNase I (Promega) with a minor modification to the manufacturer's protocol. In brief, the same quantity of total RNA was ethanol coprecipitated by using excess purified RNA probe and the same quantity of a loading control (a [γ-³³P] end-labeled single-strand foreign DNA probe [see Table S1 in the supplemental material]). The precipitate was dissolved, hybridized overnight at 45°C, and digested according to the manufacturer's protocol. Finally, the RNase-protected fragments were resolved through an 8 to 10% denaturing polyacrylamide gel, dried, and visualized by exposure to a PhosphorImager screen or an X-ray film. The signal strength was quantified according to the formula [(protected signal - background)/loading control] using Image Gauge Software V4.22 soft (Fuji film).

Bioinformatic analysis.

All of the sequences of the restriction genes were collected from the REBASE database (http://rebase.neb.com/rebase/rebase.html). In silico promoter prediction was carried out with the tools available at http://www.softberry.com/all.htm. The RNA secondary structure was predicted by Mfold (http://www.bioinfo.rpi.edu/applications/mfold/rna/form1.cgi) and RNA structure V4.4 (http://rna.urmc.rochester.edu/rnastructure.html).

RESULTS

Defining a promoter upstream of the restriction enzyme gene.

The EcoRI R-M gene complex was found on a small set of bacterial plasmids related to the ColE1 plasmid (4). In this system, the restriction gene (ecoRIR) lies 29 bp upstream of the modification gene (ecoRIM) in the same orientation (Fig. 1A). To identify the transcription initiation site of the promoter, an ecoRIR gene-specific primer (1114r; see Table S1 in the supplemental material) was synthesized and end labeled with [γ-³³P]ATP. Extension of this primer annealed to the total RNA from E. coli MC1061 harboring the ecoRIRM operon (pIK163, Table 1) revealed that the transcription initiation site is located 38 bp upstream of the translation start codon of the ecoRIR gene (Fig. 1B and C).

Sequence analysis of upstream of this site revealed two overlapping candidate sequences for −10 box of promoter P_R (8, 29) (Fig. 1C). One is TATGTT, identical with that of promoter P_lac, located 11 bp upstream of the initiation site. The other is TGtTAatcT, similar to a −10-extended promoter consensus (TGnTATAAT) (19), located 6 bp upstream of the initiation site. Substitutions in the 3 bp affecting both of these two putative −10 box candidates (TATgtt→GCCgtt) resulted in a significant reduction in gene expression in a translational fusion (Fig. 1D). We further constructed two 2-bp substitution mutants. One is only to affect the first candidate −10 sequence (TAtgtt→CCtgtt); the other is only to affect the second candidate −10 sequence (tgttAatcT→tgttCatcC). Interestingly, both mutations resulted in a similar reduction in gene expression in the translational fusion (Fig. 1D). This result revealed that both of these −10-like sequences are required for the promoter activity.

Negative regulation of the restriction gene by an intragenic region with reverse promoters.

To investigate the regulation of the ecoRIRM operon, ecoRIR′::′lacZ translational fusions with different deletions in the ecoRIR gene were examined (unpublished data). A region within the ecoRIR gene (+390 to +471) was found to play a role in the negative regulation (Fig. 2A). Within this region, computer analysis revealed two overlapping promoter-like elements in the reverse orientation (Fig. 2B). A transcriptional lacZ fusion with the putative reverse promoter sequence in the reverse orientation (+455→+381) showed a higher activity than that in the forward orientation (+381→+455) (Fig. 2C, pLY91 to pLY92). We designed these putative promoters as P_REV1 and P_REV2 (Fig. 1A and 2B).

FIG. 2. — Intragenic region containing two overlapping reverse promoters associated with negative regulation of the *ecoRIR* gene. (A) Effect of deletion of an intragenic region (+390 to +471) on *ecoRIR* gene expression. (B) DNA sequence involved in the negative regulation. The putative −35 sequence of promoter P_REV1 is in lowercase and boxed, while its putative −10 sequence is in lowercase and shaded. The putative −35 sequence of promoter P_REV2 is in uppercase and boxed, while its putative −10 sequence is in uppercase and shaded. The two putative transcription initiation sites are in bold and italics. The deleted region (+390 to +471) is underlined. (C) Detection of reverse promoter activity in vivo by *lacZ* transcriptional fusion. An average of four measurements is shown with the standard deviation. (D) In vitro transcription with *E. coli* RNA polymerase for identification of putative reverse promoters. The regions from positions +333 to +458 of the *ecoRIR* gene were used as the WT template; its mutant derivative carried substitution mutations at the putative −10 sequence in both P_REV1 and P_REV2 (TATAAT→CTGCAG and TATATT→ CCCGGG). M, single-strand RNA markers with sizes of 80, 70, 60, 50, and 40 nucleotides (from top to bottom). (E) Primer extension assay to identify transcription initiation sites of P_REV1 and P_REV2 with the probe lacp. The plasmid (pLY117) containing an upmutation in the −35 sequence of P_REV1 was used. The strong signal was from this mutant P_REV1 promoter, while the very weak one was from P_REV2.

Our transcription in vitro with E. coli RNA polymerase generated two transcripts of expected size and confirmed that this element indeed encoded two active promoters (Fig. 2D). In the primer extension experiments, the signals were too weak to accurately map the transcription initiation sites. As shown in Fig. 2E, however, a 2-bp substitution in the putative −35 box—CTAACA by TTGACA—of P_REV1 allowed detection of a strong signal for transcription initiation from an adenine at position +387. A weak signal with extension of 6 bp, at position +393 (Fig. 2E), should be from the WT promoter P_REV2. This result was consistent with the analysis of transcription in vitro (Fig. 2D).

Mutations in the reverse promoters affect the negative regulation of the restriction gene.

To test whether these reverse promoters are involved in the negative regulation of P_R or not, the ecoRIR was fused with the lacZ gene at the translational level with various mutations in the putative −10 and −35 boxes of P_REV1 and P_REV2. Increased lacZ activity was observed with a substitution mutation in −10 boxes of P_REV1 and P_REV2 (Fig. 3A). Simultaneous mutations in the two −35 boxes increased β-Gal activity twofold (Fig. 3A). Simultaneous mutations in the two −10 boxes showed fivefold activity (Fig. 3A, pLY60 to pLY58).

To exclude possible effects of fused peptides, we constructed transcriptional fusions. As shown in Fig. 3B, only the addition of the reverse promoter P_REV1 region (+400 to +431) was sufficient to have an effect of negative regulation (pLY66 to pLY116); however, this effect was lost by a mutation in the reverse promoter (pLY116 to pLY116mut in Fig. 3B).

Effects of the reverse promoter mutation on the transcript level in its upstream and downstream regions.

In order to probe the mechanism of the reverse promoter-associated regulation, we used an RNase protection assay, a sensitive and accurate method for measuring mRNA expression (12, 35), to examine the effects of the reverse promoter mutation on the transcript levels along the ecoRIR gene. Three probes to detect the sense transcripts were prepared: one located upstream of the reverse promoters and another two located downstream of the reverse promoters (Fig. 4A). The relative transcript level of the WT to that of the reverse promoter mutant located in the downstream region was much lower than the level in the upstream regions, as detected by the same probe in each region (Fig. 4B and C).

FIG. 4. — Effect of reverse-promoter mutations on the transcript level along the *ecoRIR* gene. (A) Reverse RNA probes designed for detection of transcript abundance in the upstream and downstream regions of the reverse promoters. (B) RNase protection assay. The same quantity of purified total RNA from *E. coli* MC1061 harboring plasmid pLY2 (vector), pLY58 (WT), or pLY60 (mutation in the reverse promoters) was divided into three aliquots, respectively. Each aliquot was annealed with an excess amount of one of the three probes (left, right, and further right, Fig. 4A) and digested with RNase I (Promega). The “RNase-protected” fragments were resolved by electrophoresis on a 10% denaturing polyacrylamide gel. (C) Relative transcript levels between the WT and its reverse-promoter mutant. The signals in panel B were quantified in two independent experiments.

To exclude the possibility that the observed relative transcript decrease resulted from differential mRNA degradation between the WT and the reverse promoter mutant, we analyzed mRNA stability after inhibition of transcription with rifampin. Quantification of the RNase protection signals with the “further downstream probe” (Fig. 4A) revealed no difference in mRNA stability between them (Fig. 5).

FIG. 5. — Transcript stability in the WT and reverse-promoter mutant. (A) RNase protection assay. Total RNA was extracted for analysis at different time intervals after the addition of rifampin, an inhibitor of transcription. Total RNAs from *E. coli* MC1061 harboring plasmid pLY58 (WT) and pLY60 (a mutation in the reverse promoters) were annealed with the “further downstream” probe (Fig. 4A). (B) Quantification of the protected signals.

DISCUSSION

In this study, we identified a promoter for transcription of the ecoRIR gene and two intragenic reverse promoters within the ecoRIR gene. The latter were associated with negative regulation of the ecoRIR gene.

Mode of negative regulation by the reverse promoters.

Our RNase protection assay revealed (Fig. 4B and C) that the relative transcript level of the WT to that of the reverse promoter mutant at the downstream region was much lower than the level at the upstream regions. This suggests that these reverse promoter-like elements affect their downstream transcript level. We can exclude or limit some of the possible mechanisms underlying the negative regulation. One possibility is that a terminator or an attenuator overlapped with the reverse promoters. The mutation of the reverse promoters may also have affected this terminator or attenuator. However, computer analysis of the secondary structure of the ecoRIR mRNA revealed no typical terminator or attenuator structure, such as a stem-loop, in this region, which made this possibility unlikely. A second possibility is that an RNase cleavage site may be present in this region, in which the mutation affected. If this were true, then the mutation of this cleavage site should result in differential mRNA stability between the WT and the reverse promoter mutant. However, we observed similar stabilities for them for the transcript downstream of this region (Fig. 5), which argues against this possibility. Third, during translation elongation, ribosome stalling caused by a nascent polypeptide could induce cleavage of mRNA at a specific arrested site (38). Our observation of similar negative regulation phenomena in both the lacZ transcriptional and the translational fusions (Fig. 3) excluded this possibility because the transcriptional fusion junction lay downstream of the reverse promoters. The fourth possibility is that antisense RNAs transcribed from these reverse promoters may lead to cleavage of the mRNA. This model is not consistent with the following observations: (i) there was no difference between the mRNA stability in the WT and the reverse promoter mutant (Fig. 5) (5); (ii) the activity of the reverse promoters is much weaker than that of the promoter P_R in vivo; and (iii) we have not detected negative regulation in trans by the reverse promoters (unpublished observations).

One of the remaining possibilities for the negative regulation is that the truncated mRNA is generated by pause or arrest of the transcription elongation complex caused by a roadblock at the reverse promoters. Such a roadblock can be formed by DNA-binding proteins (10, 32). In the case of the ecoRIR gene, binding of RNA polymerase to the reverse promoters may change the DNA conformation and generate such a roadblock, influencing the transcription elongation complex of another RNA polymerase. Indeed, a significant number of “poised” RNA polymerase binding promoters were recently identified (21, 31). RNA polymerase-induced DNA conformational change was demonstrated in the gal promoter of E. coli (18), and recent high-resolution atomic force microscopy revealed that the promoter DNA is wrapped around the RNA polymerase in the transcription initiation complex (33). Our in vitro transcription with E. coli RNA polymerase and in vivo assay with lacZ activity demonstrated that the reverse promoters can be bound by RNA polymerase. Because only transcription from weak promoters has been reported to be interfered with by a strong convergent promoter thus far (6), it would be interesting to analyze factors other than promoter strength in the reverse promoter-associated negative regulation of the ecoRIR gene.

Possible role of the negative regulation in the biology of R-M systems.

Recent ChIP-chip studies of genomewide analysis in E. coli cells revealed that more than 100 σ⁷⁰ and σ³² promoters were located within the coding sequence of genes, suggesting a previously uncharacterized regulatory function for these new promoters (42). Various types of evolutionary analyses suggested that R-M genes have undergone extensive horizontal transfer between different groups of microorganisms (17, 24). During this process, tight regulation of R-M genes likely plays an important role during their establishment in a new host and their subsequent maintenance. For the EcoRI case, the first possible role of this reverse promoter-associated regulation may be to delay expression of R.EcoRI and provide additional time for a sufficient amount of M.EcoRI enzyme expressed from a separate ecoRIM gene promoter to methylate the chromosomal DNA. The second possible role is in its maintenance. In the EcoRI plasmid-containing cells, the reverse promoter-associated regulator might control the excessive mRNA expression from ecoRIR gene promoter P_R.

The resistance of R-M gene complexes to their loss from a host cell through postsegregational host killing was observed in the EcoRI R-M system (13, 25). Postsegregational killing by classical proteic toxin-antitoxin systems relies on differential stability between the toxin and antitoxin (16). However, for the EcoRI R-M system, postsegregational killing did not rely on differential stability between the toxin (R) and the antitoxin (M) molecules (14). Our present analysis revealed that an ecoRIR transcript fused to a lacZ transcript is moderately stable. Further analysis of effects of reverse promoters mutation on the transformation and host cell killing is necessary to clarify the biological role of the reverse promoter-associated negative regulation in the ecoRIRM operon.

More than 4,000 type II R-M systems have been identified at this time (34). If this kind of reverse promoter plays a regulatory role in the ecoRI operon, we might expect to find similar reverse promoters in other R-M gene complexes. Indeed, our in silico analysis revealed many reverse promoter candidates in other restriction genes (unpublished observations).

Supplementary Material

[Supplemental material]

jbacter_189_19_6928__index.html^{(781B, html)}

Acknowledgments

We thank M. Kawai, E. Fukuda, N. Handa, and especially A. Ichige for advice and help. We thank the National Institute for Genetics for gifts of plasmids.

This study was supported by the National Project on Protein Structural and Functional Analyses (Protein 3000), the 21st Century COE Project of Elucidation of Language Structure and Semantic behind Genome and Life System, Grants-in-Aid for Scientific Research from JSPS (13141201, 15370099, and 17310113) to I.K. and Postdoctoral Fellowships for Foreign Researchers from JSPS to Y.L.

Footnotes

^▿

Published ahead of print on 6 July 2007.

^†

Supplemental material for this article may be found at http://jb.asm.org/.

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18.Kuhnke, G., C. Theres, H. J. Fritz, and R. Ehring. 1989. RNA polymerase and gal repressor bind simultaneously and with DNA bending to the control region of the Escherichia coli galactose operon. EMBO J. 8:1247-1255. [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Kumar, A., R. A. Malloch, N. Fujita, D. A. Smillie, A. Ishihama, and R. S. Hayward. 1993. The minus 35-recognition region of Escherichia coli sigma 70 is inessential for initiation of transcription at an “extended minus 10” promoter. J. Mol. Biol. 232:406-418. [DOI] [PubMed] [Google Scholar]
20.Kusano, K., T. Naito, N. Handa, and I. Kobayashi. 1995. Restriction-modification systems as genomic parasites in competition for specific sequences. Proc. Natl. Acad. Sci. USA 92:11095-11099. [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Lee, S. J., and J. D. Gralla. 2004. Osmo-regulation of bacterial transcription via poised RNA polymerase. Mol. Cell 14:153-162. [DOI] [PubMed] [Google Scholar]
22.Magnuson, R., and M. B. Yarmolinsky. 1998. Corepression of the P1 addiction operon by Phd and Doc. J. Bacteriol. 180:6342-6351. [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
24.Naderer, M., J. R. Brust, D. Knowle, and R. M. Blumenthal. 2002. Mobility of a restriction-modification system revealed by its genetic contexts in three hosts. J. Bacteriol. 184:2411-2419. [DOI] [PMC free article] [PubMed] [Google Scholar]
25.Naito, T., K. Kusano, and I. Kobayashi. 1995. Selfish behavior of restriction-modification systems. Science 267:897-899. [DOI] [PubMed] [Google Scholar]
26.Nakayama, Y., and I. Kobayashi. 1998. Restriction-modification gene complexes as selfish gene entities: roles of a regulatory system in their establishment, maintenance, and apoptotic mutual exclusion. Proc. Natl. Acad. Sci. USA 95:6442-6447. [DOI] [PMC free article] [PubMed] [Google Scholar]
27.O'Connor, C. D., and G. O. Humphreys. 1982. Expression of the EcoRI restriction-modification system and the construction of positive-selection cloning vectors. Gene 20:219-229. [DOI] [PubMed] [Google Scholar]
28.O'Driscoll, J., G. F. Fitzgerald, and D. Van Sinderen. 2005. A dichotomous epigenetic mechanism governs expression of the LlaJI restriction/modification system. Mol. Microbiol. 57:1532-1544. [DOI] [PubMed] [Google Scholar]
29.Oliphant, A. R., and K. Struhl. 1988. Defining the consensus sequences of Escherichia coli promoter elements by random selection. Nucleic Acids Res. 16:7673-7684. [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Pingoud, A. (ed.). 2004. Restriction endonucleases. Springer-Verlag, Berlin, Germany.
31.Reppas, N. B., J. T. Wade, G. Church, and K. Struhl. 2006. The transition between transcriptional initiation and elongation in Escherichia coli is highly variable and often rate limiting. Mol. Cell 24:747-757. [DOI] [PubMed] [Google Scholar]
32.Ring, B. Z., W. S. Yarnell, and J. W. Roberts. 1996. Function of Escherichia coli RNA polymerase sigma factor sigma 70 in promoter-proximal pausing. Cell 86:485-493. [DOI] [PubMed] [Google Scholar]
33.Rivetti, C., M. Guthold, and C. Bustamante. 1999. Wrapping of DNA around the Escherichia coli RNA polymerase open promoter complex. EMBO J. 18:4464-4475. [DOI] [PMC free article] [PubMed] [Google Scholar]
34.Roberts, R. J., T. Vincze, J. Posfai, and D. Macelis. 2007. REBASE-enzymes and genes for DNA restriction and modification. Nucleic Acids Res. 35:D269-D270. [DOI] [PMC free article] [PubMed] [Google Scholar]
35.Rottman, J. B. 2002. The ribonuclease protection assay: a powerful tool for the veterinary pathologist. Vet. Pathol. 39:2-9. [DOI] [PubMed] [Google Scholar]
36.Sarkar, G., and S. S. Sommer. 1990. The “megaprimer” method of site-directed mutagenesis. BioTechniques 8:404-407. [PubMed] [Google Scholar]
37.Sneppen, K., I. B. Dodd, K. E. Shearwin, A. C. Palmer, R. A. Schubert, B. P. Som, and S. Friedman. 1994. Regulation of EcoRII methyltransferase: effect of mutations on gene expression and in vitro binding to the promoter region. Nucleic Acids Res. 22:5347-5353. [DOI] [PMC free article] [PubMed] [Google Scholar]
38.Sunohara, T., K. Jojima, H. Tagami, T. Inada, and H. Aiba. 2004. Ribosome stalling during translation elongation induces cleavage of mRNA being translated in Escherichia coli. J. Biol. Chem. 279:5368-15375. [DOI] [PubMed] [Google Scholar]
39.Tam, J. E., and B. C. Kline. 1989. The F plasmid ccd autorepressor is a complex of CcdA and CcdB proteins. Mol. Gen. Genet. 219:26-32. [DOI] [PubMed] [Google Scholar]
40.Tao, T., J. C. Bourne, and R. M. Blumenthal. 1991. A family of regulatory genes associated with type II restriction-modification systems. J. Bacteriol. 173:1367-1375. [DOI] [PMC free article] [PubMed] [Google Scholar]
41.Vijesurier, R. M., L. Carlock, R. M. Blumenthal, and J. C. Dunbar. 2000. Role and mechanism of action of PvuII, a regulatory protein conserved among restriction-modification systems. J. Bacteriol. 182:477-487. [DOI] [PMC free article] [PubMed] [Google Scholar]
42.Wade, J. T., D. C. Roa, D. C. Grainger, D. Hurd, S. J. W. Busby, K. Struhl, and E. Nudler. 2006. Extensive functional overlap between σ factors in Escherichia coli. Nat. Struct. Mol. Biol. 13:806-814. [DOI] [PubMed] [Google Scholar]

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[r17] 17.Kobayashi, I. 2004. Genetic addiction: a principle in symbiosis of genes in a genome, p. 105-144. In B. E. Funnel and G. J. Phillips (ed.), Plasmid biology. ASM Press, Washington, DC.

[r18] 18.Kuhnke, G., C. Theres, H. J. Fritz, and R. Ehring. 1989. RNA polymerase and gal repressor bind simultaneously and with DNA bending to the control region of the Escherichia coli galactose operon. EMBO J. 8:1247-1255. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r19] 19.Kumar, A., R. A. Malloch, N. Fujita, D. A. Smillie, A. Ishihama, and R. S. Hayward. 1993. The minus 35-recognition region of Escherichia coli sigma 70 is inessential for initiation of transcription at an “extended minus 10” promoter. J. Mol. Biol. 232:406-418. [DOI] [PubMed] [Google Scholar]

[r20] 20.Kusano, K., T. Naito, N. Handa, and I. Kobayashi. 1995. Restriction-modification systems as genomic parasites in competition for specific sequences. Proc. Natl. Acad. Sci. USA 92:11095-11099. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r21] 21.Lee, S. J., and J. D. Gralla. 2004. Osmo-regulation of bacterial transcription via poised RNA polymerase. Mol. Cell 14:153-162. [DOI] [PubMed] [Google Scholar]

[r22] 22.Magnuson, R., and M. B. Yarmolinsky. 1998. Corepression of the P1 addiction operon by Phd and Doc. J. Bacteriol. 180:6342-6351. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r23] 23.Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

[r24] 24.Naderer, M., J. R. Brust, D. Knowle, and R. M. Blumenthal. 2002. Mobility of a restriction-modification system revealed by its genetic contexts in three hosts. J. Bacteriol. 184:2411-2419. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r25] 25.Naito, T., K. Kusano, and I. Kobayashi. 1995. Selfish behavior of restriction-modification systems. Science 267:897-899. [DOI] [PubMed] [Google Scholar]

[r26] 26.Nakayama, Y., and I. Kobayashi. 1998. Restriction-modification gene complexes as selfish gene entities: roles of a regulatory system in their establishment, maintenance, and apoptotic mutual exclusion. Proc. Natl. Acad. Sci. USA 95:6442-6447. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r27] 27.O'Connor, C. D., and G. O. Humphreys. 1982. Expression of the EcoRI restriction-modification system and the construction of positive-selection cloning vectors. Gene 20:219-229. [DOI] [PubMed] [Google Scholar]

[r28] 28.O'Driscoll, J., G. F. Fitzgerald, and D. Van Sinderen. 2005. A dichotomous epigenetic mechanism governs expression of the LlaJI restriction/modification system. Mol. Microbiol. 57:1532-1544. [DOI] [PubMed] [Google Scholar]

[r29] 29.Oliphant, A. R., and K. Struhl. 1988. Defining the consensus sequences of Escherichia coli promoter elements by random selection. Nucleic Acids Res. 16:7673-7684. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r30] 30.Pingoud, A. (ed.). 2004. Restriction endonucleases. Springer-Verlag, Berlin, Germany.

[r31] 31.Reppas, N. B., J. T. Wade, G. Church, and K. Struhl. 2006. The transition between transcriptional initiation and elongation in Escherichia coli is highly variable and often rate limiting. Mol. Cell 24:747-757. [DOI] [PubMed] [Google Scholar]

[r32] 32.Ring, B. Z., W. S. Yarnell, and J. W. Roberts. 1996. Function of Escherichia coli RNA polymerase sigma factor sigma 70 in promoter-proximal pausing. Cell 86:485-493. [DOI] [PubMed] [Google Scholar]

[r33] 33.Rivetti, C., M. Guthold, and C. Bustamante. 1999. Wrapping of DNA around the Escherichia coli RNA polymerase open promoter complex. EMBO J. 18:4464-4475. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r34] 34.Roberts, R. J., T. Vincze, J. Posfai, and D. Macelis. 2007. REBASE-enzymes and genes for DNA restriction and modification. Nucleic Acids Res. 35:D269-D270. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r35] 35.Rottman, J. B. 2002. The ribonuclease protection assay: a powerful tool for the veterinary pathologist. Vet. Pathol. 39:2-9. [DOI] [PubMed] [Google Scholar]

[r36] 36.Sarkar, G., and S. S. Sommer. 1990. The “megaprimer” method of site-directed mutagenesis. BioTechniques 8:404-407. [PubMed] [Google Scholar]

[r37] 37.Sneppen, K., I. B. Dodd, K. E. Shearwin, A. C. Palmer, R. A. Schubert, B. P. Som, and S. Friedman. 1994. Regulation of EcoRII methyltransferase: effect of mutations on gene expression and in vitro binding to the promoter region. Nucleic Acids Res. 22:5347-5353. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r38] 38.Sunohara, T., K. Jojima, H. Tagami, T. Inada, and H. Aiba. 2004. Ribosome stalling during translation elongation induces cleavage of mRNA being translated in Escherichia coli. J. Biol. Chem. 279:5368-15375. [DOI] [PubMed] [Google Scholar]

[r39] 39.Tam, J. E., and B. C. Kline. 1989. The F plasmid ccd autorepressor is a complex of CcdA and CcdB proteins. Mol. Gen. Genet. 219:26-32. [DOI] [PubMed] [Google Scholar]

[r40] 40.Tao, T., J. C. Bourne, and R. M. Blumenthal. 1991. A family of regulatory genes associated with type II restriction-modification systems. J. Bacteriol. 173:1367-1375. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r41] 41.Vijesurier, R. M., L. Carlock, R. M. Blumenthal, and J. C. Dunbar. 2000. Role and mechanism of action of PvuII, a regulatory protein conserved among restriction-modification systems. J. Bacteriol. 182:477-487. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r42] 42.Wade, J. T., D. C. Roa, D. C. Grainger, D. Hurd, S. J. W. Busby, K. Struhl, and E. Nudler. 2006. Extensive functional overlap between σ factors in Escherichia coli. Nat. Struct. Mol. Biol. 13:806-814. [DOI] [PubMed] [Google Scholar]

PERMALINK

Negative Regulation of the EcoRI Restriction Enzyme Gene Is Associated with Intragenic Reverse Promoters^▿^†

Yaoping Liu

Ichizo Kobayashi

Abstract