Negative regulation of the EcoRI restriction enzyme gene is associated with intragenic reverse promoters

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.

FIG. 1.

Defining the promoter P_R. (A) Organization and map of ecoRIRM gene complexes. In addition to promoter P_R, two reverse promoters, P_REV1 and P_REV2, were identified in the present study. The +1 designates the transcription initiation site of P_R; the coordinates are numbered accordingly. (B) Primer extension assay to identify the transcription initiation site (indicated by an arrow and numbered as +1) in the ecoRIRM operon (pIK163). (C) DNA sequence upstream of the ecoRIR gene. The putative −35 sequence of promoter P_R is boxed, while its putative −10 sequence is shaded. The transcription initiation site of P_R is in boldface and indicated by an arrow. The start codon of the ecoRIR gene is in italics, lower case and bold. The ribosome binding site (RBS) is underlined. (D) Verification of promoter P_R by mutation analysis with ecoRIR-lacZ translational fusion strains. An average of four measurements is shown with the standard deviation.

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.

FIG. 3.

Negative regulation of the ecoRIR gene associated with the reverse promoters. (A) Effect of reverse-promoter mutations on ecoRIR gene expression in ecoRIR-lacZ translational fusion. pLY58, translational fusion of a fragment (−67 to +669) of ecoRIRM operon to lacZ into pLY2 (WT); pLY60, pLY58 derivative with a TATAAT-to-CTGCAG mutation in the −10 box of P_REV1 and a TATATT-to-CCCGGG mutation in the −10 box of P_REV2; pLY77, pLY58 derivative with a TATAAT-to-CTGCAG mutation in the −10 box of P_REV1; pLY79, pLY58 derivative with a TATATT-to-GATATC mutation in −10 box of P_REV2; pLY85, pLY58 derivative with a CTAACA-to-CGAGGA mutation in the −35 box of P_REV1 and a TTCCCA-to GGCCGC mutation in the −35 box of P_REV2. (B) Effect of reverse-promoter mutations on ecoRIR gene expression in ecoRIR-lacZ transcriptional fusion. pLY116 contained the reverse promoter of P_REV1; the pLY116mut was derived from pLY116 with mutation in −10 box of P_REV1 (TATAAT→CTGCAG).

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.

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.