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Link to original content: https://pubmed.ncbi.nlm.nih.gov/16260475/
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. 2005 Oct 31;33(19):6268-76.
doi: 10.1093/nar/gki928. Print 2005.

Detection of low-level promoter activity within open reading frame sequences of Escherichia coli

Mitsuoki Kawano  1 , Gisela Storz, B Sridhar Rao, Judah L Rosner, Robert G Martin
Affiliations

Detection of low-level promoter activity within open reading frame sequences of Escherichia coli

Mitsuoki Kawano et al. Nucleic Acids Res. .

Abstract

The search for promoters has largely been confined to sequences upstream of open reading frames (ORFs) or stable RNA genes. Here we used a cloning approach to discover other potential promoters in Escherichia coli. Chromosomal fragments of approximately 160 bp were fused to a promoterless lacZ reporter gene on a multi-copy plasmid. Eight clones were deliberately selected for high activity and 105 clones were selected at random. All eight of the high-activity clones carried promoters that were located upstream of an ORF. Among the randomly-selected clones, 56 had significantly elevated activity. Of these, 7 had inserts which also mapped upstream of an ORF, while 49 mapped within or downstream of ORFs. Surprisingly, the eight promoters selected for high activity matched the canonical sigma70 -35 and -10 sequences no better than sequences from the randomly-selected clones. For six of the nine most active sequences with orientations opposite to that of the ORF, chromosomal expression was detected by RT-PCR, but defined transcripts were not detected by northern analysis. Our results indicate that the E.coli chromosome carries numerous -35 and -10 sequences with weak promoter activity but that most are not productively expressed because other features needed to enhance promoter activity and transcript stability are absent.

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Figures

Figure 1
Figure 1
The distribution of promoter (β-galactosidase) activities of 105 random fragments of E.coli as assayed in the promoterless lacZ tester plasmid, pRS551. We defined a sequence as having potential promoter activity when the β-galactosidase activity of the strain was increased by 3-fold or more over that of the strain with the parental plasmid pRS551 (i.e. to >10 MU). We found that 56 (53%) of the inserts (tested in only one orientation) exhibited promoter activity by this criterion. However, some of the inserts may contain more than one promoter. Applying the Poisson distibution, P(0) = e−λ, where P(0) is the probability of finding no activity (i.e. the null class of plasmids expressing 0–10 MU = 47%), and solving for λ (the average number of potential promoters per insert), we calculate there are on average 0.76 promoters per fragment (in one of the two possible orientations). We therefore conclude that there are ∼1.52 (twice 0.76) promoters per 163 bp or one full promoter in either direction per ∼107 bp.
Figure 2
Figure 2
Detection of antisense transcripts by RT–PCR and northern analysis. (A) RT–PCR was performed with primer sets listed in Supplementary Table S1 and with (plus) and without (minus) RT. The products were analyzed by electrophoresis on 2% agarose gels. Lanes 1 and 2, ygeH antisense transcript (expected size, 124 bp); lanes 3 and 4, rhsE antisense transcript (expected size, 95 bp); lanes 5 and 6, yfjN antisense transcript (expected size, 92 bp); lanes 7 and 8, yiaO antisense transcript (expected size, 87 bp); lanes 9 and 10, yjcE antisense transcript (expected size, 103 bp); lanes 11 and 12, ydiM antisense transcript (expected size, 114 bp); lanes 13 and 14, ecpD antisense transcript (expected size, 119 bp); lanes 15 and 16, topA antisense transcript (expected size, 101 bp); lanes 17 and 18, yehI antisense transcript (expected size, 108 bp); lane M for 50 bp ladder size marker and lanes 19 and 20, RyjC RNA (expected size, 77 bp). The lack of correlation between the RT–PCR signal and β-galactosidase activity may be due to differences in the hybridization efficiencies for the primer pairs used. (B) Total RNA separated on 1% agarose gels and transferred to nylon membranes was probed with primers to the antisense strands of rhsE, yfjN, yiaO and yjcE as well as to the 77 nt antisense RNA RyjC. RNA molecular weight markers were run with each set of samples for direct estimation of RNA transcript length, but the RNA marker lane for only one of the panels is shown.
Figure 3
Figure 3
Identification of the transcriptional start site of the antisense RNA from yfjN by primer extension analysis. (A) Lane 1, control host strain (DJ480); lane 2, DJ480 carrying single-copy λ prophage with the pRS551-derived anti-yfjN–lacZ fusion; lane 3, DJ480 carrying single-copy λ prophage with the pRS552-derived anti-yfjN–lacZ fusion; lane 4, DJ480 harboring the pRS551-anti-yfjN-lacZ plasmid; lane 5, DJ480 harboring the pRS552-anti-yfjN –lacZ plasmid. Reverse transcription reactions were performed by using 30 µg of total RNA from the parent and prophage-containing strains (lanes 1–3) and 5 µg of total RNA from plasmid-containing strains (lanes 4 and 5); The sequence ladder was generated using the primer used in the primer extension reactions. Although the levels of the primer extension products for the prophage strains are extremely low, the same relative levels were seen in all three repetitions of the experiment. (B) Top strand sequence from nt 2 763 939 to 2 764 368 of the E.coli chromosome (1–430 of yfjN). The yfjN ORF extends to 2 765 012. The ATG start codon of yfjN is boxed. The sequence cloned in pRS551 and pRS552 is surrounded by brackets (between 2 764 127 and 2 764 357). The +1 transcriptional start site is shown by an arrow. The −10 and −35 sequences of the yfjN antisense promoter are underlined.
Figure 4
Figure 4
Correlation between match to optimal σ70 promoter (−35 and −10 sequences) measured by the pftools 2.2 program (homology score, X) and log10 of the promoter (β-galactosidase, Y) activity. The regression line for the 39 randomly isolated promoters that have homology scores of 45 or greater (open circles) has the formula Y = −5.5496 + 0.14772X and an R2 of 0.417. Two SDs are indicated by the dashed lines. The eight inserts selected on the basis of high activity are shown by filled circles.
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