Sensitive detection of bacterial transcription initiation sites and differentiation from RNA processing sites in the pheromone-induced plasmid transfer system of Enterococcus faecalis.

Abstract

A method was developed to detect 5' ends of bacterial RNAs expressed at low levels and to differentiate newly initiated transcripts from processed transcripts produced in vivo. The procedure involves use of RNA ligase to link a specific oligoribonucleotide to the 5' ends of cellular RNAs, followed by production of cDNA and amplification of the gene of interest by PCR. The method was used to identify the precise sites of transcription initiation within a 10-kb region of the pheromone-inducible conjugative plasmid pCF10 of Enterococcus faecalis. Results confirmed the 5' end of a very abundant, constitutively produced transcript (fromprgQ) that had been mapped previously by primer extension and defined the initiation point of a less abundant, divergently transcribed message (from prgX). The method also showed that the 5' end of a pheromone-inducible transcript (prgB) that had been mapped by primer extension was generated by processing rather than new initiation. In addition, the results provided evidence for two promoters, 3 and 5 kb upstream ofprgB, and indicated that only the transcripts originating 5 kb upstream may be capable of extending to prgB.

Pheromone cCF10 and plasmid pCF10‐encoded regulatory molecules act post‐transcriptionally to activate expression of downstream conjugation functions

“…The 2.3 kb fragment corresponds to transcripts spanning prgQ through the 5Ј region of prgA, since it is also evident in overexposed prgQ blots (not shown), whereas the 1.5 kb fragment is probably derived by endonucleolytic cleavage of the 2.3 kb fragment in the prgS coding region. The 3 kb fragment probably corresponds to transcripts originating at the prgT promoter that do not extend to prgB (Bensing et al, 1996). The 3Ј QL, prgS and prgT probes each detected a species greater than 10 kb that was only present after induction with pheromone.…”

“…First, translation of ORFs downstream of the pro-inhibitor coding region is very inefficient in the absence of cCF10 induction. The only exception is a low level of constitutive translation of Sec10 (Mori et al, 1988), possibly from the separate prgTA transcript (Bensing et al, 1996). Inhibition of downstream ORF translation is not entirely due to cis-action of iCF10, but may be due to action of other negative control molecules, such as PrgX.…”

“…Also shown is a schematic representation of the two prgS gene products involved in prgB expression. Arrows indicate endonucleolytic processing sites mapped in previous studies (Bensing et al, 1996;Chung, 1993). The 5Ј region encodes a 10.5 kDa basic protein (PrgS).…”

“…1), but can function at distances of up to 12 kb. RNA ligase-mediated polymerase chain reaction (PCR) analysis of prgB transcripts and transcriptional fusion analysis have indicated that the monocistronic prgB transcript detected in Northern blots (Chung and Dunny, 1992) results from processing of transcripts that originate 5 kb upstream at the prgQ promoter (Bensing et al, 1996). A region of pCF10 adjacent to the positive control region includes genes, prgN, prgY and prgX, required for negative control of prgB expression , which suppress positive control unless cultures are induced with pheromone.…”

Esre: A novel essential non‐coding RNA in Escherichia coli

“…5 0 RACE assay were carried out essentially as described [21] with minor following modifications. 300 ng of total RNA was reverse-transcribed with 2 pmol of esre-specific primer P005 with 20 units of reverse transcriptase (Promega).…”