SENSITIVE DETECTION OF BACTERIAL TRANSCRIPTION INITIATION SITES AND DIFFERENTIATION FROM RNA PROCESSING SITES IN THE PHEROMONE-INDUCED PLASMID TRANSFER SYSTEM OF ENTEROCOCCUS-FAECALIS

A method was developed to detect 5' ends of bacterial RNAs expressed a t low levels and to differentiate newly initiated transcripts from pro cessed transcripts produced in vivo. The procedure involves use of RNA ligase to link a specific oligoribonucleotide to the 5' ends of cellu lar 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-i nducible conjugative plasmid pCF10 of Enterococcus faecalis. Results c onfirmed the 5' end of a very abundant, constitutively produced transc ript (from prgQ) that had been mapped previously by primer extension a nd defined the initiation point of a less abundant, divergently transc ribed message (from prgX). The method also showed that the 5' end of a pheromone-inducible transcript (prgB) that had been mapped bg primer extension was generated by processing rather than new initiation. In a ddition, the results provided evidence for two promoters, 3 and 5 kb u pstream of prgB, and indicated that only the transcripts originating 5 kb upstream may be capable of extending to prgB.