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 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 (from prgQ) 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 of prgB, and indicated that only the transcripts originating 5 kb upstream may be capable of extending to prgB.
Bensing
B A
BA
Department of Microbiology, University of Minnesota, Minneapolis 55455,USA.
Meyer
B J
BJ
Dunny
G M
GM
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003227:Conjugation, Genetic; D017931:DNA Primers; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D013293:Enterococcus faecalis / Q000235:genetics* / Q000378:metabolism*; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010675:Pheromones / Q000502:physiology*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012329:RNA, Bacterial / Q000096:biosynthesis* / Q000302:isolation & purification; D015183:Restriction Mapping; D012680:Sensitivity and Specificity; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic
D017931:DNA Primers; D009838:Oligodeoxyribonucleotides; D010675:Pheromones; D012329:RNA, Bacterial; D012321:DNA-Directed RNA Polymerases