Early transcriptional arrest at Escherichia coli rplN and ompX promoters.
Bacterial transcription elongation factors GreA and GreB stimulate the intrinsic RNase activity of RNA polymerase (RNAP), thus helping the enzyme to read through pausing and arresting sites on DNA. Gre factors also accelerate RNAP transition from initiation to elongation. Here, we characterized the molecular mechanism by which Gre factors facilitate transcription at two Escherichia coli promoters, PrplN and PompX, that require GreA for optimal in vivo activity. Using in vitro transcription assays, KMnO(4) footprinting, and Fe(2+)-induced hydroxyl radical mapping, we show that during transcription initiation at PrplN and PompX in the absence of Gre factors, RNAP falls into a condition of promoter-proximal transcriptional arrest that prevents production of full-length transcripts both in vitro and in vivo. Arrest occurs when RNAP synthesizes 9-14-nucleotide-long transcripts and backtracks by 5-7 (PrplN) or 2-4 (PompX) nucleotides. Initiation factor sigma(70) contributes to the formation of arrested complexes at both promoters. The signal for promoter-proximal arrest at PrplN is bipartite and requires two elements: the extended -10 promoter element and the initial transcribed region from positions +2 to +6. GreA and GreB prevent arrest at PrplN and PompX by inducing cleavage of the 3'-proximal backtracked portion of RNA at the onset of arrested complex formation and stimulate productive transcription by allowing RNAP to elongate the 5'-proximal transcript cleavage products in the presence of substrates. We propose that promoter-proximal arrest is a common feature of many bacterial promoters and may represent an important physiological target of regulation by transcript cleavage factors.
Department of Cell Biology, School of Osteopathic Medicine at Stratford, University of Medicine and Dentistry of New Jersey, Stratford, New Jersey 08084, USA.
Ekaterina
E
Stepanova
Minshi
M
Wang
Konstantin
K
Severinov
Sergei
S
Borukhov
D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics; D002474:Cell-Free System / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D006867:Hydrolases / Q000096:biosynthesis* / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000502:physiology*; D012329:RNA, Bacterial / Q000096:biosynthesis / Q000235:genetics; D012269:Ribosomal Proteins / Q000096:biosynthesis* / Q000235:genetics; D012808:Sigma Factor / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic / Q000502:physiology*; D035602:Transcriptional Elongation Factors / Q000235:genetics / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; C079668:GreA protein, E coli; C079667:GreB protein, E coli; D012329:RNA, Bacterial; D012269:Ribosomal Proteins; D012808:Sigma Factor; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; C041174:ribosomal protein L14; C482905:OmpX protein, E coli; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases; D006867:Hydrolases