title: Mechanisms of Very Long Abortive Transcript Release during Promoter Escape.
abstract: A phage T5 N25 promoter variant, DG203, undergoes the escape transition at the +16 to +19 positions after transcription initiation. By specifically examining the abortive activity of the initial transcribing complex at position +19 (ITC19), we observe the production of both GreB-sensitive and GreB-resistant VLAT19. This suggests that ITC19, which is perched on the brink of escape, is highly unstable and can achieve stabilization through either backtracking or forward translocation. Of the forward-tracked fraction, only a small percentage escapes normally (followed by stepwise elongation) to produce full-length RNA; the rest presumably hypertranslocates to release GreB-resistant VLATs. VLAT formation is dependent not only on consensus -35/-10 promoters with 17 bp spacing but also on sequence characteristics of the spacer DNA. Analysis of DG203 promoter variants containing different spacer sequences reveals that AT-rich spacers intrinsically elevate the level of VLAT formation. The AT-rich spacer of DG203 joined to the -10 box presents an UP element sequence capable of interacting with the polymerase α subunit C-terminal domain (αCTD) during the escape transition, which in turn enhances VLAT release. Utilization of the spacer/-10 region UP element by αCTD subunits requires a 10-15 bp hypertranslocation. We document the physical occurrence of hyper forward translocation using ExoIII footprinting analysis.
authors.affiliation: Biology Department, Bryn Mawr College , Bryn Mawr, Pennsylvania 19010, United States.
authors.forename: Monica
authors.initials: M
authors.lastname: Chander
authors.affiliation: Program in Biochemistry, Mount Holyoke College , South Hadley, Massachusetts 01075, United States.
authors.forename: Ahri
authors.initials: A
authors.lastname: Lee
authors.affiliation: Program in Biochemistry, Mount Holyoke College , South Hadley, Massachusetts 01075, United States.
authors.forename: Tenaya K
authors.initials: TK
authors.lastname: Vallery
authors.affiliation: Program in Biochemistry, Mount Holyoke College , South Hadley, Massachusetts 01075, United States.
authors.forename: Mya
authors.initials: M
authors.lastname: Thandar
authors.affiliation: Program in Biochemistry, Mount Holyoke College , South Hadley, Massachusetts 01075, United States.
authors.forename: Yunnan
authors.initials: Y
authors.lastname: Jiang
authors.affiliation: Program in Biochemistry, Mount Holyoke College , South Hadley, Massachusetts 01075, United States.
authors.forename: Lilian M
authors.initials: LM
authors.lastname: Hsu
mesh_terms: D001692:Biological Transport; D018983:DNA Footprinting; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics*; D017906:Siphoviridae / Q000235:genetics
chemical_list: D012333:RNA, Messenger; D012321:DNA-Directed RNA Polymerases