title: Mechanism of transcription initiation and promoter escape byE.coliRNA polymerase.
abstract: To investigate roles of the discriminator and open complex (OC) lifetime in transcription initiation byEscherichia coliRNA polymerase (RNAP; α2ββ'ωσ70), we compare productive and abortive initiation rates, short RNA distributions, and OC lifetime for the λPRand T7A1 promoters and variants with exchanged discriminators, all with the same transcribed region. The discriminator determines the OC lifetime of these promoters. Permanganate reactivity of thymines reveals that strand backbones in open regions of long-lived λPR-discriminator OCs are much more tightly held than for shorter-lived T7A1-discriminator OCs. Initiation from these OCs exhibits two kinetic phases and at least two subpopulations of ternary complexes. Long RNA synthesis (constrained to be single round) occurs only in the initial phase (<10 s), at similar rates for all promoters. Less than half of OCs synthesize a full-length RNA; the majority stall after synthesizing a short RNA. Most abortive cycling occurs in the slower phase (>10 s), when stalled complexes release their short RNA and make another without escaping. In both kinetic phases, significant amounts of 8-nt and 10-nt transcripts are produced by longer-lived, λPR-discriminator OCs, whereas no RNA longer than 7 nt is produced by shorter-lived T7A1-discriminator OCs. These observations and the lack of abortive RNA in initiation from short-lived ribosomal promoter OCs are well described by a quantitative model in which ∼1.0 kcal/mol of scrunching free energy is generated per translocation step of RNA synthesis to overcome OC stability and drive escape. The different length-distributions of abortive RNAs released from OCs with different lifetimes likely play regulatory roles.
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706.
authors.forename: Kate L
authors.initials: KL
authors.lastname: Henderson
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706.
authors.forename: Lindsey C
authors.initials: LC
authors.lastname: Felth
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706.
authors.forename: Cristen M
authors.initials: CM
authors.lastname: Molzahn
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706.|Department of Chemistry, University of Wisconsin-Madison, Madison, WI 53706.
authors.forename: Irina
authors.initials: I
authors.lastname: Shkel
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison, Madison, WI 53706.
authors.forename: Si
authors.initials: S
authors.lastname: Wang
authors.affiliation: Program in Biophysics, University of Wisconsin-Madison, Madison, WI 53706.
authors.forename: Munish
authors.initials: M
authors.lastname: Chhabra
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison, Madison, WI 53706.
authors.forename: Emily F
authors.initials: EF
authors.lastname: Ruff
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706.
authors.forename: Lauren
authors.initials: L
authors.lastname: Bieter
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706.|Department of Chemistry, University of Wisconsin-Madison, Madison, WI 53706.
authors.forename: Joseph E
authors.initials: JE
authors.lastname: Kraft
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706; mtrecord@wisc.edu.|Department of Chemistry, University of Wisconsin-Madison, Madison, WI 53706.|Program in Biophysics, University of Wisconsin-Madison, Madison, WI 53706.
authors.forename: M Thomas
authors.initials: MT
authors.lastname: Record
mesh_terms: D004269:DNA, Bacterial / Q000235:genetics* / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D024363:Transcription Initiation Site; D014158:Transcription, Genetic
chemical_list: D004269:DNA, Bacterial; D012321:DNA-Directed RNA Polymerases
keywords: RNA polymerase; abortive RNA; hybrid length; open complex lifetime; transcription initiation