Interactions Between Phage T7 Early Promoters And E Coli Rna Polymerase

Author : Hao-Che Wang
Publisher :
ISBN 13 :
Total Pages : 0 pages
Book Rating : 4.:/5 (138 download)

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Download or read book Kinetics and Mechanism of Open Complex Formation, Stabilization, and Initiation by E. Coli RNA Polymerase at [lambda]PR and T7A1 Promoters written by Hao-Che Wang and published by . This book was released on 2022 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: The flow of genetic information in cells starts with transcription by the enzyme RNA polymerase (RNAP) of DNA information to synthesize a wide variety of structural and functional RNAs which collectively participate in most cellular processes. Transcription is therefore a highly regulated step in gene expression. Research discussed here focuses on transcription initiation, the earliest phase of transcription, and on questions of how differences in promoter DNA sequences and length affect initiation thermodynamics and kinetics. During transcription initiation, RNAP binds to and unwinds the transcription start site region of promoter DNA, opening 13 bp to form an open complex (OC). Chapter 2 reports a detailed kinetic-mechanistic comparison of OC formation for the model λPR and T7A1 promoters, and Chapter 3 does the same for OC dissociation kinetics and mechanisms. For OC formation, kinetics of 2-amino purine (-4) fluorescence, Cy3 (-100) - Cy5 (+14) FRET, and single-dye PIFE were determined and compared with filter binding kinetics of formation of long-lived promoter complexes. A range of temperatures and of glycine betaine concentrations was examined. From these comparisons, we conclude that early steps in the mechanism of OC formation are similar for the two promoters while later steps including DNA opening differ significantly. Our data are consistent with previous reports that opening of the T7A1 bubble occurs in two adjacent kinetic steps vs. one step for λPR. After the DNA opening transition state, the kinetics and mechanisms of OC formation at these two promoters become similar again. In Chapter 3, we investigated the roles of clamp closing and of allosteric effects of discriminator and upstream interactions on downstream elements in stabilizing E. coli RNA polymerase- λPR promoter open complexes (OC). Our approach is to determine dissociation rate constants for the stable OC (kd) and for the unstable I2 intermediate OC (k-2) at λPR and T7A1 promoters and promoter variants with exchanged discriminators, core promoters or UP elements by filter binding assays. Values of kd are also determined for downstream-truncated promoters (at +12, +6) and for a jaw deletion RNAP variant. Analysis yields free energy changes 8́6G3^o for the conversion of unstable I2 to a stable OC, and the large contribution to 8́6G3^o for all promoters, from clamp closing on the promoter from +6 upstream, as well as the significant contributions to 8́6G3^o for promoters with the λPR discriminator from downstream interactions with the Îø lobe and Îø' jaw that are allosterically controlled by interactions of the discriminator with ϳ1.2 and the Îø gate loop. Lastly, in chapter 4, we further test how promoters with different OC stability affect the escaping of the RNAP from promoter. Preliminary results indicate the OC lifetime mainly alters the rate for converting the OC into initial complex (IC) for NTP incorporations. Taken together, our studies provide a more complete understanding of how promoter sequence and length influences different phases of transcription initiation.