Text

theses

The N-terminal conserved region of σ (σR1.1) is a highly negatively charged segment with 90-100 amino acids. In free σ, it interacts with DNA-binding determinants on σ, preventing free σ from association with promoter DNA. In RNA polymerase (RNAP) holoenzyme, σR1.1 no longer masks DNA-binding determinants on σ, therefore enabling association with promoter DNA. Deletion of σR1.1 has significant effects on rates of formation of RNAP-promoter open complex (RPo). The solution structure of σR1.1 has been solved by NMR. Ensemble fluorescence resonance energy transfer (FRET) analysis indicate that σR1.1 acts as a “molecular-mimic” of DNA by occupying the active-center cleft in RNAP holoenzyme, and must be displaced out of the active-center cleft upon formation of RPo. However, the precise positions and orientations of σR1.1 in holoenzyme and RPo have remained uncertain. Recent crystal structures of σR1.1 in RNAP holoenzyme from two groups—Murakami and co-workers and Darst and co-workers—place σR1.1 in RNAP holoenzyme inside the RNAP active-center cleft, consistent with the ensemble FRET analysis, but the folds and the rotational orientations of σR1.1 in the crystal structures from the two groups are different. In this work, I have used systematic single-molecule FRET and distance-restrained docking to define the positions and rotational orientations of σR1.1 in RNAP holoenzyme in solution and in RPo in solution. The results for RNAP holoenzyme indicate that, in RNAP holoenzyme in solution, σR1.1 is located inside the active-center cleft, in a position and rotational orientation consistent with the crystal structure from Darst and co-workers. The results for RPo indicate that, in RPo in solution, σR1.1 is located outside the active-center cleft—at least 40 Å away from its position in holoenzyme—is positioned between the RNAP β’ jaw and β dispensable region 1 (βDR1), and potentially makes direct protein-protein interactions with βDR1. Deletion of βDR1 affects the rate and temperature-dependence of formation of RPo and alters the footprint of RPo. My results suggest that, upon formation of RPo, σR1.1 is displaced from the RNAP active-center cleft to a binding site on βDR1, and, as such, provide an explanation for previously detected effects of deleting βDR1.

ETD_5119

Ph.D.

Includes bibliographical references

Includes vita

by Qumiao Xu

doi:10.7282/T3DV1GXQ

ETD doctoral

The author owns the copyright to this work.