Student Theses and Dissertations

Date of Award

2008

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Muir Laboratory

Abstract

Although the multi-subunit RNA Polymerase (RNAP) structures have revolutionized our understanding of transcription, we still do not fully understand the molecular details of bacterial promoter recognition and melting. In addition, our understanding is generally limited to highly conserved elements of the structure, with little focus on the bacterial lineage-specific domain insertions. Furthermore, we lack information about the hidden functional residue networks that underlie the activities of this complex multi-subunit molecular machine. By combining structural and computational methods we: (1) Used X-ray crystallography to investigate promoter -35 element recognition by domain 4 of the Group IV sigma factors, revealing that conserved positions within -35 element induce an AA/TT-tract like DNA geometry, allowing for indirect promoter recognition despite the absence of direct protein/DNA interactions with several highly conserved DNA bases. (2) Created comprehensive multiple sequence alignments for the two bacterial large subunits (β/β′) and their homologues from the following multisubunit RNAPs: bacterial, eukaryotic pol I/II/III, Nuclear-Cytoplasmic Large double-stranded DNA Viruses, archeal, and plant plastid. To aid in the creation of the alignments we also developed a sequence retrieval and processing system termed BlaFA (BLAST to FASTA File to Alignment). As a result of our analysis we gained insights into shared sequence regions, the bacterial large subunit intergenic spacing, and the bacterial lineage-specific domain insertions. (3) Used our multi-subunit RNAP alignments and Statistical Coupling Analysis (SCA) to determine co-evolving residue networks within and between the two large subunits, as well as with omega/Rpb6. In addition, we uncovered a previously unidentified principle of co-evolution, namely the role of adapter groups in bridging and coordinating the independently evolving main group networks which were responsible for key aspects of transcription including: catalysis and RNAP interactions with DNA and RNA during initiation, elongation, and termination. (4) Used structure based modeling to computationally trap and understand promoter -10 element recognition by the Group I sigma factors. Our results revealed an unexpected upstream shift of the -10 element during recognition possibly validating a previously proposed twist and melt mechanism of DNA melting. (5) Determined the conditions necessary to express and purify a Group IV sigma factor from Thermus Thermophilus.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

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