Graduation Year
2021
Document Type
Thesis
Degree
M.S.
Degree Name
Master of Science (M.S.)
Degree Granting Department
Biology (Cell Biology, Microbiology, Molecular Biology)
Major Professor
Lindsey N. Shaw, Ph.D.
Committee Member
Prahathees Eswara, Ph.D.
Committee Member
Zhiming Ouyang, Ph.D.
Keywords
Omega subunit, Staphylococcus aureus, Structure-function analysis, Transcription, RpoZ, RNA Polymerase
Abstract
In bacteria, RNA polymerase (RNAP) is a well-characterized and highly conserved multi-subunit enzyme complex responsible for transcription of DNA into RNA. The ω subunit (rpoZ/RpoZ), one of the smaller accessory subunits of RNAP, is often overlooked and under-studied, however, particularly in Gram-positive species. We have previously shown that deleting ω impacts the integrity of RNA polymerase in S. aureus, especially the β' subunit, and alters preference of the core enzyme for sigma factors, skewing heavily towards σB instead of the housekeeping σ factor. Consequently, this causes deregulation of myriad transcriptional processes, strongly rewiring gene expression circuits, and ultimately impairing the virulence of rpoZ mutants. Despite these findings, we have little understanding of the key structural features of the S. aureus (and Gram-positive) ω subunit, and the molecular mechanism behind how it influences complex stability and specificity. Herein, we perform a comprehensive structure-function analysis of ω in S. aureus using alanine scanning mutagenesis. A library of his6-tagged, mutated complements was created in a rpoZ mutant whereby all residues were converted to alanine (the 6 native alanines were changed to serines). These were then assessed for their ability to complement known phenotypes of the S. aureus rpoZ mutant, and to associate with, and pull-down, core-RNAP. In so doing, we garnered exquisite insight into key residues/regions of the ω subunit that functionally impacts its ability to associate with RNA polymerase and influence its pleotropic role governing S. aureus transcriptional specificity and disease causation.
Scholar Commons Citation
Patil, Shrushti B., "A Functional Characterization of the Omega (ω) subunit of RNA Polymerase in Staphylococcus aureus" (2021). USF Tampa Graduate Theses and Dissertations.
https://digitalcommons.usf.edu/etd/9707
