MECHANISMS OF INTRINSIC TRANSCRIPTION TERMINATION AND RIBONUCLEOLYTIC DECAY IN Bacillus subtilis
Open Access
- Author:
- Mandell, Zachary
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- May 22, 2022
- Committee Members:
- Paul Babitzke, Chair & Dissertation Advisor
Istvan Albert, Major Field Member
Timothy Meredith, Outside Field Member
Philip Bevilacqua, Outside Unit Member
Katsuhiko Murakami, Major Field Member
Wendy Hanna-Rose, Program Head/Chair - Keywords:
- transcription
genomics
transcriptomics
RNA biology
translation
Microbiology - Abstract:
- Gene expression is regulated at many levels, including both the transcription and decay of RNA. Transcription terminates at discrete locations across the genome. After release, the resultant transcripts are degraded. This thesis outlines recent studies that utilized biochemical, molecular, genetic, genomic, physiological, and computational techniques to further our understanding of how transcription termination and RNA decay are regulated in the Gram+ organism Bacillus subtilis. Transcription termination is known to occur via two mechanisms in bacteria, intrinsic termination (also frequently referred to as Rho-independent, or factor-independent termination), and Rho-dependent termination. Based primarily on in vitro studies using Escherichia coli RNA polymerase, it was generally assumed that intrinsic termination and Rho-dependent termination are distinct mechanisms, and that the signals required for intrinsic termination are present primarily within the nascent RNA. In this dissertation, I detail experiments that I conducted in B. subtilis, which show that intrinsic termination in this organism is highly stimulated by NusA, NusG, and even Rho. In NusA-stimulated intrinsic termination, NusA facilitates the formation of weak terminator hairpins and compensates for distal U-rich tract interruptions. In NusG-stimulated intrinsic termination, NusG stabilizes a sequence-dependent pause at the point of termination, which extends the timeframe for RNA hairpins with weak terminal base pairs to form in either a NusA-stimulated or a NusA-independent fashion. Rho stimulates intrinsic termination by preventing the formation of antiterminator-like RNA structures that could otherwise compete with the terminator hairpin. Combined, NusA, NusG, and Rho stimulate approximately 97% of all intrinsic terminators in B. subtilis. Thus, the historical distinction between Rho-dependent and intrinsic termination is overly simplistic and needs to be modernized. Moreover, the general view that intrinsic termination is primarily a factor-independent process needs to be revised to account for recent findings. The B. subtilis genome encodes for four known 3’ to 5’ exoribonucleases; polynucleotide phosphorylase (PNPase), RNase R, RNase PH, and YhaM. In E. coli, PNPase functions as a member of a multi-protein complex dedicated to the decay of mRNA. This complex is known as the degradosome. One function of the degradosome is to ensure that PNPase remains physically associated with RhlB, an RNA helicase. Whether PNPase operates in the context of a degradosome in B. subtilis remains controversial. In this dissertation I describe experiments that I conducted in B. subtilis, showing that PNPase cooperates with CshA, an RNA helicase, to degrade particularly structured mRNAs on a genome-wide level. Moreover, I obtained evidence that this cooperation occurs independently of a degradosome. In addition, I detail experiments that point towards the presence of a fifth, as-yet unidentified, 3’ exoribonuclease.