Date of Award

12-1-2021

Language

English

Document Type

Master's Thesis

Degree Name

Master of Science (MS)

College/School/Department

Department of Biomedical Sciences

Content Description

1 online resource (x, 119 pages) : color illustrations.

Dissertation/Thesis Chair

Joseph T Wade

Committee Members

Keith Derbyshire , Pallavi Ghosh

Keywords

antitermination, Cas6, CRISPR, Rho, transcription termination, Genetic transcription, RNA polymerases, Transcription factors, Escherichia coli

Subject Categories

Molecular Biology

Abstract

Transcription termination factor Rho is an essential protein in many bacteria. Rho is an ATP-dependent RNA helicase that accounts for 20-50% of termination events in Escherichia coli and plays an important role in preventing pervasive transcription that otherwise might be lethal to bacteria. Rho termination usually occurs in the 3’ UTRs and non-coding regions of the DNA, and the actively transcribing RNA is believed to be protected by translating ribosomes from Rho termination since both transcription and translation are a simultaneous process in prokaryotes. However, in reality, Rho terminates transcription of some protein-coding genes, and fails to terminate transcription of some long non-coding CRISPR array transcripts. These apparent anomalies are the focus of this thesis.Premature Rho termination occurs in 5’ UTRs and ORFs, which account for more than 3% of Rho termination events. We used complementary RNA-sequencing approaches to detect 3´ ends in 5´ UTRs and ORFs and discovered novel regions of premature termination in the protein coding regions. Additionally, characterization of these prematurely terminated genes revealed the abundance of cis- and trans- acting elements in the 5’UTRs that are involved in modulation of Rho termination. CRISPR Cas systems serve as adaptive immune systems in bacteria. CRISPR Cas systems function by acquiring new foreign DNA sequence when infected, storing them in the form of spacers that will be used upon reinfection. CRISPR arrays consist of short repeats arranged alternative to the spacers and often yield long non-coding transcripts making them susceptible to Rho termination. It is important for bacteria that CRISPR arrays are protected from Rho termination and recently a widespread BoxA-mediated antitermination mechanism was identified, which involves the Nus factor protein complex. Previously, we found that there are a major set of CRISPR arrays in various bacterial species that lack a boxA sequence upstream of CRISPR arrays. Here, we investigated two scenarios for how CRISPR arrays are protected from premature Rho termination. First, looking specifically into species that have CRISPR type II-C system revealed that type II-C systems have internal promoters in individual repeats, obviating the need for an antitermination mechanism. Second, we looked at a type I-E CRISPR system that has a boxA upstream of the CRISPR array. Our results indicate that the Cas6 ribonuclease regulates the expression of an engineered reporter constructs, independent of Rho; rather, Cas6 prevents formation of a repressive structure in the RNA. Additionally, our genome-wide study showed that Cas6 is involved in global regulation of genes in E. coli.

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