In Escherichia coli, growth is rate-limited by translation capacity . Stalled ribosomes have profound effects on a cell such as altered mRNA abundance, decreased ribosome availability, and an imbalanced proteome. The absence of elongation factor P (EF-P), a universally conserved transpeptidation enhancer, presents an extreme example of this scenario, wherein ribosomes accumulate disproportionately onto messages that are more slowly translated and cell growth becomes notably impaired. We discovered that faster-growing cells arise spontaneously in Δefp cultures, suggesting that translation defects could be circumvented by mutating other genes. This thesis presents a genetic and biochemical analysis of a mechanism Δefp cells employ to overcome translation stress. Using a dual luciferase reporter system, we found that transpeptidation remained hindered in the faster growing Δefp cells. Whole genome sequencing of several fast-growing strains revealed mutations in a poorly characterized RNA helicase called HrpA. We determined that deletion of hrpA, or mutations at several conserved residues critical for HrpA's function, was sufficient to improve the fitness of Δefp cells. HrpA is a DEAH-box RNA helicase and represents a large class of enigmatic proteins that use ATP to restructure cellular RNAs; however, it's direct function in cellular physiology has yet to be clearly demonstrated [2, 3]. Several HrpA mutants were engineered to interrogate the molecular mechanism of HrpA and how its function impairs Δefp cells. Complementation in Δefp ΔhrpA cells showed that a number of these mutants were unable to restore sickness, suggesting they were defective in key aspects of RNA processing. It was discovered that wild-type HrpA is associated with actively translating ribosomes and several of the inactive HrpA mutants impose substantial deleterious effects on translation and ribosome production. In sum, the work presented here describes a mechanism by which cells overcome translation stress involving a novel genetic and biochemical relationship between EF-P and HrpA.
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Master of Science (M.S.)
College of Medicine
Length of Campus-only Access
Masters Thesis (Open Access)
Wingo, Robert, "Exacerbation of ΔEFP Sickness in Escherichia coli By an Uncharacterized RNA Helicase" (2018). Electronic Theses and Dissertations. 6034.
Restricted to the UCF community until August 2021; it will then be open access.