Date of Award

2019

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biotechnology Science and Engineering

Committee Chair

Luis R. Cruz-Vera

Subject(s)

Genetic regulation., Gene expression., Genetic translation.

Abstract

The metabolism of bacteria is finely tuned through differential regulation of gene expression, which allows bacteria to adapt to changes in the external environment. Oftentimes, expression of genes is regulated during protein synthesis. Changes in concentration and chemical composition of factors involved in protein synthesis contribute to the regulation of the expression of genes. Such differences are often exploited by researchers to control cell growth of microorganism of health and industrially relevant. Regulation of protein synthesis often produces the machinery called ribosome to stall (also known as ribosome arrest), where interactions between ribosomal nucleotides, translation factors, the nascent peptide and an activating ligand result in transient pausing. The tna operon serves as a model system to study gene regulation through translation arrest. Translation termination inhibition at tnaC controls the expression of structural genes tnaA and tnaB, which express tryptophanase and an L-Tryptophan (L-Trp) transporter, respectively. Free L-Trp molecules interact with the nascent peptide and the nucleotides of the exit tunnel and cause conformational changes in the exit tunnel. These changes are relayed to the peptidyl transferase center (PTC), which results in its inactivation. Two paralog proteins, Release Factor 1 (RF1) and Release Factor 2 (RF2), function at the PTC by promoting hydrolysis of the ester bond between the nascent peptide and peptidyl-tRNA using a conserved GGQ motif. The objective of this study was to determine how L-Trp and TnaC inhibit the function of release factors in the ribosome and determine the structural features of the factors that facilitate this. Our findings, through biochemical and genetic analyses, indicate that ribosome release aided by RF2 is preferentially inhibited over RF1, such difference is determined by two non-conserved residues surrounding the functional GGQ motif of the RF paralogs. Altogether, our results indicate the accommodation at the ribosomal PTC of the GGQ motif of RF2, unlike RF1, is not compatible with the conformational changes at the PTC when L-Trp and TnaC are bound in the ribosome leading to termination arrest. This difference between both RF paralogs could be exploited in the future to control differentially the expression of bacterial genes.

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