Investigating the mechanism of mRNA cleavage in the ribosomal A-site

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DESCRIPTION (provided by applicant): The specific goal of this project is to elucidate the catalytic mechanism of A-site mRNA cleavage induced by RelE. Under stressful conditions, RelE induces codon specific cleavage of mRNA in the A-site, inhibiting translation in eubacteria and archae. A-site mRNA cleavage is not unique to RelE. This cleavage has been observed upon ribosome stalling and is thought to be required for ribosome recycling of stalled ribosomes; however, the factors required for cleavage are unknown. Codon specific recognition of A-site mRNA by RelE is similar to that observed by the release factors. The broad goals of this project are to determine how A-site mRNA cleavage occurs to gain insight into this form of translation regulation. The specific goals of this proposal are to determine the chemical nature of A-site cleavage (aim 1), investigate the presence of catalytic metal-ions (aim 2), and explore the catalytic role of residues in RelE and the ribosome (aims 3 and 4). Because RelE cannot cleave naked mRNA and does not contain the expected catalytic acid or base and the ribosome does not cleave mRNA in the A-site in the absence of external factors, it is unclear if the ribosome or RelE contains the catalytic components necessary for mRNA cleavage. Stalled 70S ribosomes complexes with an open A-site will be used to monitor mRNA cleavage in vitro. The specific 5' and 3' products of the cleaved mRNA will be determined (aim 1), identifying the reaction chemistry and providing clues to possible catalytic mechanism. Metal-ion specificity switch experiments will be used to identify the presence of catalytic metal-ions. Alanine mutants of conserved residues in RelE, as well as non-lethal ribosome mutants of residues in the A-site, will be assayed using single turnover kinetics to identify if catalytic residues reside on RelE or the ribosome (aim 3 and 4). Information on binding and catalysis will illuminate the individual contributions of the ribosome and RelE in cleavage of mRNA. PUBLIC HEALTH RELEVANCE: This proposal outlines the investigation of initial step in ribosomal recycling, A-site mRNA cleavage. Several antibiotics work by stalling the ribosome and blocking translation and A-site cleavage has been linked to antibiotic resistance. Understanding the mechanism of A-site mRNA cleavage may add insight into how to more efficiently use antibiotics to block translation.