All cells, from bacterial to human, use ribosome pausing as a general strategy to regulate protein expression. Regulated ribosome pausing appears to be particularly common during the synthesis of proteins that are important for controlling cell growth and preventing cancer. The long-term goal is to understand how some paused ribosomes elicit a unique ribonuclease activity that cleaves messenger RNA (mRNA) in a position corresponding to the ribosome A-site. This A-site cleavage activity destabilizes mRNAs and may represent a novel mechanism of gene regulation. The specific hypothesis is that the newly-synthesized nascent protein elicits a ribosome arrest, which induces the ribosome to catalyze A-site mRNA cleavage. The hypothesis is based on the observations that 1) A-site mRNA cleavage requires specific nascent peptide sequences, 2) the ribosome and translational pausing are required for mRNA cleavage, and 3) the ribosome usually protects A-site mRNA from ribonucleases. Based on these observations, the experimental focus of this proposal is to determine the molecular requirements of A-site mRNA cleavage.
The specific aims are to: 1. Determine the substrate requirements for A-site mRNA cleavage. Nascent peptide sequences will be screened for those that support efficient cleavage, and A-site codon sequence specificity will be determined. 2. Identify ribosomal components that are important for A-site mRNA cleavage. Ribosomal RNA is critical for decoding A-site mRNA. Ribosomal RNA is hypothesized to play an important role in the cleavage of the A- site codon during translational pause events. Known mutations and antibiotics that modulate the fidelity of ribosome decoding will be studied for effects on A-site mRNA cleavage. In addition, directed and unbiased mutagenic approaches will be used to produce ribosome variants that have altered A-site cleavage properties. 3. Reconstitute the A-site mRNA cleavage reaction in vitro. If the ribosome catalyzes A-site cleavage, then it should be possible to reconstitute the A-site cleavage reaction in a defined in vitro translation system comprised of highly-purified components. If the ribosome does not catalyze A-site mRNA cleavage, the defined translation system will be used in a biochemical approach to identify the trans-acting A-site nuclease. ? ? ?
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