Codon Choice and Gene Expression in Saccharomyces Cerevisiae

University Of Rochester, Rochester NY

Investigators

Abstract

Intellectual Merit: Translation of mRNA into protein is a fundamental process requiring massive energy expenditure and a large fraction of the cell's components. Translation in all kingdoms is regulated both by signal transduction pathways, and by codon-mediated signals. Translation of different genes occurs with stunning differences in efficiency in that steady state expression levels of proteins vary by as much as six orders of magnitude relative to their mRNA abundance. Moreover, the genetic code itself plays a major role in modulating the efficiency of translation: The 61 codons that specify insertion of the twenty amino acids into proteins include many synonymous codons that specify insertion of the same amino acid, and these synonymous codons are used with vastly different efficiencies. There is a wealth of evidence that the particular choice of codons used to encode a protein influences its translation efficiency, as well as the accuracy of amino acid insertion, and protein folding. Although the identities of a set of 25 optimal codons that result in rapid and accurate translation in yeast have been known for over a quarter of a century, there has been no systematic description of how codons affect expression. Crucial questions that remain unresolved include the identity and properties of codons or codon combinations that cause reduced expression in eukaryotes, whether it is the arrangement and/or location of these codons within a gene that results in reduced expression, and how or why usage of these codons might affect expression. The goal of this project is to systematically determine how codon usage impacts gene expression in a eukaryote, the yeast Saccharomyces cerevisiae. In particular, codons and codon combinations that impair expression will be identified, the parameters of codon density, arrangement, and location that are important to impair expression will be defined, and the mechanisms by which codons and codon combinations modulate gene expression will be examined. The results obtained from this research are likely to yield crucial new insights into the perplexing problem of how translation efficiency is modulated to such a large extent by codon choice. This is an essential first step to begin to understand the importance of this regulation for the survival and biology of organisms. Broader Impacts: Thorough understanding of the codon-based factors that contribute to expression of proteins in yeast will provide a framework to understand the fundamental processes by which the genetic code modulates gene expression and may uncover physiologically important regulatory processes that depend on the particular choice of codons used in encoding some genes. These studies also have a practical benefit of guiding pharmaceutical, biochemical and structural biologists in their efforts to design synthetic genes to produce high levels of protein. Moreover, the project offers ideal opportunities to expose both undergraduate and high school students to scientific research, since the proposed work uses both robust methodology (involving yeast genetics and luciferase assays) and a non-hazardous organism (baker's yeast), both of which are optimal for hands-on research experience for undergraduates and high school students. Undergraduate students will be encouraged to join the laboratory, and high school students will be involved in collaboration with a colleague with a Masters of Education.