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.
Biological systems expend enormous resources to obtain both the desired amount of each protein and accuracy in its production, with the result that the concentration of individual proteins within a cell varies between 50 copies per cell to over 1 million copies per cell. To achieve this end, every step in the pathway of gene expression from transcription of DNA into RNA, through translation of RNA into protein, as well as mRNA and protein degradation, is highly regulated. Translation, which entails converting the genetic code in the mRNA into the amino acid sequence found in proteins, occurs with substantial differences in efficiency, that is, the amount of protein per mRNA varies by at least three orders of magnitude among different genes. The genetic code, which is comprised of 61 codons (three adjacent nucleotides) that specify insertion of the twenty amino acids into proteins, includes many synonymous codons that specify insertion of the same amino acid. The particular choice of codons used to encode a protein influences its translation efficiency. Although the identities of 25 optimal codons that result in rapid and accurate translation in yeast have been known for more than thirty years, there had been no systematic description of how suboptimal codons affect expression. The goal of this project was to systematically determine how codon usage impacts gene expression in a eukaryote, the yeast Saccharomyces cerevisiae. 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 upon codon choice. This systematic study in the yeast S. cerevisiae resulted in the identification of a particular codon, CGA, that causes a remarkable reduction in translation efficiency; this represented the first identification of a strongly inhibitory codon in a eukaryote. Three new insights into the ways in which translation efficiency is modulated by codon choice emerged from studies with the CGA codon. First, profound differences in translation efficiency can stem from decoding interactions within the ribosome, rather than differences in the amount of tRNA available to decode the codon. The primary cause of reduced expression by CGA codons is wobble decoding, a specific interaction between the tRNA and the codon. Wobble decoding is universal. Second, two adjacent codons, rather than individual codons, are the true effectors that modulate translation, an idea for which there was prior evidence, but not a consensus view. Adjacent CGA codons are far more potent inhibitors of expression than separated CGA codons. Third, the magnitude of codon-mediated effects can be much greater than previously suspected. As few as two adjacent CGA codons near the beginning of the gene can cause more than a 50% reduction in expression, overturning the notion that codon-mediated regulation was a sum of many very small effects. Since adjacent codons modulate expression, there are likely to be combinations of non-identical codons that inhibit translation. The need to find such inhibitory codon combinations lead us to develop the RNA-ID method, in which sequences are inserted into or upstream of GFP, and changes in expression result in changes in GFP fluorescence, which is monitored relative to a constant RFP signal using flow cytometry. The system is highly reproducible, can detect small differences in expression, has a 250-fold dynamic range and is designed for facile cloning of libraries of sequences. This method has general applications in the analysis of regulatory sequences within or upstream of the coding sequences. Studies aimed at discerning the mechanism of CGA-mediated inhibition and the cellular response to CGA codon repeats have connected CGA-mediated inhibition of translation to known quality control systems and demonstrated that defects in decoding CGA-CGA codon pairs involve two distinct defects in translation, one of which is best explained as failure in the initial step in decoding, acceptance of the tRNA by the ribosome. This project provided opportunities for the education of three graduate students, five undergraduates, and two high school students, who worked on the project. Both graduate and undergraduate students acquired expertise in translation regulation, and acquired technical expertise in genetic, RNA and fluorescence detection methods. Graduate students developed communication skills by presenting their work in multiple forms; undergraduates presented both a talk and either a poster or a paper. One undergraduate was awarded the President’s Award for Undergraduate Research in the Natural Sciences and Mathematics and earned a Degree with Distinction in Research. The high school students, one of whom was a student from an inner city school, acquired hands-on experience of genetic concepts and of laboratory environment. Two graduate students participated in teaching high school students, an opportunity both for them and the high school students. One graduate extended her involvement in education by taking a graduate level course in science education.
