Intellectual Merit: Translation of the genetic code from triplet codons in mRNA into the amino acid sequences of proteins ultimately determines the protein composition of the cell. Translation affects the amount of each protein, is responsible for errors within the protein, and is regulated in response to energy needs, nutrient availability, and hormonal changes. During translation, sixty-one codons specify insertion of the twenty amino acids into proteins, and thus include many synonymous codons that specify insertion of the same amino acid. There is overwhelming evidence that the efficiency of translation is influenced by the choice of synonymous codons used to encode a polypeptide. However, neither the identity, nor the properties, of codons or codon combinations that reduce translation efficiency are known. The long-term goal of this project is to identify the codons and codon combinations that inhibit translation in the yeast Saccharomyces cerevisiae and to determine how they do so. An initial systematic analysis of the effects of individual codon repeats on translation showed that the CGA codon was translated inefficiently due to I-A wobble interactions with tRNA(Arg-ICG), and that adjacent CGA-CGA codon pairs were far more inhibitory than individual CGA codons. This result implies that there are likely other codon pairs or triplets that inhibit translation. To find inhibitory codon combinations, a robust and sensitive method, called RNAID, was developed, which uses fluorescence activated cell sorting (FACS) to identify GFP variants with altered expression. One aim of the current project is to comprehensively identify all codon pairs that have a significant inhibitory effect on translation and to assess their biological importance. Inhibitory codon combinations will be identified using RNA-ID, coupled with deep sequencing and computational analysis. The biological roles of these inhibitory codon combinations will be examined using bioinformatics approaches to test hypotheses about their location, representation, conservation, and effects on ribosome decoding. The biological consequences of inhibitory codon combinations will then be evaluated, by examining cellular fitness and individual protein amounts in cells expressing tRNA variants that suppress the inhibitory effects of these codon combinations. A second aim of the project is to determine the mechanisms by which these inhibitory codon combinations exert their effects. Cellular components that mediate effects of inhibitory codon combinations will be defined using genetic selections, as well as directed screens to assess the effects of known translational quality control factors and elongation factors. Study of these inhibitory codon pairs will lead to new insights into the translation machinery and its quality control mechanisms, including: the definition of common decoding properties of inhibitory codon combinations, definition of how codon interactions affect elongation, and knowledge of the biological functions of inhibitory codon combinations.

Broader Impacts: From the education and training perspective, this project will enable graduate students, undergraduates and high school students to connect biological research to the educational process. These students will employ the cutting edge technology of FACS combined with the simplicity of yeast genetics to participate in investigations of fundamental questions of gene expression. Both the robust methodology of yeast genetics and flow cytometry, and the non-hazardous organism (baker's yeast) are optimal for students' efforts. A program of study will be developed, including an introductory series of exercises, an individual research project and inclusion in scientific discourse. Undergraduates will be recruited from the 50-75 University of Rochester (UR) biochemistry majors for whom the PI is the faculty advisor and from the UR Summer Scholars Program, which recruits many students from primarily undergraduate colleges. High school students will be recruited through direct contact with high school counselors. From an applied scientific perspective, the comprehensive understanding of how codon choice affects translation produced from this project will be immensely useful for scientists interested in engineering and producing large amounts of protein for biochemistry, structural biology or biotechnology.

Agency
National Science Foundation (NSF)
Institute
Division of Molecular and Cellular Biosciences (MCB)
Application #
1329545
Program Officer
william eggleston
Project Start
Project End
Budget Start
2013-09-15
Budget End
2016-06-30
Support Year
Fiscal Year
2013
Total Cost
$555,000
Indirect Cost
Name
University of Rochester
Department
Type
DUNS #
City
Rochester
State
NY
Country
United States
Zip Code
14627