Define the identity and properties of inhibitory codon combinations

Project Collaborators:

Dr. Mark Dumont, Dr. Eric Phizicky, Dr. Edward Snell Hauptman-Woodward Institute, Dr. Michael Snyder Stanford University

Although accurate and efficient translation of mRNA into proteins is crucially important for the cell, not all of the 61 codons that specify insertion of amino acids into nascent polypeptides behave equally in this process. In fact, almost all synonymous codons, i.e. those that specify the same amino acid, are used at very different frequencies within most organisms, including the yeast S. cerevisiae. Although codon use has been implicated for over 25 years as a determinant of expression levels, neither the identity, nor the defining characteristics of codons that impair expression are known. Studies in the Grayhack laboratory are designed to define the rules and mechanisms by which specific codons, and arrangements of codons, impair gene expression, and to identify regulatory models dictated by codon usage in the yeast genome. We performed the first systematic analysis of the effects of codons on expression and implicated wobble decoding of the CGA codon pairs as the effecter of reduced expression.

To deduce the scope and magnitude of codon-mediated effects, we are identifying additional codon combinations that are responsible for reduced translational efficiency and defining the parameters that modulate codon-mediated regulation. We are extending the identification of codon combinations that cause reduced expression in yeast, using a GFP reporter in which randomized codon combinations are inserted at amino acid 4. Insertions that cause low expression of GFP are selected with Fluorescent Activated Cell Sorting. The resulting definition of inhibitory codons may illuminate the relationship between translation, codon properties and impaired expression. Second, we are determining the parameters of codon density, arrangement, and location that are important to impair expression to learn whether it is the arrangement and/or their location within a gene that results in reduced expression. Delineation of these factors may reveal the role of interactions between sites on the ribosome in codon-mediated modulation of expression.

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