Dissecting fundamental mechanisms of protein translation in Saccharomyces cerevisiae
- Dustin Howard Hite.
- Aug. 2013.
- Physical description
- online resource (x, 109 pages) : illustrations (some color)
- Hite, Dustin Howard.
- Brown, Patrick O'Reilly, 1954- thesis advisor (primary).
- Das, Rhiju. thesis advisor.
- Herschlag, Daniel thesis advisor.
- Straight, Aaron, 1966- thesis advisor.
- Stanford University. Department of Biochemistry.
- Stanford University. Committee on Graduate Studies. degree grantor.
- Includes bibliographical references (p. 102-109). 125 refs.
- The central dogma of biology states that DNA, the genetic information, is transcribed into RNA, an information containing intermediate, which is then translated into proteins, actionable molecules which perform the majority of tasks required for life. To synthesize proteins, the cell employs a massive, macromolecular machine, the ribosome, and a myriad of protein factors to successfully translate an mRNA. My graduate studies have focused both on the ribosome and the protein translation factors that interact with the ribosome to facilitate translation initiation, elongation, and termination. First, utilizing recent advances in high throughput sequencing, we discovered that sequencing of ribosome protected fragments could illuminate in vivo dynamics of ribosome structural changes in Saccharomyces cerevisiae. We demonstrated that the ribosome protects two distinct sizes of fragments and assigned each fragment population to approximate stages of the translation elongation cycle where large structural rearrangements of the ribosome are known to occur. Once these assignments were made, we were able to model elongation speed and demonstrated that, contrary to previous reports, tRNA abundance and codon optimality were not the major determinants of elongation speed; surprisingly our data indicated that the polarity of the amino acid being decoded dictated elongation rates under these conditions, with polar amino acids acting to slow elongation rates. This study also implicated Dom34, a known NO GO decay factor, as a novel component of canonical translation termination and ribosome recycling. Second, we used another genome-wide assay of translation, "gradient encoding" microarray analysis, to interrogate the genome-wide effects of depleting five individual translation factors. Based on the current understanding of the molecular mechanisms of each translation factor, we hypothesized that the depletion of each factor would result in differential translation of mRNAs based on the physical properties of each mRNA species. However, we were startled to observe that the translational program of S. cerevisiae was relatively unperturbed by the depletion of three initiation factors, one elongation factor, and one termination factor. Further investigation revealed that yeast were actively compensating for the deficiency of each factor by either increasing or decreasing translation initiation rates such that the depleted factor was no longer limiting. This tuning was mediated by changes in eIF2[alpha] phosphorylation levels, a known modulator of translation initiation. Overall, we have leveraged high throughput technologies to provide novel understanding of in vivo structural dynamics of the ribosome and reveal a novel, unexpected robustness of the translational program in S. cerevisiae.
- Peptide Chain Elongation, Translational
- RNA, Messenger > metabolism
- Ribosomes > metabolism
- High-Throughput Nucleotide Sequencing
- Peptide Elongation Factors > metabolism
- Saccharomyces cerevisiae > metabolism
- Publication date
- Submitted to the Department of Biochemistry and the Committee on Graduate Studies of Stanford University.
- Thesis (Ph.D.)--Stanford University, 2013.