Analysis of the Regulation of Translation Elongation Factor 2 Levels and Posttranslational Modifications in Saccharomyces Cerevisiae PDF Download
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Author: Pedro A. Ortiz
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Category :
Languages : en
Pages : 268
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Author: Pedro A. Ortiz
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ISBN:
Category :
Languages : en
Pages : 268
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Author: Pamela Anne Zobel-Thropp
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Category :
Languages : en
Pages : 190
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Author: Dustin Howard Hite
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Category :
Languages : en
Pages :
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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.
Author:
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Category : Medicine
Languages : en
Pages : 866
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Author: Karen Renee Christie
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Category :
Languages : en
Pages : 600
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Regulation of the process of transcriptional elongation is an important control mechanism in the expression of some genes. To fully understand this form of regulation will require better understanding of the functions of transcription elongation factors. The goal of this work was to characterize the transcription elongation factor TFIIS from Saccharomyces cerevisiae, originally called P37. I demonstrated that, like the mammalian TFIIS proteins, the yeast protein stimulates RNA polymerase II to cleave the nascent RNA transcript and to read-through an intrinsic block to elongation. Investigation of the protein-protein contacts between TFIIS and RNA polymerase II indicated that the carboxyl-terminal domain of the largest subunit, subunit four, and subunit seven of the polymerase are not required for TFIIS to promote cleavage and read-through by the polymerase. In addition the carboxyl-terminal half of the yeast TFIIS protein is sufficient for both of these in vitro activities. This result is consistent with the previous results demonstrating the carboxyl-terminus of mouse TFIIS was sufficient to activate RNA polymerase in vitro.
Author: Felix Schirmaler
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Category :
Languages : en
Pages : 238
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Author: Boris Zinshteyn
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Category :
Languages : en
Pages : 159
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The goal of this thesis is to elucidate the mechanisms that govern translational efficiency (TE) - the amount of protein produced from each molecule of mRNA. While the mechanisms regulating the TE of a few specific messages are well understood, the general contribution of translational control to differences in cellular protein levels is currently unclear. Recent advances have enabled the direct measurement of protein levels and translation rates genome-wide, and studies in multiple organisms have found varying degrees of translation regulation, both at steady state, and in response to stress or developmental cues. Despite this influx of high-throughput data, the mechanisms underlying the differences in gene-specific and condition-dependent TE remain largely unknown. In this thesis, I describe the roles of two different components of the translational machinery in regulating translational efficiency. In Chapter 1, I discuss the features of mRNA coding sequences that can affect TE, thereby introducing Chapter 2, in which I investigate the role of a conserved anticodon tRNA modification in determining the rate of translation elongation and the phenotypic consequences of its loss for budding yeast. In Chapter 3, I discuss the regulation of translation initiation to introduce Chapter 4, in which I explore how the RNA binding specificity of the core translation factor, yeast eukaryotic initiation factor 4G (eIF4G), contributes to genome-wide competition between mRNAs. Finally, I will discuss future directions for this work.
Author: Caren Jody Stark
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ISBN:
Category :
Languages : en
Pages : 538
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Author: Chi-Ting Huang
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Category : Saccharomyces cerevisiae
Languages : en
Pages : 244
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Author: Vince Harjono
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Category :
Languages : en
Pages : 111
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Post-transcriptional regulation represents a powerful and versatile mechanism to fine-tune gene expression to meet cellular and environmental demands. One important aspect of post-transcriptional regulation involves regulation of protein translation, the process of building proteins from a messenger RNA. In this dissertation, I use biochemical and molecular biology techniques to study how translation is mechanistically regulated by both mRNA and protein factors. In chapter 2, I discuss the development of a quantitative method in eukaryotes to measure ribosomal stalls of cis-mRNA factors on protein elongation. We find that different distributions of nonoptimal codons trigger different surveillance and rescue pathways despite similar levels of elongation delay. In chapter 3, I explore the relationship between translatability and mRNA localization during glucose starvation and investigate potential factors that influence this relationship. We find that a complex made from Rvb1 and Rvb2 is involved in promoter-directed cytoplasmic fate in a subset of stress response genes in glucose starvation. Furthermore, we use carefully designed reporters to interrogate how translatability determines cytoplasmic localization and find that active translation is linked to exclusion from stress-induced cytoplasmic granules. Finally in chapter 4, I discuss improvements on the method we have developed, possible future directions for the work described in this dissertation, and my concluding remarks.
