Examining the role yeast initiator tRNA sequence and structure plays in communicating start codon recognition

Translation;tRNA;yeast;scanning;initiation;initiator;ribosome;Biophysics

Munoz, Antonio MichaelBailey, Scott ;

Johns Hopkins University

关键词: Translation;    tRNA;    yeast;    scanning;    initiation;    initiator;    ribosome;    Biophysics;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/37204/MUNOZ-DISSERTATION-2014.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

Translation initiation accuracy in the cell is of paramount importance, as beginning polypeptide synthesis at the wrong location can lead to the production of toxic proteins. In vivo, altering elements in various regions of tRNAi results in either decreased (Sui- phenotype) or increased (Ssu- phenotype) fidelity of start codon recognition. We have characterized the behaviors of mutant initiator tRNAs as they may affect the transition between the scanning-competent Pout/Open state of the PIC and the scanning-arrested Pin/Closed state. Single mutations in the acceptor stem produce both Sui- phenotypes and defects in forming the eIF2-GTP-Met-tRNAi ternary complex (TC). Sui- mutations also reduce off rates, consistent with their stabilizing the closed state of the PIC and decreasing fidelity. The Sui- acceptor stem mutation G70A also dramatically reduces the TC binding rate, but this is completely rescued by an Ssu- mutation in the N-terminal tail of eIF1A. Anticodon stem mutants conferring the Ssu- phenotype exhibit defects in the affinity of TC for the 40S subunit. Other Sui- mutations in the T loop and ASL display slightly slowed rates of association, indicating a distinct mechanism in blocking rearrangement to the Pin/Closed state. We demonstrate that the initiator tRNA sequence and structure are finely tuned to allow accurate start codon recognition by the preinitiation complex, highlighting the role of tRNA as an active player in translation. Over the course of this study a new protocol for purifying yeast ribosomes was established wherein a monolithic anion exchange column was employed as an initial separation and concentration step. This method results in an increase in yield, while also decreasing preparation variability and reducing reagent usage. Lastly, additional experiments were performed to understand the roles and interplay of factors eIF5, eIF1, and eIF1A. This work furthered the understanding of how the C-terminal tail (CTT) of eIF1A comes into closer proximity with the N-terminal domain of eIF5 and also linking this event to eIF1 dissociation, the rate of movement of the eIF1A-CTT and eIF5-NTD towards each other, and phosphate release from eIF2.