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Characterization of Fidelity Mechanisms in Protein Translation

Vargas-Rodriguez, Oscar E

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2014, Doctor of Philosophy, Ohio State University, Chemistry.
Decoding of the genetic information is established by aminoacyl-tRNA synthetases (ARSs), which are responsible for pairing amino acids with tRNA adaptors carrying nucleotide triplets matching the corresponding codon on the messenger RNA. The accuracy in amino acid selection by ARSs is challenged by the structural similarities shared by some genetically encoded amino acids, which leads to the formation of mis-matched aminoacyl-tRNA (aa-tRNA) pairs. Accumulation of such aa-tRNA species results in mis-incorporation of amino acids into protein sequences, which may induce protein misfolding and aggregation leading to a myriad of cellular phenotypes including apoptosis and neurodegeneration. Thus, some synthetases have adopted alternative catalytic functions to prevent mistranslation. In most bacterial prolyl-tRNA synthetases (ProRSs), which mischarge Cys and Ala onto tRNAPro, an editing domain (INS) functions to deacylate Ala-tRNAPro. In contrast, a dedicated trans-acting editing enzyme, known as YbaK, homologous to INS, is responsible for the hydrolysis of Cys-tRNAPro. However, in approximately 30% of bacteria, ProRSs lack an INS domain, and instead many of these organisms encode a single-domain INS-like protein known as ProXp-ala, which deacylates Ala-tRNAPro in trans. INS, YbaK, and ProXp-ala are part of a larger family of deacylases known as the INS superfamily that also includes three proteins of unknown function: ProXp-x, ProXp-y, and ProXp-z. In this work, we used biochemical approaches to investigate the function of the INS superfamily in the fidelity of protein translation. We first analyzed the distribution of INS and INS-like domains to establish the phylogenetic relationship between the six families. This study revealed the species-specific distribution of these proteins, which are proposed to be involved in preventing mistranslation. We focused on Caulobacter crescentus, which encodes a ProRS with a truncated INS domain that lacks catalytic activity, as well as YbaK and ProXp-ala. The in vitro characterization of these three enzymes highlighted the diversity of approaches used to prevent protein mistranslation and revealed a novel triple-sieve mechanism of editing that relies exclusively on trans-acting enzymes. Furthermore, investigation of the tRNA specificity of INS, YbaK, and ProXp-ala showed three distinct strategies for tRNA recognition. Whereas YbaK lacks inherent tRNA specificity, ProXp-ala and INS require tRNAPro acceptor stem elements G72/A73 and anticodon bases G35/G36, respectively. Furthermore, we show that evolutionary changes of the tRNAPro operational code acted as a selective pressure on ProXp-ala, which allowed co-adaptation of human ProXp-ala to recognize cytosolic tRNAPro. Finally, we investigated ProXp-z activity and showed that although this enzyme is homologous to INS, its catalytic activity is unrelated to ProRS aminoacylation errors. Using in vitro deacylation assays with a series of aa-tRNA substrates, we determined that ProXp-z is a dual specific deacylase that preferentially hydrolyzes Ser- and Thr-tRNA species. Based on this activity, we renamed it as ProXp-ST, and proposed that the function of this enzyme is responsible for preventing global mistranslation of multiple codon.
Karin Musier-Forsyth, PhD (Advisor)
Juan Alfonzo, PhD (Committee Member)
Dehua Pei, PhD (Committee Member)
199 p.

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Citations

  • Vargas-Rodriguez, O. E. (2014). Characterization of Fidelity Mechanisms in Protein Translation [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1397685427

    APA Style (7th edition)

  • Vargas-Rodriguez, Oscar. Characterization of Fidelity Mechanisms in Protein Translation. 2014. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1397685427.

    MLA Style (8th edition)

  • Vargas-Rodriguez, Oscar. "Characterization of Fidelity Mechanisms in Protein Translation." Doctoral dissertation, Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1397685427

    Chicago Manual of Style (17th edition)