University of Illinois – Urbana-Champaign
Black Pyrkosz, Alexis A.
Evolution and dynamic behavior of transfer RNA in the first two steps of translation.
Degree: PhD, 0335, 2010, University of Illinois – Urbana-Champaign
In protein synthesis, a key component of the cellular machinery is transfer RNA (tRNA).
This small nucleic acid is crucial to the maintenance of the genetic code because it discriminately binds the messenger RNA codon at the ribosome and adds the cognate
amino acid to the growing polypeptide chain. The role of tRNA as an adaptor molecule has been understood for decades, but details about the charging of tRNA with cognate amino acids prior to entering the ribosome are still emerging. Aminoacyl-tRNA synthetases (aaRSs) are enzymes that recognize specific tRNAs and amino acids from the cellular pool and facilitate the charging of the correct amino acids on tRNAs. Following aminoacylation, tRNAs dissociate from the aaRSs and bind the elongation factor Tu (EF-Tu) for delivery to the ribosome.
The recognition of specific tRNA species by the aaRSs, EF-Tu, and other enzymes along the translation pathway is based on sets of highly conserved nucleotides within different
groups of tRNA species. Previous work to identify these recognition elements has focused on experimental studies of single organisms. Here, bioinformatic analyses are used to predict recognition elements for groups of tRNA organized by domain of life and specificity. Shannon entropy differences between evolutionary profiles of tRNA domain/specificity
groups and the representatives of all tRNA species reveal the uniquely conserved nucleotides within each tRNA domain/specificity, consistent with experiment. Comparative analysis of consensus sequences for these evolutionary profiles is used to locate tuning elements, also consistent with experiment. The discriminator base and the
G53:C63 base pair are identified as conserved in several tRNA domain/specificities, particularly among Archaea. Both sets of predictions expand on the current knowledge of recognition elements, providing suggestions for new mutation studies.
AaRS:tRNA complex formation and the aminoacylation reaction have been well-characterized through many high resolution crystal structures and biochemical assays, but dissociation of the charged tRNA with subsequent binding to EF-Tu is not well understood. Using
molecular modeling and molecular dynamics simulations, the effects of protonation states and the presence/absence of substrates and EF-Tu on tRNA release are explored. Using multiple dynamics and energetics analyses, the migration of protons from the 3' end of the tRNA and the alpha-ammonium group on the charging amino acid is shown to accelerate
tRNA dissociation. The presence of AMP has only a minimal effect. Further, pKa calculations predict that Glu41, a conserved residue binding the alpha-ammonium group of the charging amino acid, is part of a proton relay system for releasing the charging amino acid
upon transfer. This system is conserved both in structure and sequences across homologous aaRSs and may represent a universal handle for binding and releasing the charging amino acid. Addition of EF-Tu to the aaRS:tRNA complex stimulates tRNA dissociation.
Knowledge of the…
Advisors/Committee Members: Luthey-Schulten, Zaida A. (advisor), Luthey-Schulten, Zaida A. (Committee Chair), van der Donk, Wilfred A. (committee member), Mitchell, Douglas A. (committee member), Tajkhorshid, Emad (committee member).
Subjects/Keywords: transfer RNA; aminoacyl-tRNA synthetase; identity elements; bioinformatics; Shannon information entropy; molecular dynamics; dynamical networks; mmpbsa; free energies of binding; dissociation mechanism; molecular modeling; GluRS; CysRS
to Zotero / EndNote / Reference
APA (6th Edition):
Black Pyrkosz, A. A. (2010). Evolution and dynamic behavior of transfer RNA in the first two steps of translation. (Doctoral Dissertation). University of Illinois – Urbana-Champaign. Retrieved from http://hdl.handle.net/2142/15505
Chicago Manual of Style (16th Edition):
Black Pyrkosz, Alexis A. “Evolution and dynamic behavior of transfer RNA in the first two steps of translation.” 2010. Doctoral Dissertation, University of Illinois – Urbana-Champaign. Accessed November 27, 2020.
MLA Handbook (7th Edition):
Black Pyrkosz, Alexis A. “Evolution and dynamic behavior of transfer RNA in the first two steps of translation.” 2010. Web. 27 Nov 2020.
Black Pyrkosz AA. Evolution and dynamic behavior of transfer RNA in the first two steps of translation. [Internet] [Doctoral dissertation]. University of Illinois – Urbana-Champaign; 2010. [cited 2020 Nov 27].
Available from: http://hdl.handle.net/2142/15505.
Council of Science Editors:
Black Pyrkosz AA. Evolution and dynamic behavior of transfer RNA in the first two steps of translation. [Doctoral Dissertation]. University of Illinois – Urbana-Champaign; 2010. Available from: http://hdl.handle.net/2142/15505