Author: John Eargle
Publisher:
Published: 2010
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Translation, the process of reading genetic information and synthesizing the corresponding proteins, is universal and found throughout the three domains of life. The flow of information in translation involves a series of distinct but highly conserved RNA·protein complexes with the ribosome being the largest ribonucleoprotein complex in the cell. As the molecular instantiation of the genetic code, tRNA plays a central role in the translational machinery where it interacts with several proteins and other RNAs during the course of protein synthesis. We use molecular dynamics (MD) simulations informed by evolutionary analysis to investigate the dynamics of several RNA·protein complexes involved in translation. Many analysis methods and tools were developed during the course of this study. We present the evolutionary analysis environment MultiSeq, dynamical network analysis and visualization, and a protocol for the preparation of RNA·protein MD simulations. For several Class I aminoacyl-tRNA synthetases (aaRSs), the rate determining step in aminoacylation is the dissociation of the charged tRNA from the enzyme. Through molecular modeling, internal pKa calculations, and MD simulations, distinct, mechanistically relevant post-transfer states with the charged tRNA (Glu-tRNA(Glu)) bound to glutamyl-tRNA synthetase are considered. The behavior of these nonequilibrium states is characterized as a function of time using dynamical network analysis, local energetics, and changes in free energies to estimate transitions that occur during the release of the tRNA. Dynamical network analysis reveals that there are a large number of suboptimal paths through the protein·RNA complex that can be used for communication between the identity elements on the tRNAs and the catalytic site in the aaRS·tRNA complexes. Residues and nucleotides in the majority of pathways bridging communities, local substructures that are highly intraconnected but loosely interconnected, are evolutionarily conserved and are predicted to be important for allosteric signaling. The same monomers are also found in a majority of the suboptimal paths. Modifying these residues or nucleotides has a large effect on the communication pathways in the protein·RNA complex consistent with kinetic data. The highly conserved general base Glu41 is proposed to be a part of a proton relay system for destabilizing the bound charging amino acid following aminoacylation. Addition of elongation factor Tu (EF-Tu) to the aaRS·tRNA complex stimulates the dissociation of the tRNA core and acceptor stem. We use MD simulations to investigate the dynamics of the EF-Tu·GTP·aa-tRNA(Cys) complex and the roles played by Mg2+ ions and modified nucleosides on the free energy of RNA·protein binding. Combined energetic and evolutionary analyses identify the coevolution of residues in EF-Tu and aa-tRNAs at the binding interface. Highly conserved EF-Tu residues are responsible for both attracting aa-tRNAs as well as providing nearby nonbonded repulsive energies which help fine-tune molecular attraction at the binding interface. The trend in EF-Tu·Cys-tRNA(Cys) binding energies observed as the result of mutating the tRNA agrees with experimental observation. We also predict variations in binding free energies upon misacylation of tRNA(Cys) with D-cysteine or O-phosphoserine and upon changing the protonation state of L-cysteine. Finally, ongoing work is presented on the role of ribosomal signatures in the first steps of ribosomal assembly. Ribosomal signatures are features that are completely conserved within one domain of life but absent from the other domains. Correlations between rRNA signatures and signatures in the ribosomal proteins (r-proteins) show that the rRNA signatures coevolved with both domain specific r-proteins and inserts in universal r-proteins. The largest bacterial structural rRNA signature with such a coevolutionary protein partner is found in the five-way junction of the 16S rRNA 5' domain, which is held together by the universal r-protein S4. We characterize the dynamics and flexibility of the free S4 structural signature and rRNA signature helix 16 (h16) as well as the S4·h16 complex. Investigation into the folding and binding of these components will be carried out using Go-like potentials to bias the complex structure towards its native state.
