John Perona, PhD
Aminoacyl-tRNA synthetases: Intensive protein engineering studies, involving pre-steady state kinetics coupled with Xray crystallography, are directed towards understanding the origins of specific amino acid-tRNA pairing by Escherichia coli glutaminyl-tRNA synthetase (GlnRS). We are particularly invested in elucidating a novel role for tRNA in assisting the protein active site to select against noncognate amino acids. This long-term project includes detailed mapping of the intramolecular communication pathways connecting the amino acid and tRNA binding sites, and the use of bioinformatics approaches to uncover correlations between protein and tRNA sequences across evolutionary time. A second tRNA synthetase that we study is the phosphoseryl-tRNA synthetase (SepRS) derived from the methanogen Methanosarcina mazei. This highly complex tetrameric enzyme is of interest because it is likely to reveal novel catalytic principles within the tRNA synthetase family, and because of its potential for protein engineering towards expansion of the genetic code in vivo.
Two-step pathway for glutamine incorporation into protein: Most organisms in Nature lack GlnRS. Instead, these organisms glutaminylate tRNA by a two-step pathway. First, a novel class of glutamyl-tRNA synthetase (GluRS-ND) misacylates glutamine tRNA with glutamate. Next, a tRNA-dependent amidotransferase enzyme (AdT) converts the misacylated glutamate into glutamine, while the amino acid remains covalently joined to the tRNA. Bioinformatic analysis suggests that some GluRS enzymes carry out both misacylation and correct aminoacylation of glutamate tRNAs, while others may be dedicated solely to misacylation. Using enzymology and crystallography approaches, we seek to uncover the molecular determinants that functionally differentiate GluRS enzymes. Study of the AdT enzyme concentrates first on developing a kinetic and thermodynamic framework for the complex set of activities. These include amidolysis of asparagine, transfer of the released ammonia through an intramolecular enzyme tunnel, phosphorylation of misacylated glutamate, and finally displacement of phosphate by reaction with ammonia, producing glutaminylated tRNA.
tRNA methylases: tRNA methylases form an important subset of the large class of RNA modifying enzymes. The importance of methylation relates to its role in conferring enhanced thermal stability, in providing positive or negative recognition determinants for tRNA synthetases and other enzymes, and in enhancing the efficiency and fidelity of codon-anticodon pairing on the ribosome. We are studying the kinetic mechanism of the archaeal Trm5 methylase, which delivers a methyl group to the N1 position of guanine at position 37 of tRNA, directly to the anticodon. Methylation at position G37 is crucial to aminoacylation by both SepRS and cysteinyl-tRNA synthetase (CysRS) in methanogens. We also use bioinformatics to identify novel, previously uncharacterized tRNA methylases from methanogens that may also be crucial in translation. Molecular cloning and characterization of these enzymes is followed by detailed enzymological and crystallographic studies to provide a general framework for catalysis in this enzyme family.
Novel metabolism in methanogens: We are studying the redundant pathways for incorporation of cysteine into proteins in the mesophilic anaerobe Methanosarcina mazei. This organism features three cysteine tRNA isoacceptors, and two pathways for aminoacylation: the canonical CysRS enzyme, and a two step pathway involving phosphoserylation of cysteine-tRNA by SepRS. A second enzyme, SepCysS, converts phosphoserylated cysteine-tRNA into the correct cysteinylated species. Both pathways are dependent on tRNA modification, and we have obtained in vitro evidence for functional partitioning in the cell. We have begun study of this pathway in vivo to evaluate this hypothesis. This project also encompasses characterization of novel tRNA modifying enzymes and of companion enzymes to SepCysS, which likely requires persulfidation at a conserved cysteine residue for optimal function. More broadly, we are interested in connections between methanogenesis and protein synthesis in methanogens generally. In Methanococcus jannaschii, we are studying a critical hydrogenase in the methanogenesis pathway as well as several homologs of this enzyme. At least one of these proteins interacts with tRNA and with tRNA synthetases, suggesting novel, unexplored linkages between methanogenesis and protein synthesis that could provide additional levels of regulatory control. The role of methanogens in processing most methane in the biosphere suggests some significance to the phenomena of greenhouse warming.