? Aminoacyl-tRNA synthetases establish the genetic code by linking specific amino acids to tRNAs that bear triplet anticodon sequences. The universal distribution of these enzymes across the phylogenetic tree suggests that they are among the oldest proteins to have developed extraordinary specificity towards amino acids and tRNAs. However, while extensive studies of synthetases of bacterial and eucaryotic domains have led to general principles of substrate specificity, these are not sufficient to understand synthetases of the archaea domain, which consists of organisms that thrive in extreme environments. For example, recent studies have identified a dual specific prolyl-tRNA synthetase (ProRS) of the archaeon Methanococcus jannaschii, which has the ability to activate both proline and cysteine and catalyze aminoacylation of tRNA with proline and cysteine. The dual specificity has challenged the traditional view of one synthetase for one amino acid and raised many fundamental questions about synthetase specificity. In addition, the discovery of the dual-specific ProRS suggests that it is associated with a multi-synthetase complex that builds a network of synthetase-synthetase interactions to enhance binding of general tRNA. This raises the possibility of a previously un-recognized evolutionary relationship with the multi-synthetase complex of eucarya. Further, recent studies show that the specificity of tRNA aminoacylation by the dual-specific ProRS requires modification, which is a major departure from ProRS of bacterial and eucarya domains. To understand tRNA recognition of the dual-specific ProRS, the identification of the necessary modifications is necessary. ? ? Aim 1 of this project is to elucidate the molecular and structural basis of the dual-specificity of M. jannaschii ProRS.
Aim 2 is to test the networking ability of the dual-specific ProRS in the organization of a multi-synthetase complex.
Aim 3 is to identify the modification required for the dual-specific ProRS to achieve specificity of tRNA recognition.
These aims are to be integrated with the functional genomics of M. jannaschii to gain a broader insight into its aminoacyl-tRNA synthetases as a basis for understanding the origin and evolution of contemporary mRNA-encoded protein synthesis in extant organisms. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM066267-03
Application #
6883921
Study Section
Biochemistry Study Section (BIO)
Program Officer
Jones, Warren
Project Start
2003-05-01
Project End
2007-04-30
Budget Start
2005-05-01
Budget End
2006-04-30
Support Year
3
Fiscal Year
2005
Total Cost
$276,320
Indirect Cost
Name
Thomas Jefferson University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
053284659
City
Philadelphia
State
PA
Country
United States
Zip Code
19107
Hauenstein, Scott I; Hou, Ya-Ming; Perona, John J (2008) The homotetrameric phosphoseryl-tRNA synthetase from Methanosarcina mazei exhibits half-of-the-sites activity. J Biol Chem 283:21997-2006
Zhang, Chun-Mei; Liu, Cuiping; Christian, Thomas et al. (2008) Pyrrolo-C as a molecular probe for monitoring conformations of the tRNA 3'end. RNA 14:2245-53
Zhang, Chun-Mei; Liu, Cuiping; Slater, Simon et al. (2008) Aminoacylation of tRNA with phosphoserine for synthesis of cysteinyl-tRNA(Cys). Nat Struct Mol Biol 15:507-14
Christian, Thomas; Hou, Ya-Ming (2007) Distinct determinants of tRNA recognition by the TrmD and Trm5 methyl transferases. J Mol Biol 373:623-32
Betteridge, Thu; Liu, Hanqing; Gamper, Howard et al. (2007) Fluorescent labeling of tRNAs for dynamics experiments. RNA 13:1594-601
Hou, Ya-Ming; Li, Zhi; Gamper, Howard (2006) Isolation of a site-specifically modified RNA from an unmodified transcript. Nucleic Acids Res 34:e21
Christian, Thomas; Evilia, Caryn; Hou, Ya-Ming (2006) Catalysis by the second class of tRNA(m1G37) methyl transferase requires a conserved proline. Biochemistry 45:7463-73
Ougland, Rune; Zhang, Chun-Mei; Liiv, Aivar et al. (2004) AlkB restores the biological function of mRNA and tRNA inactivated by chemical methylation. Mol Cell 16:107-16
Zhang, Chun-Mei; Hou, Ya-Ming (2004) Synthesis of cysteinyl-tRNACys by a prolyl-tRNA synthetase. RNA Biol 1:35-41
Christian, Thomas; Evilia, Caryn; Williams, Sandra et al. (2004) Distinct origins of tRNA(m1G37) methyltransferase. J Mol Biol 339:707-19