It has previously been established that alternative, indirect pathways of formation of aminoacyl-tRNAs exist in some organisms for the amino acids glutamine, asparagine and selenocysteine. The goal of this proposal is to investigate the means by which the archaea (archaebacteria), one of the three lines of descent, generate cysteinyl- tRNA given the inability to detect cysteinyl-tRNA synthetase in archaeal genome sequences. Evidence for direct and indirect pathways will be examined by assaying cysteinyl-tRNA formation in cell extracts of the methanogenic archaea. The enzyme(s) involved in the activity will then be purified and the respective gene(s) cloned using N-terminal protein sequences. An overexpression system in E. coli will then be used to produce sufficient material for biophysical and biochemical characterizations. In particular, the RNA:protein interactions required for translational fidelity will be investigated using both site-directed mutagenesis and X-ray crystallography of the tRNA and the identified enzyme(s). The results should provide insight into the evolution of the genetic code and into the limits on the flexibility of the translational apparatus in all three domains of life.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
5F32GM019278-03
Application #
6138295
Study Section
Special Emphasis Panel (ZRG3-BIO (01))
Program Officer
Wolfe, Paul B
Project Start
1997-12-15
Project End
Budget Start
1999-12-15
Budget End
2000-12-14
Support Year
3
Fiscal Year
2000
Total Cost
$37,516
Indirect Cost
Name
Yale University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
082359691
City
New Haven
State
CT
Country
United States
Zip Code
06520