The accuracy of protein synthesis depends on the fidelity of the joining of amino acids to their cognate tRNAs, a reaction catalyzed by the structurally diverse aminoacyl-tRNA synthetases. Each aminoacyl-tRNA synthetase selects its correct transfer RNA and amino acid substrates from a pool of chemically similar molecules. The precise chemical basis of this discrimination is not well understood. The studies proposed here will examine the tRNA and amino acid recognition functions of histidine-tRNA synthetase, one of the smallest and simplest aminoacyl-tRNA synthetases. The approach is to use genetic selections to isolate mutants in the hiss gene that confer phenotypic changes in ligand recognition on the protein. These include reduced affinity for histidine, leading to histidine auxotrophy, and the ability to aminoacylate defective histidine tRNAs in vivo. In a separate approach, crystals of histidine-tRNA synthetase will be prepared as part of a collaborative effort to obtain the three-dimensional structure of the protein. This combined genetic and biochemical approach will provide details of the interaction of an aminoacyl-tRNA synthetase and its TRNA and amino acid ligands. This system is a valuable model of complex protein-RNA interactions where sequence discrimination is imposed during catalytic steps, and may help to understand human autoimmune diseases such as myositis, in which synthetase-tRNA complexes have been implicated as antigens.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29GM048146-05
Application #
2185610
Study Section
Biochemistry Study Section (BIO)
Project Start
1992-08-01
Project End
1998-07-31
Budget Start
1996-08-01
Budget End
1998-07-31
Support Year
5
Fiscal Year
1996
Total Cost
Indirect Cost
Name
University of Vermont & St Agric College
Department
Biochemistry
Type
Schools of Medicine
DUNS #
066811191
City
Burlington
State
VT
Country
United States
Zip Code
05405
Yan, W; Francklyn, C (1994) Cytosine 73 is a discriminator nucleotide in vivo for histidyl-tRNA in Escherichia coli. J Biol Chem 269:10022-7
Francklyn, C; Harris, D; Moras, D (1994) Crystallization of histidyl-tRNA synthetase from Escherichia coli. J Mol Biol 241:275-7