The long range objective of this proposal is to understand the relationship between structure and function of eukaryotic initiator tRNAS, including the molecular mechanisms underlying their specific recognition by components of the protein synthesis machinery. Because of their unique role in initiation of protein biosynthesis, initiator tRNAS from prokaryotic and eukaryotic sources possess biochemical properties which are distinct from those of non-initiator tRNAS. These include (i) specific binding to the initiation factor eIF-2, (ii) direct binding to the P site on the ribosome and (iii) exclusion from binding to the A site on the ribosome. Along with these specific properties, initiator tRNAS possess unique sequence and/or structural features which are not found in non-initiator tRNAS. The immediate objective of this proposal is to understand the relationship between these features and the above properties of eukaryotic initiator tRNAS. The approach to be used involves isolation and analysis of properties of mutant human tRNAS. Site specific mutations will be used to generate different classes of mutant tRNAs: (a) those derived from initiator tRNAs which have lost one, two or all three of the features unique to initiator tRNAs and (b) those derived from the non-initiator methionine tRNA which now possess one, two or all three of the structural features unique to initiator tRNA. The function and detailed properties of these and other mutant tRNAs will be studied in vivo, in the yeast S. cerevisiae and in mammalian cells, and in vitro.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM046942-02
Application #
3306455
Study Section
Molecular Biology Study Section (MBY)
Project Start
1992-02-01
Project End
1996-01-31
Budget Start
1993-02-01
Budget End
1994-01-31
Support Year
2
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Massachusetts Institute of Technology
Department
Type
Schools of Arts and Sciences
DUNS #
City
Cambridge
State
MA
Country
United States
Zip Code
02139
Drabkin, H J; RajBhandary, U L (1998) Initiation of protein synthesis in mammalian cells with codons other than AUG and amino acids other than methionine. Mol Cell Biol 18:5140-7
Park, H J; RajBhandary, U L (1998) Tetracycline-regulated suppression of amber codons in mammalian cells. Mol Cell Biol 18:4418-25
Drabkin, H J; Estrella, M; Rajbhandary, U L (1998) Initiator-elongator discrimination in vertebrate tRNAs for protein synthesis. Mol Cell Biol 18:1459-66
Drabkin, H J; Park, H J; RajBhandary, U L (1996) Amber suppression in mammalian cells dependent upon expression of an Escherichia coli aminoacyl-tRNA synthetase gene. Mol Cell Biol 16:907-13
Farruggio, D; Chaudhuri, J; Maitra, U et al. (1996) The A1 x U72 base pair conserved in eukaryotic initiator tRNAs is important specifically for binding to the eukaryotic translation initiation factor eIF2. Mol Cell Biol 16:4248-56
RajBhandary, U L (1994) Initiator transfer RNAs. J Bacteriol 176:547-52
Drabkin, H J; Helk, B; RajBhandary, U L (1993) The role of nucleotides conserved in eukaryotic initiator methionine tRNAs in initiation of protein synthesis. J Biol Chem 268:25221-8