The biogenesis of tRNA from primary gene transcripts to their final functional form in E. coli and mammalian cells will be investigated and, in particular, the function of RNase P, a ribonucleoprotein essential for the processing of the 5' termini of tRNAs, will be examined in detail. Procedures for the selection in vivo of mutations in the genes for the subunits of RNase P and its substrate will be employed, along with site- directed mutagenesis, to study the interactions between subunits of the RNase P themselves and with the substrate. New strains will be constructed to facilitate the analysis of mutant derivatives of M1 RNA, the catalytic subunit of RNase P from E. coli, and its substrates in vivo. An attempt will be made to define the minimal structure recognized by RNase P in its substrates. Hypotheses concerning the nature of a tRNA binding site in M1 RNA, its resemblance to a similar site in large ribosome subunit rRNA, and the role of the 3' terminal CCA sequence in substrates will be explored with site-directed mutagenesis. The nature of the enzyme, and its cleavage site, responsible for the processing of the precursor to M1 RNA will be elucidated using strains deficient in various ribonucleases and with deletion mutagenesis at the site of processing. The effects of alterations in the repeated sequences downstream from gene coding for M1RNA, on expression of the upstream region, will also be examined. Human and other mammalian genomic DNA libraries will be screened with a cDNA probe encoding the RNA subunit of RNase P from human cells in an attempt to characterize the nucleotide sequences and the regulation of the corresponding genes and to provide data for a comparison of the hypothetical two-dimensional structures of these analogs of M1 RNA with models of analogs from other organisms.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM019422-20
Application #
3484295
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1981-07-01
Project End
1994-08-31
Budget Start
1991-09-01
Budget End
1992-08-31
Support Year
20
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Yale University
Department
Type
Schools of Arts and Sciences
DUNS #
082359691
City
New Haven
State
CT
Country
United States
Zip Code
06520