The long-term goals of this research are to comprehend RNA processing in the E. coli cell.
The specific aims are to: isolate mutants defective in RNase f and in other RNA processing enymes; to construct strains, using transposons which carry mutations in all the possible combinations, in the rnc, rne, rnf, and rnp genes; to clone all the structual genes for the RNA processing enzymes, RNase III, RNase E, Rnase F, and Rnase P; to purify to homogeneity RNase E, and RNase F; to investigate the mechanism of action of RNase III, RRNase E, and RNase F; to sequence the DNA for the rnc, rne, rnf, and rnpA, rnpB genes; to sequence, at least partially, the RNase III, RNase E, and RNase F proteins; to comprehend the contribution of each of the four processing enzymes, RNase III, RNase E, RNase F, and RNase P to the metabolism of rRNA, tRNA and mRNA; to prepare minigenes from which substrates for RNase III, RNase E, and RNase F can be transcribed; to modify by genetic engineering and directed mutagenesis the genes for the substrates of these enzymes and to find out which of them are still substrates; to study the regulation of the rnc, rne, rnf, and rnp genes. These studies will be carried out by using genetical, biochemical, physiological, and molecular biology approaches, such as genetic mapping of genes, purification and characterization of RNA molecules, purification of enzymes, sequencing of nucleic acids and proteins. The experiments will be carried out by using columns, gel electrophoresis, chromatography and various other techniques.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM019821-10A1
Application #
3269769
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1978-12-01
Project End
1991-03-31
Budget Start
1987-04-01
Budget End
1988-03-31
Support Year
10
Fiscal Year
1987
Total Cost
Indirect Cost
Name
Washington University
Department
Type
Schools of Medicine
DUNS #
062761671
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
Makarov, E M; Apirion, D (1992) 10Sa RNA: processing by and inhibition of RNase III. Biochem Int 26:1115-24
Srivastava, R A; Srivastava, N; Apirion, D (1992) Characterization of the RNA processing enzyme RNase III from wild type and overexpressing Escherichia coli cells in processing natural RNA substrates. Int J Biochem 24:737-49
Taraseviciene, L; Miczak, A; Apirion, D (1991) The gene specifying RNase E (rne) and a gene affecting mRNA stability (ams) are the same gene. Mol Microbiol 5:851-5
Miczak, A; Srivastava, R A; Apirion, D (1991) Location of the RNA-processing enzymes RNase III, RNase E and RNase P in the Escherichia coli cell. Mol Microbiol 5:1801-10
Chauhan, A K; Apirion, D (1991) The rne gene is the structural gene for the processing endoribonuclease RNase E of Escherichia coli. Mol Gen Genet 228:49-54
Chauhan, A K; Miczak, A; Taraseviciene, L et al. (1991) Sequencing and expression of the rne gene of Escherichia coli. Nucleic Acids Res 19:125-9
Oh, B K; Apirion, D (1991) 10Sa RNA, a small stable RNA of Escherichia coli, is functional. Mol Gen Genet 229:52-6
Miczak, A; Chauhan, A K; Apirion, D (1991) Two new genes located between 2758 and 2761 kilobase pairs on the Escherichia coli genome. J Bacteriol 173:3271-2
Srivastava, R K; Miczak, A; Apirion, D (1990) Maturation of precursor 10Sa RNA in Escherichia coli is a two-step process: the first reaction is catalyzed by RNase III in presence of Mn2+. Biochimie 72:791-802
Oh, B K; Chauhan, A K; Isono, K et al. (1990) Location of a gene (ssrA) for a small, stable RNA (10Sa RNA) in the Escherichia coli chromosome. J Bacteriol 172:4708-9

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