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.
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