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Functions and properties of RNase G and RNase E from Escherichia coli Briant, Douglas James


Ribonucleic acid (RNA) is a vital molecule in the cell. Messenger RNA (mRNA) serves as the intermediate between DNA and protein while ribosomal RNA (rRNA) and transfer RNA (tRNA) catalyze translation. RNA is processed and ultimately degraded by ribonucleases. The majority of the endoribonucleolytic activity in Escherichia coli is derived from RNase E. This 1061 amino acid protein associates with at least three other proteins to form a complex called the RNA degradosome. This work established that the degradosome does not assemble de novo with successive rounds of degradation. It also determined that RNase E lacks 5'-phosphatase activity. Studies into the activity of RNase E are confounded by the fact that it associates into a complex with other RNA processing enzymes. We therefore utilized RNase G, which shares 35% amino acid sequence identity (50% similarity) to the catalytic domain of RNase E, as a model for RNase E. RNase G is the endonuclease responsible for forming the mature 5'-end of 16S rRNA. Non-denaturing purifications for RNase G were developed and the correct Nterminal sequence of the protein unambiguously identified. Through cross-linking studies, sucrose gradient centrifugation and gel filtration, it was determined that RNase G, and by inference, RNase E, exists primarily as a dimer. Site-directed mutagenesis was utilized to elucidate the role of six cysteine residues, including two highly conserved cysteines, of RNase G. None of the mutations resulted in a loss of activity, although subtle influences on structure and activity were observed with the RNase G variants. The S1 domain, which potentially binds RNA, was deleted without inactivating the enzyme. Further studies are required to determine if the S1 domain plays a role in substrate recognition. Finally, examinations using synthetic, chimeric RNA-DNA oligonucleotides revealed the chemical requirements for recognition and cleavage of a substrate by RNase E or RNase G. I concluded that a single 2'- OH group 5' to the site of cleavage was sufficient for endoribonucleic activity. This work established RNase G as a model for investigating the activity and structure of the catalytic domain of RNase E.

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