Ribonuclease III mechanism of double-strand RNA cleavage
Principal Investigator: ALLEN NICHOLSON
Abstract: The enzymatic cleavage of double-stranded RNA is an essential step in the maturation and decay of many eukaryotic and prokaryotic RNAs. Double-stranded (ds) RNA cleavage is carried out by members of the ribonuclease Ill superfamily of endoribonucleases. RNase III orthologues participate in mRNA and rRNA maturation, mRNA degradation, and antisense RNA action. An important recent finding is the central involvement of the RNase III orthologue "Dicer" in RNA interference (RNAi). RNAi is proposed to suppress viral infection, inhibit retroposon movement, and participate in specific developmental pathways in eukaryotes, including humans. The occurrence of highly conserved sequences and structural elements indicate that RNase Ill orthologues use the same catalytic mechanism, which is unknown. The most studied member of the RNase III superfamily is Escherichia coli ribonuclease III and there has been recent significant progress in determining the E. coil RNase Ill mechanism of action. The long-range goal of this project is to determine the mechanism of dsRNA cleavage by E. coil RNase Ill. The specific aims of the project are to: (1) determine the functional roles of conserved amino acids in the catalytic domain. To accomplish this, RNase Ill mutants with substitutions of conserved catalytic domain residues will be tested in substrate binding and cleavage assays; (2), determine divalent metal ion stoichiometry and function in catalysis. To accomplish this, kinetic assays of cleavage, and metal ion binding assays using RNase Ill and selected mutants will be performed; (3), identify sequence and structural features in the RNase Ill double-stranded-RNA-binding domain (dsRBD) that confer binding energy and optimize catalytic efficiency. This will be accomplished by analyzing RNA binding of specific dsRBD mutants, and by phage display; (4), determine the stoichiometry of an RNase Ill-substrate complex, and identify intersubunit interactions important for RNase Ill function. Gel filtration, centrifugation, and substrate binding and cleavage assays using artificial heterodimers will be performed. Accomplishing these aims not only will provide important information on E. coil RNase Ill, but will also provide mechanistic insight on RNase Ill orthologue function in eukaryotic RNA processing and degradation pathways, including RNAi.
Funding Period: 1997-08-01 - 2005-07-31
more information: NIH RePORT
- Catalytic mechanism of Escherichia coli ribonuclease III: kinetic and inhibitor evidence for the involvement of two magnesium ions in RNA phosphodiester hydrolysisWeimei Sun
Department of Chemistry, Temple University 1901 North 13th Street, Philadelphia, PA 19122, USA
Nucleic Acids Res 33:807-15. 2005..A preliminary model is presented for functional roles of two divalent metal ions in the RNase III catalytic mechanism...
- Characterization of RNA sequence determinants and antideterminants of processing reactivity for a minimal substrate of Escherichia coli ribonuclease IIIAlexandre V Pertzev
Department of Chemistry, Temple University, Philadelphia, PA 19122, USA
Nucleic Acids Res 34:3708-21. 2006..The base pair sequence control of reactivity is discussed within the context of new structural information on a post-catalytic complex of a bacterial RNase III bound to the cleaved minimal substrate...
- Heterodimer-based analysis of subunit and domain contributions to double-stranded RNA processing by Escherichia coli RNase III in vitroWenzhao Meng
Department of Chemistry, Temple University, 1901 North 13th Street, Philadelphia, PA 19122, USA
Biochem J 410:39-48. 2008..These findings support a reaction pathway involving the largely independent action of the dsRBDs and the catalytic sites in substrate recognition and cleavage respectively...