The recognition and cleavage of double-helical RNA is essential for the maturation, function, and decay of cellular and viral RNA molecules. These processes are catalyzed by double-stranded(ds)-RNA-specific ribonucleases. The long-range goal of this project is to determine the enzymatic mechanism of dsRNA cleavage and its role in regulating cellular and viral RNA metabolism and function. The short-term goal is to analyze Escherichia coli ribonuclease III, a dsRNA-specific nuclease that participates in rRNA, tRNA and mRNA maturation, function and decay. RNase III contains a domain that specifically binds dsRNA, as well as a second, catalytic domain which can bind and site-specifically cut dsRNA. dsRNA-specific ribonucleases with similar domains are found in all cells examined, and perform similar functional roles. A model for RNase III action has been developed that involves the coordinated action of the dsRNA-binding and catalytic domains. To test and refine this model the specific aims are to: 1. Determine how the dsRNA-binding domain enhances substrate cleavage by the catalytic domain. Interactions between the catalytic domain, dsRNA-binding domain, and dsRNA substrate required for efficient cleavage will be identified by crosslinking, protection and interference assays on specific protein-dsRNA complexes. 2. Identify catalytic domain components essential for dsRNA cleavage. The conserved, charged or polar amino acids in the catalytic domain will be mutated and mutant enzyme cleavage activities measured by kinetic assays. The dsRNA-binding and cleavage activities of genetically-selected RNase III mutants (provided by collaborator R. W. Simons) also will be measured. The minimum number of metal ions required for the cleavage step will be determined by kinetic and equilibrium binding experiments. 3. Identify dsRNA-binding domain components directly involved in dsRNA recognition. Conserved residues in the putative recognition surface of the dsRNA-binding domain will be mutated and the dsRNA-binding and cleavage activities measured by gel mobility shift and filter binding assays. The dsRNA-binding activities of mutants obtained by genetic screens will be similarly measured. Mutations which disrupt functional interactions between the dsRNA-binding and catalytic domains will be identified and characterized.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM056457-03
Application #
6019359
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1997-08-01
Project End
2001-07-31
Budget Start
1999-08-01
Budget End
2000-07-31
Support Year
3
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Wayne State University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
City
Detroit
State
MI
Country
United States
Zip Code
48202
Meng, Wenzhao; Nicholson, Allen W (2008) Heterodimer-based analysis of subunit and domain contributions to double-stranded RNA processing by Escherichia coli RNase III in vitro. Biochem J 410:39-48
Pertzev, Alexandre V; Nicholson, Allen W (2006) Characterization of RNA sequence determinants and antideterminants of processing reactivity for a minimal substrate of Escherichia coli ribonuclease III. Nucleic Acids Res 34:3708-21
Sun, Weimei; Pertzev, Alexandre; Nicholson, Allen W (2005) Catalytic mechanism of Escherichia coli ribonuclease III: kinetic and inhibitor evidence for the involvement of two magnesium ions in RNA phosphodiester hydrolysis. Nucleic Acids Res 33:807-15
Sun, Weimei; Li, Gang; Nicholson, Allen W (2004) Mutational analysis of the nuclease domain of Escherichia coli ribonuclease III. Identification of conserved acidic residues that are important for catalytic function in vitro. Biochemistry 43:13054-62
Zhang, Yuanzheng; Calin-Jageman, Irina; Gurnon, James R et al. (2003) Characterization of a chlorella virus PBCV-1 encoded ribonuclease III. Virology 317:73-83
Nicholson, Rhonda H; Nicholson, Allen W (2002) Molecular characterization of a mouse cDNA encoding Dicer, a ribonuclease III ortholog involved in RNA interference. Mamm Genome 13:67-73
Amarasinghe, A K; Calin-Jageman, I; Harmouch, A et al. (2001) Escherichia coli ribonuclease III: affinity purification of hexahistidine-tagged enzyme and assays for substrate binding and cleavage. Methods Enzymol 342:143-58
Sun, W; Jun, E; Nicholson, A W (2001) Intrinsic double-stranded-RNA processing activity of Escherichia coli ribonuclease III lacking the dsRNA-binding domain. Biochemistry 40:14976-84
Calin-Jageman, I; Amarasinghe, A K; Nicholson, A W (2001) Ethidium-dependent uncoupling of substrate binding and cleavage by Escherichia coli ribonuclease III. Nucleic Acids Res 29:1915-25
Sun, W; Nicholson, A W (2001) Mechanism of action of Escherichia coli ribonuclease III. Stringent chemical requirement for the glutamic acid 117 side chain and Mn2+ rescue of the Glu117Asp mutant. Biochemistry 40:5102-10

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