The site-specific cleavage of double-stranded RNA by ribonuclease III of Escherichia coli is a key step in the maturation, function and decay of cellular and viral RNAs. Nucleases similar to RNase III occur in eukaryotic cells, and perform similar functional roles. The long-term objective is to determine the enzymatic mechanism of double-stranded RNA recognition and cleavage and its role in RNA maturation, function and decay. Specific Watson- Crick base-pair sequences determine the RNase III binding site, and an internal loop determines whether one or both RNA strands are cleaved The pattern of cleavage in turn controls RNA function and half-life.
The Specific Aims are to: 1. Determine the Watson-Crick base-pair sequence features which confer specific binding of RNase III. RNase III recognition of substrate is dependent upon specific Watson-Crick base-pair (W-C bp) sequences near the cleavage site. To determine W-C bp sequence features which confer specific binding, substrates containing bp substitutions or base analogues lacking functional groups will be tested for their in vitro binding and cleavage activities. In vitro genetic selection will determine the range of W-C bp sequences that confers specific binding. 2. Determine the RNA structural features that confer single-strand cleavage. An internal loop switches the pattern of double-strand cleavage to single-strand cleavage. To determine how this motif allows RNase III binding, but confers single-strand cleavage, mutant substrates with altered internal loop sequences will be tested for their binding affinities and cleavage reactivities. In vitro genetic selection will be used to determine the range of internal loop structures that confer binding and/or cleavage. 3. Determine 2'-hydroxyl group involvement in binding, and the energetic contribution of ionic and hydrophobic interactions. A specific set of substrate 2'-hydroxyls and phosphodiester oxygens may directly contact RNase III and contribute to overall binding energy. To identify the 2'-hydroxyl groups important for binding 2'-deoxyphosphorothioate-substituted RNAs will be used in modification-interference assays, and site-directed 2'-deoxy- substituted substrates will be tested for their in vitro binding and cleavage reactivities. The ionic and hydrophobic contributions to binding will be determined by measuring substrate binding affinities as a function of salt concentration and temperature, respectively.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
7R01GM056772-05
Application #
6727003
Study Section
Physiological Chemistry Study Section (PC)
Program Officer
Rhoades, Marcus M
Project Start
1999-05-01
Project End
2003-04-30
Budget Start
2003-01-01
Budget End
2003-04-30
Support Year
5
Fiscal Year
2002
Total Cost
$12,255
Indirect Cost
Name
Temple University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
City
Philadelphia
State
PA
Country
United States
Zip Code
19122
Gone, Swapna; Alfonso-Prieto, Mercedes; Paudyal, Samridhdi et al. (2016) Mechanism of Ribonuclease III Catalytic Regulation by Serine Phosphorylation. Sci Rep 6:25448
Paudyal, Samridhdi; Alfonso-Prieto, Mercedes; Carnevale, Vincenzo et al. (2015) Combined computational and experimental analysis of a complex of ribonuclease III and the regulatory macrodomain protein, YmdB. Proteins 83:459-72
Nicholson, Allen W (2014) Ribonuclease III mechanisms of double-stranded RNA cleavage. Wiley Interdiscip Rev RNA 5:31-48
Redhu, Shiv K; Castronovo, Matteo; Nicholson, Allen W (2013) Digital imprinting of RNA recognition and processing on a self-assembled nucleic acid matrix. Sci Rep 3:2550
Castronovo, M; Stopar, A; Coral, L et al. (2013) Effects of nanoscale confinement on the functionality of nucleic acids: implications for nanomedicine. Curr Med Chem 20:3539-57
Alla, Nageswara R; Nicholson, Allen W (2012) Evidence for a dual functional role of a conserved histidine in RNA·DNA heteroduplex cleavage by human RNase H1. FEBS J 279:4492-500
Gone, Swapna; Nicholson, Allen W (2012) Bacteriophage T7 protein kinase: Site of inhibitory autophosphorylation, and use of dephosphorylated enzyme for efficient modification of protein in vitro. Protein Expr Purif 85:218-23
Shi, Zhongjie; Nicholson, Rhonda H; Jaggi, Ritu et al. (2011) Characterization of Aquifex aeolicus ribonuclease III and the reactivity epitopes of its pre-ribosomal RNA substrates. Nucleic Acids Res 39:2756-68
Nathania, Lilian; Nicholson, Allen W (2010) Thermotoga maritima ribonuclease III. Characterization of thermostable biochemical behavior and analysis of conserved base pairs that function as reactivity epitopes for the Thermotoga 23S rRNA precursor. Biochemistry 49:7164-78
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

Showing the most recent 10 out of 15 publications