Ribonucleases (RNases) play a central role in a number of vital RNA cellular processes in all living cells. One of these processes is mRNA degradation, which is an important mechanism for post-transcriptional control of gene expression. RNases are also required for maturation and turnover of structural RNAs. E. coli has served as a model system for understanding the role of ribonucleases in cellular RNA metabolism, and eight distinct exoribonucleases have been identified in this bacterium. Of these, three (RNase T, Rnase D, and oligoribonuclease) are members of a larger exonuclease superfamily that includes the proof-reading domains of DNA polymerases. These three proteins share similar sequence motifs and have been dubbed the DEDD family exoribonucleases. However, functionally these exoribonuclease are quite distinct. We have initiated structural studies of this family of exoribonucleases, in collaboration with the laboratory of Dr. Murray Deutscher at the University of Miami, to structurally characterize these proteins. The structure of oligoribonuclease has been solved and we have diffraction quality crystals of RNase T. Specifically, we propose to: 1. Initiate detailed structure-function studies of oligoribonuclease to better understand its active site, metal requirements, and dimeric state. We also propose to look for differences between the prokaryotic and eukaryotic forms of this enzyme, by obtaining the structure of the human homologue of oligoribonuclease. 2. Optimize the crystals obtained for RNase T, and derive its atomic structure. 3. Determine the atomic structure of RNase D. 4. Understand the similarities and differences between these three enzymes in terms of substrate specificity, quarternary structure, metal requirements and catalytic mechanism, to better characterize this family of enzymes. The long term goals of this research are to understand the structures and mechanisms of action of all the exoribonucleases in a single organism (E. coli); these studies will complement a parallel study to completely determine and characterize the physiological role of all the exoribonucleases in E. coli, now underway in the Deutscher laboratory.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM069972-03
Application #
6937081
Study Section
Physical Biochemistry Study Section (PB)
Program Officer
Ikeda, Richard A
Project Start
2003-09-30
Project End
2008-08-31
Budget Start
2005-09-01
Budget End
2006-08-31
Support Year
3
Fiscal Year
2005
Total Cost
$292,397
Indirect Cost
Name
University of Miami School of Medicine
Department
Biochemistry
Type
Schools of Medicine
DUNS #
052780918
City
Miami
State
FL
Country
United States
Zip Code
33146
Schmier, Brad J; Nelersa, Claudiu M; Malhotra, Arun (2017) Structural Basis for the Bidirectional Activity of Bacillus nanoRNase NrnA. Sci Rep 7:11085
Schmier, Brad J; Seetharaman, Jayaraman; Deutscher, Murray P et al. (2012) The structure and enzymatic properties of a novel RNase II family enzyme from Deinococcus radiodurans. J Mol Biol 415:547-59
Malhotra, Arun (2011) Reprint of: Tagging for Protein Expression. Protein Expr Purif :
Nelersa, Claudiu M; Schmier, Brad J; Malhotra, Arun (2011) Purification and crystallization of Bacillus subtilis NrnA, a novel enzyme involved in nanoRNA degradation. Acta Crystallogr Sect F Struct Biol Cryst Commun 67:1235-8
Steen, Kady-Ann; Malhotra, Arun; Weeks, Kevin M (2010) Selective 2'-hydroxyl acylation analyzed by protection from exoribonuclease. J Am Chem Soc 132:9940-3
Malhotra, Arun (2009) Tagging for protein expression. Methods Enzymol 463:239-58
Larrea, Andres A; Pedroso, Ilene M; Malhotra, Arun et al. (2008) Identification of two conserved aspartic acid residues required for DNA digestion by a novel thermophilic Exonuclease VII in Thermotoga maritima. Nucleic Acids Res 36:5992-6003
Tolun, Adviye A; Dickerson, Ian M; Malhotra, Arun (2007) Overexpression and purification of human calcitonin gene-related peptide-receptor component protein in Escherichia coli. Protein Expr Purif 52:167-74
Zuo, Yuhong; Zheng, Heping; Wang, Yong et al. (2007) Crystal structure of RNase T, an exoribonuclease involved in tRNA maturation and end turnover. Structure 15:417-28
Lalonde, Maureen S; Zuo, Yuhong; Zhang, Jianwei et al. (2007) Exoribonuclease R in Mycoplasma genitalium can carry out both RNA processing and degradative functions and is sensitive to RNA ribose methylation. RNA 13:1957-68

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