David E. Kennell Professor Phone, office: (314)-362-2751 Phone, lab: (314)-362-2750 FAX: (314)-362-1232 email: kennell@borcim.wustl.edu 586 McDonnell Science Building Box 8230 Department of Molecular Microbiology Washington University School of Medicine 660 South Euclid Avenue St. Louis, MO 63110-1093.

Research Interests | CV | Publications | Technology for licensing

Research Interests

We are studying processive exonucleases as a model system to understand the mechanics of protein movement on a nucleic acid molecule. These studiesarose from our long term work on mRNA degradation in E. coli. We concluded that the inactivation and initiation of mass decay of most messages occurs near the 5'-end at any one of a number of specific targets. Specific cleavages in lac mRNA were usually between pyrimidine-adenosine residues and suggested an RNase with that specificity. We identified and purified such an endonuclease and called it RNase M. In the total cell population of oligonucleotides resulting from cleavages, most start with 5'-0H-Adenosine. RNase M is the only endoRNase known that would enrich for that residue.

Another endoRNase was identified that degraded RNA oligonucleotides rapidly but polymer RNA only very slowly, and we concluded that it helps degrade the terminal oligonucleotides of mRNA decay. An insertion mutant in the gene for the periplasmic endoRNase I did not synthesize the oligo RNase or RNase I. The former is a cytoplasmic form of RNase I--called RNase I*. These enzymes, each with unique specificity, could have arisen during evolution by gene duplication. Studies are underway to understand their relationships.

The 3' exoRNase II probably accounts for most of the degradation of the oligonucleotide fragments released by cleavages of mRNA. It moves processively (remains attached) while releasing mononucleotides from the 3' end of the RNA. RNase II recognition differs for poly(A), poly(C) and poly(U). At lower reaction temperatures poly(C) was degraded more slowly than the other polymers and the enzyme dissociated from poly(C) at every ~12 nt. This anomolous pattern of degradation only occurred when poly(C) was a helix-coil structure and has led to a model of the reaction mechanics. Physical studies of the protein and site-directed mutations are part of long term projects to understand how this protein and its nucleic acid substrate move relative to each other.

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Publications

1. Cannistraro, V.J. and Kennell, D. (1997). RNase YI* and RNA structure studies. Nucleic Acids Research 25: 1405-1411.
2. Cannistraro, V.J. and Kennell, D. (1995). Identifying ends of infrequent RNA molecules in bacteria. In Methods in Molecular Genetics vol. 6 (ed. K.W. Adolph). Academic Press, Orlando, FL. pp 280-300.
3. Cannistraro, V.J. and Kennell, D. (1994). The processive reaction mechanism of ribonuclease II. J. Mol. Biol. 243: 930-943.
4. Lim, L.W., Mathur, S., Cannistraro, V.J. and Kennell, D. (1993). Preliminary X-ray crystallographic studies of ribonuclease I from Escherichia coli.. J. Mol. Biol. 234: 499-501.
5. Mathur, S., Cannistraro, V.J. and Kennell, D. (1993). Identification of an intracellular pyrimidine-specific endoribonuclease in Bacillus subtilis. J. Bacteriol. 175: 6717-6720.
6. Cannistraro, V.J. and Kennell, D. (1993). The 5'ends of RNA oligonucleotides in Escherichia coli and mRNA degradation. Europ. J. Biochem. 213: 285-293.
7. Srivastava, S.K., Cannistraro, V.J. and Kennell, D. (1992). Broad-specificity endoribonucleases and mRNA degradation in Escherichia coli. J. Bacteriol. 174: 56-62.
8. Cannistraro, V.J. and Kennell, D. (1991). RNase I*, a form of RNase I, and mRNA degradation in Escherichia coli. J. Bacteriol. 173:4653-4659.
9. Meador, J. III and Kennell, D. (1990). Cloning and sequencing the gene encoding Escherichia coli ribonuclease I: exact physical mapping using the genome library. Gene 95:1-7.
10. Meador, J. III, Cannon, B., Cannistraro, V.J. and Kennell, D. (1990). Purification and characterization of E. coli RNase I: comparisons with RNase M. Europ. J. Biochem. 187:549-553.
11. Cannistraro, V.J. and Kennell, D. (1989). Purification and characterization of ribonuclease M and mRNA degradation in E. coli. Europ. J. Biochem. 181: 363-370.
12. Subbarao, M.N. and Kennell, D. (1988). Evidence for endonucleolytic cleavages in decay of lacZ and lacI messenger RNAs. J. Bacteriol. 170: 2860-2865.
13. Cannistraro, V.J., Hwang, P. and Kennell, D.E. (1987). Isolating and sequencing the predominant 3'-ends of a specific mRNA. J. Biochem. Biophys. Methods 14: 211-221.
14. Cannistraro, V.J., Subbarao, M.N. and Kennell, D.E. (1986). Specific endonucleolytic cleavage sites for decay of E. coli mRNA. J. Mol. Biol. 192: 257-274.
15. Kennell, D.E. (1986). The instability of messenger RNA in bacteria. In Maximizing Gene Expression (eds. W.S. Reznikoff and L. Gold) Butterworth Publ., Stoneham, MA., 101-140.
16. Cannistraro, V.J. and Kennell, D. (1985). Evidence that the 5'-end of lac mRNA starts to decay as soon as it is synthesized. J. Bacteriol. 161: 820-822.
17. Cannistraro, V.J. and Kennell, D.E. (1985). The 5'-ends of Escherichia coli lac mRNA. J. Mol. Biol. 182: 241-248.
18. Cannistraro, V.J., Wice, B.M. and Kennell, D.E. (1985). Isolating and sequencing the predominant 5'-ends of a specific mRNA in cells. II. End-labeling and sequencing. J. Biochem. Biophys. Methods 11: 163-175.
19. Cannistraro, V.J., Strominger, M.B., Wice, B.M. and Kennell, D.E. (1985). Isolating and sequencing the predominant 5'-ends of a specific mRNA in cells. I. Purification by filter hybridization. J. Biochem. Biophys. Methods 11: 153-161.

Technology for Licensing

• E.coli strain DK523. overproducing for RNase I.