Abstract

We shall attempt to identify the major 5' (and possible 3') ends of mRNA from the lactose (lac) operon of E. coli that are present in growing cells. From earlier studies we expect unique 5'-ends from specific cleavages near the start of lac z and between the lac zy and ya junctions. Specific lac RNA regions will be purified and 5' (or 3') end labeled using gamma-32P-ATP. Large 32P-oligonucleotides will be isolated after partial RNase treatment and separation on 2D polyacrylamide gels. Their positions will be identified by autoradiography before they are eluted and partially digested in alkali and the sequences determined by 2D separations with electrophoresis on cellulose acetate and TLC homochromatography. We are constructing plasmids that carry specific segments of the lac operon for these and other studies. The same methods will be used to identify the 5' ends of lac mRNA in certain lac Oc mutants which we showed had altered decay and ribosome initiation frequencies for the z message. Other work by us suggested an involvement of RNase III in processing lac z message for translation. The lac mRNA from RNase III-cells will be analyzed similarly.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
9R01GM034127-09
Application #
3284657
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1985-03-01
Project End
1988-02-29
Budget Start
1985-03-01
Budget End
1986-02-28
Support Year
9
Fiscal Year
1985
Total Cost
Indirect Cost

  Institution

Name
Washington University
Department
Type
Schools of Medicine
DUNS #
062761671
City
Saint Louis
State
MO
Country
United States
Zip Code
63130

  Publications

Cannistraro, V J; Kennell, D (1994) The processive reaction mechanism of ribonuclease II. J Mol Biol 243:930-43
Lim, L W; Mathur, S; Cannistraro, V J et al. (1993) Preliminary X-ray crystallographic studies of ribonuclease I from Escherichia coli. J Mol Biol 234:499-501
Cannistraro, V J; Kennell, D (1993) The 5' ends of RNA oligonucleotides in Escherichia coli and mRNA degradation. Eur J Biochem 213:285-93
Mathur, S; Cannistraro, V J; Kennell, D (1993) Identification of an intracellular pyrimidine-specific endoribonuclease from Bacillus subtilis. J Bacteriol 175:6717-20
Srivastava, S K; Cannistraro, V J; Kennell, D (1992) Broad-specificity endoribonucleases and mRNA degradation in Escherichia coli. J Bacteriol 174:56-62
Cannistraro, V J; Kennell, D (1991) RNase I*, a form of RNase I, and mRNA degradation in Escherichia coli. J Bacteriol 173:4653-9
Meador 3rd, J; Cannon, B; Cannistraro, V J et al. (1990) Purification and characterization of Escherichia coli RNase I. Comparisons with RNase M. Eur J Biochem 187:549-53
Meador 3rd, J; Kennell, D (1990) Cloning and sequencing the gene encoding Escherichia coli ribonuclease I: exact physical mapping using the genome library. Gene 95:1-7
Cannistraro, V J; Kennell, D (1989) Purification and characterization of ribonuclease M and mRNA degradation in Escherichia coli. Eur J Biochem 181:363-70
Subbarao, M N; Kennell, D (1988) Evidence for endonucleolytic cleavages in decay of lacZ and lacI mRNAs. J Bacteriol 170:2860-5

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