In Escherichia coli messenger RNAs are rapidly degraded with in average functional half-life of approximately 90 seconds. Based on our recent results and those from other laboratories, we have developed a working model for mRNA turnover in which functional mRNAs are degraded in a series of apparent endonucleolytic steps. The initial breakdown products are subsequently digested exonucleolytically to mononucleotides which can be recycled for the synthesis of new RNA species. By employing a multiple ams pnp rnb mutant we have been able to stabilize the initial breakdown products of a number of individual mRNA molecules. Our long range goal is to understand the important regulatory features of mRNA turnover along with its enzymology. In the previous grant period we succeeded in showing that ribonuclease II, polynucleotide phosphorylase, and the Ams gene product are essential for the later steps of mRNA turnover. In addition, we were able to identify a new locus (mrd, messenger RNA degradation), that is required for both mRNA degradation and cell viability. Our current aims are to analyze the fate of individual mRNA molecules in strains deficient in polynucleotide phosphorylase, ribonuclease II and the ams gene product. By sequencing the ends of the initial breakdown products we hope into identify the structural features associated with the cleavage sites. Additionally, we will attempt to clone and identify by the Ams and Mrd gene products. New multiple mutants carrying mutations in the ams mrd pnp rnb and rnc genes will be constructed and their mRNA turnover patterns determined. We will also purify and characterize both temperature sensitive ribonuclease II and ribonuclease III proteins. If time permits, we will attempt to gain further insights into the mechanism of mRNA turnover by examining temperature resistant revertants of ams pnp rnb and pnp rnb multiple mutants.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM028760-09
Application #
3276049
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1979-04-01
Project End
1993-06-30
Budget Start
1990-07-01
Budget End
1991-06-30
Support Year
9
Fiscal Year
1990
Total Cost
Indirect Cost
Name
University of Georgia
Department
Type
Schools of Arts and Sciences
DUNS #
City
Athens
State
GA
Country
United States
Zip Code
30602
Ow, Maria C; Liu, Qi; Mohanty, Bijoy K et al. (2002) RNase E levels in Escherichia coli are controlled by a complex regulatory system that involves transcription of the rne gene from three promoters. Mol Microbiol 43:159-71
Mohanty, B K; Kushner, S R (2000) Polynucleotide phosphorylase, RNase II and RNase E play different roles in the in vivo modulation of polyadenylation in Escherichia coli. Mol Microbiol 36:982-94
Mohanty, B K; Kushner, S R (2000) Polynucleotide phosphorylase functions both as a 3' right-arrow 5' exonuclease and a poly(A) polymerase in Escherichia coli. Proc Natl Acad Sci U S A 97:11966-71
Ow, M C; Liu, Q; Kushner, S R (2000) Analysis of mRNA decay and rRNA processing in Escherichia coli in the absence of RNase E-based degradosome assembly. Mol Microbiol 38:854-66
Mohanty, B K; Kushner, S R (1999) Analysis of the function of Escherichia coli poly(A) polymerase I in RNA metabolism. Mol Microbiol 34:1094-108
Mohanty, B K; Kushner, S R (1999) Residual polyadenylation in poly(A) polymerase I (pcnB ) mutants of Escherichia coli does not result from the activity encoded by the f310 gene. Mol Microbiol 34:1109-19
Zhang, G; Deng, E; Baugh, L et al. (1998) Identification and characterization of Escherichia coli DNA helicase II mutants that exhibit increased unwinding efficiency. J Bacteriol 180:377-87
Wang, R F; O'Hara, E B; Aldea, M et al. (1998) Escherichia coli mrsC is an allele of hflB, encoding a membrane-associated ATPase and protease that is required for mRNA decay. J Bacteriol 180:1929-38
Granger, L L; O'Hara, E B; Wang, R F et al. (1998) The Escherichia coli mrsC gene is required for cell growth and mRNA decay. J Bacteriol 180:1920-8
Zhang, G; Deng, E; Baugh, L R et al. (1997) Conserved motifs II to VI of DNA helicase II from Escherichia coli are all required for biological activity. J Bacteriol 179:7544-50

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