The objective of the present proposal is to understand the mechanisms regulating pyrimidine gene expression in Escherichia coli. In this bacterium, the de novo synthesis of UMP, the precursor of all pyrimidine nucleotides is catalyzed by six enzymes encoded by six unlinked genes and small operons: carAB, pyrBI, pyrC, pyrD, pyrE, and pyrF. The expression of these genes and operons is noncoordinately regulated by the intracellular levels of uridine or cytidine nucleotides. Recent studies have shown that pyrBI and pyrE expression is regulated primarily by an attenuation control mechanism in which transcriptional termination at a Rho-independent terminator (attenuator) immediately preceding the pyr structural gene(s) is regulated by the relative rates of UTP- sensitive transcription and coupled translation within a leader region upstream of the attenuator. At present, little is known about the mechanisms regulating the expression of the other pyrimidine genes. In this study experiments are designed to identify new regulatory elements and to test possible models for the regulation of pyrimidine gene expression. The proposed research will focus on the pyrBI operon and the pyrC and pyrF genes. Experiments are planned to complete the genetic and biochemical characterization of key regulatory elements involved in attenuation control of pyrBI expression, including nucleotide (UTP and GTP)specific transcriptional pausing. Strains exhibiting altered attenuation control will be isolated and should provide a variety of interesting mutations, including rpo mutations which will be used to characterize fundamental properties of RNA polymerase. Attenuator-independent regulation of pyrBI expression, which significantly contributes to the pyrimidine-mediated control of this operon, also will be characterized. Of particular interest is the possible involvement of UTP-sensitive abortive transcriptional initiation in attenuator-independent regulation. Recent results suggest that pyrC expression is regulated by a novel attenuation control mechanism in which transcriptional termination at a Rho-dependent erminator within the pyrC structural gene is controlled by the extent of coupled transcription and translation. Experiments are planned to test the effect of rho mutations and mutations that alter the rate of pyrC translational initiation on the regulation of pyrC expression. In addition, the possible role of the purine repressor, encoded by the purR gene, in the regulation of pyrC, pyrD, and car AB expression will be examined. The remaining experiments outlined will examine the mechanism regulating pyrF expression. The possible regulatory roles of trans- acting factors, abortive initiation, and the orF gene (the second gene in the pyrF-orfF operon) will be explored.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM029466-12
Application #
3277063
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1981-08-01
Project End
1994-07-31
Budget Start
1992-08-01
Budget End
1993-07-31
Support Year
12
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of Alabama Birmingham
Department
Type
Schools of Dentistry
DUNS #
004514360
City
Birmingham
State
AL
Country
United States
Zip Code
35294
Han, Xiaosi; Turnbough Jr, Charles L (2014) Transcription start site sequence and spacing between the -10 region and the start site affect reiterative transcription-mediated regulation of gene expression in Escherichia coli. J Bacteriol 196:2912-20
Turnbough Jr, Charles L (2011) Regulation of gene expression by reiterative transcription. Curr Opin Microbiol 14:142-7
Turnbough Jr, Charles L; Switzer, Robert L (2008) Regulation of pyrimidine biosynthetic gene expression in bacteria: repression without repressors. Microbiol Mol Biol Rev 72:266-300, table of contents
Sipos, Katalin; Szigeti, Reka; Dong, Xiuzhu et al. (2007) Systematic mutagenesis of the thymidine tract of the pyrBI attenuator and its effects on intrinsic transcription termination in Escherichia coli. Mol Microbiol 66:127-38
Mosrin-Huaman, Christine; Turnbough Jr, Charles L; Rahmouni, A Rachid (2004) Translocation of Escherichia coli RNA polymerase against a protein roadblock in vivo highlights a passive sliding mechanism for transcript elongation. Mol Microbiol 51:1471-81
Meng, Qi; Turnbough Jr, Charles L; Switzer, Robert L (2004) Attenuation control of pyrG expression in Bacillus subtilis is mediated by CTP-sensitive reiterative transcription. Proc Natl Acad Sci U S A 101:10943-8
Cheng, Y; Dylla, S M; Turnbough Jr, C L (2001) A long T. A tract in the upp initially transcribed region is required for regulation of upp expression by UTP-dependent reiterative transcription in Escherichia coli. J Bacteriol 183:221-8
Pokholok, D K; Redlak, M; Turnbough Jr, C L et al. (1999) Multiple mechanisms are used for growth rate and stringent control of leuV transcriptional initiation in Escherichia coli. J Bacteriol 181:5771-82
Han, X; Turnbough Jr, C L (1998) Regulation of carAB expression in Escherichia coli occurs in part through UTP-sensitive reiterative transcription. J Bacteriol 180:705-13
Gaal, T; Bartlett, M S; Ross, W et al. (1997) Transcription regulation by initiating NTP concentration: rRNA synthesis in bacteria. Science 278:2092-7

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