An Escherichia coli RNA polymerase defective in transcription due to its overproduction of abortive initiation products - PubMed (original) (raw)
. 1994 Feb 11;236(1):72-80.
doi: 10.1006/jmbi.1994.1119.
Affiliations
- PMID: 7508986
- DOI: 10.1006/jmbi.1994.1119
An Escherichia coli RNA polymerase defective in transcription due to its overproduction of abortive initiation products
D J Jin et al. J Mol Biol. 1994.
Abstract
One of the potential regulatory steps in procaryotic transcription is promoter clearance, a transition step in transcription initiation at which an RNA polymerase (RNAP) switches from the initial transcribing stage to the elongation stage. The biological significance of promoter clearance and the role of RNAP in this process are not understood. One approach to address these questions is to study mutant RNAPs that have altered promoter clearance. Because the antibiotic rifampicin inhibits transcription by preventing an initial transcribing complex from entering the elongation mode, mutant RNAPs which confer rifampicin (Rifr) are likely to be altered in promoter clearance. To test this hypothesis, we studied the effects of Rifr RNAPs on the pyrBI promoter, which is subject to control of promoter clearance in response to the availability of UTP. Two Rifr alleles that carry a different altered amino acid residue at position 529 of the beta subunit appeared to be defective in transcription from the pyrBI promoter in vivo. Biochemical analysis of one of these mutant RNAPs, RpoB3401 with a R529C change in the beta subunit, showed that it overproduces aborted initiation products from the pyrBI promoter and thus is defective in promoter clearance leading to reduced productive initiation. The severity of overproducing the aborted initiation products is an inverse function of the UTP concentration indicating that RpoB3401 has reduced affinity for UTP and thus is subject to a high Km barrier during promoter clearance. The defect of RpoB3401 in abortive initiation in vitro could account fully for its reduced initiation activity in vivo demonstrating the biological significance of abortive synthesis in transcription initiation. Our results indicate that at least part of the "rif region" is important for the process of abortive initiation and that promoter clearance can be regulated in part by modulating the Km of RNAP for nucleotides during initiation. The mutant enzyme is not altered in stuttering RNA synthesis at the pyrBI promoter, previously observed with wild-type RNAP. Our results also show that the mechanisms underlying the two non-productive initiation events (abortive synthesis and stuttering synthesis) at the pyrBI promoter are distinct.
Similar articles
- Molecular anatomy of the transcription complex of Escherichia coli during initiation.
Chatterji D, Kumar KP. Chatterji D, et al. Indian J Biochem Biophys. 1992 Apr;29(2):128-34. Indian J Biochem Biophys. 1992. PMID: 1398704 Review. - Promoter clearance and escape in prokaryotes.
Hsu LM. Hsu LM. Biochim Biophys Acta. 2002 Sep 13;1577(2):191-207. doi: 10.1016/s0167-4781(02)00452-9. Biochim Biophys Acta. 2002. PMID: 12213652 Review.
Cited by
- Mutations compensating for the fitness cost of rifampicin resistance in Escherichia coli exert pleiotropic effect on RNA polymerase catalysis.
Kurepina N, Chudaev M, Kreiswirth BN, Nikiforov V, Mustaev A. Kurepina N, et al. Nucleic Acids Res. 2022 Jun 10;50(10):5739-5756. doi: 10.1093/nar/gkac406. Nucleic Acids Res. 2022. PMID: 35639764 Free PMC article. - Insights into RNA polymerase catalysis and adaptive evolution gained from mutational analysis of a locus conferring rifampicin resistance.
Yurieva O, Nikiforov V Jr, Nikiforov V, O'Donnell M, Mustaev A. Yurieva O, et al. Nucleic Acids Res. 2017 Nov 2;45(19):11327-11340. doi: 10.1093/nar/gkx813. Nucleic Acids Res. 2017. PMID: 29036608 Free PMC article. - Insights into the mechanism of initial transcription in Escherichia coli RNA polymerase.
Samanta S, Martin CT. Samanta S, et al. J Biol Chem. 2013 Nov 1;288(44):31993-2003. doi: 10.1074/jbc.M113.497669. Epub 2013 Sep 18. J Biol Chem. 2013. PMID: 24047893 Free PMC article. - Isolation and characterization of RNA polymerase rpoB mutations that alter transcription slippage during elongation in Escherichia coli.
Zhou YN, Lubkowska L, Hui M, Court C, Chen S, Court DL, Strathern J, Jin DJ, Kashlev M. Zhou YN, et al. J Biol Chem. 2013 Jan 25;288(4):2700-10. doi: 10.1074/jbc.M112.429464. Epub 2012 Dec 5. J Biol Chem. 2013. PMID: 23223236 Free PMC article.
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources