Harinarayanan, R. and Gowrishankar, J. (2003) Host Factor Titration by Chromosomal R-loops as a Mechanism for Runaway Plasmid Replication in Transcription Termination-defective Mutants of Escherichia coli. Journal of Molecular Biology, 332 (1). pp. 31-46. ISSN 0022-2836

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Abstract

Two Escherichia coli genes, rnhA and recG, encode products that disrupt R-loops by hydrolysis and unwinding, respectively. It is known that the propensity for R-loop formation in vivo is increased during growth at 21 °C. We have identified several links between rnhA, recG, and R-loop-dependent plasmid replication on the one hand, and genes rho and nusG involved in factor-dependent transcription termination on the other. A novel nusG-G146D mutation phenocopied a rho-A243E mutation in conferring global deficiency in transcription termination, and both mutants were killed at 21 °C following overexpression of rnhA+. Mutant combinations rnhA-nusG or recG-rho were synthetically lethal at 21 °C, with the former being suppressed by recG+ overexpression. rho and nusG mutants were killed following transformation with plasmids such as pACYC184 or pUC19 (which have R-loop replication intermediates) even at 30 °C or 37 °C, and the lethality was correlated with greatly increased content of supercoiled monomer species of these and other co-resident R-loop-dependent plasmids. Plasmid-mediated lethality in the mutants was suppressed by overexpression of rnhA+ or recG+. Two additional categories of trans-acting suppressors of the plasmid-mediated lethality were identified whose primary effects were, respectively, a reduction in plasmid copy number even in the wild-type strain, and a restoration of the proficiency of in vivo transcription termination in the nusG and rho mutant strains. The former category of suppressors included rom+, and mutations in rpoB(Q513L), pcnB, and polA, whereas the latter included a mutation in rho (R221C) and several non-null mutations (E74K, L26P, and Δ64-137) in the gene encoding the nucleoid protein H-NS. We propose that an increased occurrence of chromosomal R-loops in the rho and nusG mutants leads to titration of a cyloplasmic host factor(s) that negatively modulates the stability of plasmid R-loop replication intermediates and consequently to runaway plasmid replication

Item Type:	Article
Depositing User:	Users 2 not found.
Date Deposited:	06 Nov 2015 09:45
Last Modified:	06 Nov 2015 09:45
URI:	http://cdfd.sciencecentral.in/id/eprint/624

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