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King, R.A. and Markov, D. and Sen, Ranjan and Severinov, K. and Weisberg, R.A. (2004) A Conserved Zinc Binding Domain in the Largest Subunit of DNA-dependent RNA Polymerase Modulates Intrinsic Transcription Termination and Antitermination but does not Stabilize the Elongation Complex. Journal of Molecular Biology, 342 (4). pp. 1143-1154. ISSN 0022-2836

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An evolutionarily conserved zinc-binding motif is found close to the amino terminus of the largest subunits of DNA-dependent RNA polymerases from bacteria, archaea, and eukaryotes. In bacterial RNA polymerase, this motif, the zinc binding domain, has been implicated in protein-DNA interactions that stabilize the transcription elongation complex and that occur downstream of the catalytic center. Here, we show that this view is incorrect, and instead, the zinc binding domain interacts with product RNA located upstream of the catalytic center and the RNA-DNA hybrid, a view consistent with structural studies of the elongation complex. We engineered mutations that alter or remove the zinc binding domain of Escherichia coli RNA polymerase. Several mutants, including one that lacked all four zinc ligands and another that lacked the entire domain, produced enzymes that were active in vitro and formed stable elongation complexes. However, they were defective in two functions that require interaction of polymerase with product RNA. First, they terminated less efficiently than the wild-type at intrinsic transcription terminators. Second, enzymes lacking the tip of the zinc binding domain or the zinc ligands did not antiterminate in response to an intrinsic antiterminator encoded by the put site of phage HK022. Termination, but not antitermination, was restored by the bacterial termination factor NusA. Surprisingly, a mutant that lacks the entire zinc binding domain regained a partial response to put. To account for this we suggest that put RNA interacts with an additional site in the elongation complex to mediate antitermination, and that this site is occluded by the wild-type zinc binding domain.

Item Type: Article
Depositing User: Dr P Divakar
Date Deposited: 01 Jul 2015 10:11
Last Modified: 01 Jul 2015 10:11
URI: http://cdfd.sciencecentral.in/id/eprint/212

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