The site of action of the antiterminator protein N from the lambdoid phage H-19B

Cheeran, Anoop ; Kolli, Nanci R. ; Sen, Ranjan (2007) The site of action of the antiterminator protein N from the lambdoid phage H-19B Journal of Biological Chemistry, 282 (42). pp. 30997-31007. ISSN 0021-9258

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Transcription antitermination by N proteins of lambdoid phages involves specific interactions of the C-terminal domain of N with the Elongation Complex (EC). The interacting surface of N on the EC is unknown, knowledge of which is essential to understand the mechanism of antitermination. Specific cleavage patterns were generated near the active site Mg2+ of the RNA polymerase of an N-modified stalled EC using iron-(S)-1-(p-bromoacetamidobenzyl)ethylenediaminetetraacetate conjugated to the only cysteine residue in the C-terminal domain of N from a lambdoid phage H-19B. Modification of EC by N also induced conformational changes around the same region as revealed from the limited trypsin digestion and in situ Fe-dithiothreitol cleavage pattern of the same EC. These data, together with the previously obtained H-19B N-specific mutations in RNA polymerase, β (G1045D) and β′ (P251S, P254L, G336S and R270C) subunits, suggest that the active center cleft of the EC could be the site of action of this antiterminator. H-19B N induced altered interactions in this region of EC, prevented the backtracking of the stalled EC at the ops pause site and destabilized RNA hairpin-β subunit flap domain interactions at the his pause site. We propose that the physical proximity of the C-terminal domain of H-19B N to the active center cleft of the EC is required for the process of transcription antitermination and that it involves both stabilization of the weak RNA-DNA hybrid at a terminator and destabilization of the interactions of terminator hairpin in the RNA exit channel.

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Source:Copyright of this article belongs to American Society for Biochemistry and Molecular Biology.
ID Code:114778
Deposited On:04 Jun 2018 05:42
Last Modified:04 Jun 2018 05:42

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