A quantitative description of the binding states and in vitro function of antitermination protein N of bacteriophage lambda.

Conant, Clarke R and Van Gilst, Marc R and Weitzel, Stephen E and Rees, William A and von Hippel, Peter H (2005) A quantitative description of the binding states and in vitro function of antitermination protein N of bacteriophage lambda. Journal of molecular biology, 348 (5). pp. 1039-1057. ISSN 0022-2836

Abstract

The N protein of bacteriophage lambda activates transcription of genes that lie downstream of termination sequences by suppressing transcription termination. N binds to specific (boxB) and non-specific sites on the transcript RNA and contacts RNA polymerase via cis-RNA looping, resulting in "antitermination" of transcription. To find the effect of N-boxB binding on antitermination, we quantitatively relate binding measurements made in isolation to in vitro antitermination activity. We measure binding of N to boxB RNA, non-specific single-stranded RNA, and non-specific double-stranded DNA fluorimetrically, and use an equilibrium model to describe quantitatively the binding of N to nucleic acids of Escherichia coli transcription elongation complexes. We then test the model by comparison with in vitro N antitermination activity measured in reactions containing these same elongation complexes. We find that binding of N protein to the nucleic acid components of transcription elongation complexes can quantitatively predict antitermination activity, suggesting that antitermination in vitro is determined by a nucleic acid binding equilibrium with one molecule of N protein per RNA transcript being sufficient for antitermination. Elongation complexes contain numerous overlapping non-specific RNA and DNA-binding sites for N; the large number of sites compensates for the low N binding affinity, so multiple N proteins are expected to bind to elongation complexes. The occupancy/activity of these proteins is described by a binomial distribution of proteins on transcripts containing multiple non-specific sites. The contribution of specific (boxB) binding to activity also depends on this distribution. Specificity is not measured accurately by measurements made in the presence and in the absence of boxB. We find that antitermination is inhibited by non-productive binding of N to non-specific sites on template DNA, and that NusA protein covers RNA sites on the transcript, limiting N access and activity. The activity and specificity of regulatory proteins that loop from high-affinity binding sites are likely modulated by multiple non-specific binding events; in vivo activity may also be regulated by the modulation of non-specific binding.

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