The Antitermin ation Activity of Bacteriophage λ N Protein is Controlled by the Kinetics of an RNA-Looping-Facilitated Interaction with the Transcription Complex

Abstract

Protein N of bacteriophage λ activates the lytic phase of phage development in infected Escherichia coli cells by suppressing the activity of transcriptional terminators that prevent the synthesis of essential phage proteins. N binds tightly to the boxB RNA hairpin located near the 5′ end of the nascent pL and pR transcripts and induces an antitermination response in the RNA polymerase (RNAP) of elongation complexes located at terminators far downstream. Here we test an RNA looping model for this N-dependent “action at a distance” by cleaving the nascent transcript between boxB and RNAP during transcript elongation. Cleavage decreases antitermination, showing that an intact RNA transcript is required to stabilize the interaction of boxB-bound N with RNAP during transcription. In contrast, an antitermination complex that also contains Nus factors retains N-dependent activity after transcript cleavage, suggesting that these host factors further stabilize the N–RNAP interaction. Thus, the binding of N alone to RNAP is controlled by an RNA looping equilibrium, but after formation of the initial RNA loop and in the presence of Nus factors the system no longer equilibrates on the transcription time scale, meaning that the “range” of antitermination activity along the template in the full antitermination system is kinetically controlled by the dissociation rate of the stabilized N–RNAP complex. Theoretical calculations of nucleic acid end-to-end contact probabilities are used to estimate the local concentrations of boxB-bound N at elongation complexes poised at terminators, and are combined with N activity measurements at various boxB-to-terminator distances to obtain an intrinsic affinity (Kd) of ∼2×10−5 M for the N–RNAP interaction. This RNA looping approach is extended to include the effects of N binding at nonspecific RNA sites on the transcript and the implications for transcription control in other regulatory systems are discussed.