Regulation of the elongation-termination decision at intrinsic terminators by antitermination protein N of phage lambda.
The mechanisms that control N-protein-dependent antitermination in the phage lambda life cycle have counterparts in the regulatory systems of other organisms. Here we examine N-dependent antitermination at the intrinsic tR' terminator of lambda to elucidate the regulatory principles involved. The tR' terminator consists of a sequence of six base-pairs along the template at which the transcription complex is sufficiently destabilized to make RNA release possible. Within this "zone of opportunity" for termination the termination efficiency (TE) at each template position is determined by a kinetic competition between alternative reaction pathways that lead either to elongation or to termination. TE values at each position within tR' have been mapped as a function of NTP concentration, and it is shown that N protein (in the presence of NusA and a nut site; the minimal system for N-dependent antitermination) can offset increases in TE that are induced by limiting the concentrations of each of the next required NTPs. By limiting NTP concentrations or working at low temperature we show that a significant effect of N within the minimal system is to increase the rate of transcript elongation three- to fivefold at most positions along the template. Assuming that a comparable increase in elongation rate applies at template positions within the terminator, we show that an increase of this magnitude is not sufficient to account for the antitermination efficiency observed and that an approximately 100-fold stabilization of the transcription complex at intrinsic termination sites as a consequence of binding the N-containing antitermination sub-assembly must be invoked as well. A general method for partitioning TE effects in antitermination between changes in elongation rate and termination complex stability is demonstrated, based on competing free energy of activation barriers for the elongation and termination reactions. The analysis and utility of such mixed modes of transcriptional regulation are considered in general terms.
Rees
W A
WA
Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, OR 97403, USA.
Weitzel
S E
SE
Das
A
A
von Hippel
P H
PH
D001426:Bacterial Proteins / Q000502:physiology; D010582:Bacteriophage lambda / Q000378:metabolism*; D001483:Base Sequence; D003080:Cold Temperature; D029968:Escherichia coli Proteins; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009711:Nucleotides / Q000378:metabolism; D010441:Peptide Chain Elongation, Translational; D010443:Peptide Chain Termination, Translational; D010445:Peptide Elongation Factors; D014157:Transcription Factors / Q000502:physiology; D014158:Transcription, Genetic; D035602:Transcriptional Elongation Factors; D054334:Viral Regulatory and Accessory Proteins / Q000737:chemistry / Q000502:physiology*
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C059417:N protein, Bacteriophage lambda; D009711:Nucleotides; D010445:Peptide Elongation Factors; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; D054334:Viral Regulatory and Accessory Proteins; C068727:nusA protein, E coli