Rees W A, Weitzel S E, Das A, von Hippel P H
Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, OR 97403, USA.
J Mol Biol. 1997 Nov 7;273(4):797-813. doi: 10.1006/jmbi.1997.1327.
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.
在噬菌体λ生命周期中控制N蛋白依赖性抗终止的机制在其他生物体的调节系统中有对应物。在这里，我们研究λ噬菌体固有tR'终止子处的N依赖性抗终止，以阐明其中涉及的调节原理。tR'终止子由模板上六个碱基对的序列组成，转录复合物在此处足够不稳定，使得RNA释放成为可能。在这个终止的“机会区域”内，每个模板位置的终止效率（TE）由导致延伸或终止的替代反应途径之间的动力学竞争决定。已将tR'内每个位置的TE值绘制为NTP浓度的函数，结果表明，N蛋白（在存在NusA和nut位点的情况下；N依赖性抗终止的最小系统）可以抵消因限制下一个所需NTP的浓度而诱导的TE增加。通过限制NTP浓度或在低温下工作，我们表明在最小系统中N的一个显著作用是在模板上的大多数位置将转录延伸速率提高三到五倍。假设在终止子内的模板位置也有类似的延伸速率增加，我们表明这种程度的增加不足以解释观察到的抗终止效率，并且还必须调用由于结合含N的抗终止亚组件而导致的转录复合物在固有终止位点处约100倍的稳定。基于延伸和终止反应的活化能垒的竞争自由能，展示了一种在抗终止中划分TE效应在延伸速率变化和终止复合物稳定性之间的通用方法。从一般角度考虑了这种转录调节混合模式的分析和实用性。
