Ellinger T, Behnke D, Knaus R, Bujard H, Gralla J D
Institut für Molekularbiologie, Universität Jena, Germany.
J Mol Biol. 1994 Jun 17;239(4):466-75. doi: 10.1006/jmbi.1994.1389.
Phased A-tract sequences were inserted in the upstream region of three synthetic promoters known to encompass different rate-limiting steps within the pathway of RNA polymerase-promoter interaction (Ellinger et al., accompanying paper). Promoter PS1, which is rate-limited in complex formation, was stimulated by A-tracts in vivo. Permanganate probing showed that the stimulation is due to an enhanced ability to compete for limiting RNA polymerase in vivo, leading to the increased formation of open complexes. By contrast, promoters PS2 and PS3, which are rate-limited in steps following open complex formation, were inhibited in vivo by A-tracts. Permanganate probing showed that the inhibition was accompanied by an A-tract-dependent accumulation of stalled initial transcribing complexes. A single A-tract was as effective as three. The phasing of the A-tracts with respect to the core promoter sequence was found to be important for promoter function. The position that caused maximal activation at one promoter caused maximal inhibition at another. These results suggest that the same molecular interaction gives rise to both inhibition and activation. This is likely to be due to facilitated RNA polymerase binding in the presence of A-tracts, which stimulates binding-limited promoters but inhibits promoter function in which polymerase escape and promoter clearance is rate limiting.
将阶段性A序列插入到三个合成启动子的上游区域，已知这些启动子在RNA聚合酶-启动子相互作用途径中包含不同的限速步骤（Ellinger等人，随附论文）。在复合物形成中受限速的启动子PS1在体内受到A序列的刺激。高锰酸钾探测表明，这种刺激是由于在体内竞争有限的RNA聚合酶的能力增强，导致开放复合物形成增加。相比之下，在开放复合物形成后的步骤中受限速的启动子PS2和PS3在体内受到A序列的抑制。高锰酸钾探测表明，这种抑制伴随着停滞的初始转录复合物的A序列依赖性积累。单个A序列与三个A序列一样有效。发现A序列相对于核心启动子序列的相位对启动子功能很重要。在一个启动子上引起最大激活的位置在另一个启动子上引起最大抑制。这些结果表明，相同的分子相互作用会导致抑制和激活。这可能是由于在A序列存在下促进了RNA聚合酶的结合，这刺激了结合受限的启动子，但抑制了聚合酶逃逸和启动子清除限速的启动子功能。
