Cheng Bo, Price David H
Department of Biochemistry, Molecular and Cellular Biology Program, University of Iowa, Iowa City, IA 52242, USA.
J Biol Chem. 2007 Jul 27;282(30):21901-12. doi: 10.1074/jbc.M702936200. Epub 2007 Jun 4.
The positive transcription elongation factor, P-TEFb, controls the fraction of initiated RNA polymerase II molecules that enter into the productive mode of elongation necessary to generate mRNAs. To better understand the mechanism of this transition into productive elongation we optimized a defined in vitro transcription system and compared results obtained with it to those obtained with a crude system. We found that controlling the function of TFIIF is a key aspect of RNA polymerase II elongation control. Before P-TEFb function, early elongation complexes under the control of negative factors are completely unresponsive to the robust elongation stimulatory activity of TFIIF. P-TEFb-mediated phosphorylation events, targeting the elongation complex containing DSIF and NELF, reverse the negative effect of DSIF and NELF and simultaneously facilitate the action of TFIIF. We also found that productive elongation complexes are completely resistant to negative elongation factors. Our data suggest that an additional factor(s) is involved in establishing the unique resistance activities of the elongation complexes before and after P-TEFb function. Furthermore, we provide evidence for the existence of another positive activity required for efficient function of P-TEFb. A model of the mechanism of P-TEFb-mediated elongation control is proposed in which P-TEFb induces the transition into productive elongation by changing the accessibility of elongation factors to elongation complexes. Our results have uncovered important properties of elongation complexes that allow a more complete understanding of how P-TEFb controls the elongation phases of transcription by RNA polymerase II.
正性转录延伸因子P-TEFb控制起始的RNA聚合酶II分子中进入生成mRNA所需的有效延伸模式的分子比例。为了更好地理解这种向有效延伸转变的机制，我们优化了一个明确的体外转录系统，并将其结果与粗制系统的结果进行比较。我们发现，控制TFIIF的功能是RNA聚合酶II延伸控制的一个关键方面。在P-TEFb发挥功能之前，受负性因子控制的早期延伸复合物对TFIIF强大的延伸刺激活性完全无反应。P-TEFb介导的磷酸化事件靶向包含DSIF和NELF的延伸复合物，逆转了DSIF和NELF的负性作用，同时促进了TFIIF的作用。我们还发现，有效延伸复合物对负性延伸因子完全有抗性。我们的数据表明，在P-TEFb发挥功能之前和之后，另外一个因子参与建立延伸复合物独特的抗性活性。此外，我们提供了证据证明存在P-TEFb有效发挥功能所需的另一种正性活性。我们提出了一个P-TEFb介导的延伸控制机制模型，其中P-TEFb通过改变延伸因子与延伸复合物的可及性来诱导向有效延伸的转变。我们的结果揭示了延伸复合物的重要特性，从而能更全面地理解P-TEFb如何控制RNA聚合酶II转录的延伸阶段。
