Moreland R J, Hanas J S, Conaway J W, Conaway R C
Program in Molecular and Cell Biology, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104, USA.
J Biol Chem. 1998 Oct 9;273(41):26610-7. doi: 10.1074/jbc.273.41.26610.
We previously identified and purified Elongin by its ability to stimulate the rate of elongation by RNA polymerase II in vitro (Bradsher, J. N., Jackson, K. W., Conaway, R. C., and Conaway, J. W. (1993) J. Biol. Chem. 268, 25587-25593). In this report, we present evidence that stimulation of elongation by Elongin requires that the early RNA polymerase II elongation complex undergoes conversion to an Elongin-activable form. We observe (i) that Elongin does not detectably stimulate the rate of promoter-specific transcription initiation by the fully assembled preinitiation complex and (ii) that early RNA polymerase II elongation intermediates first become susceptible to stimulation by Elongin after synthesizing 8-9-nucleotide-long transcripts. Furthermore, we show that the relative inability of Elongin to stimulate elongation by early elongation intermediates correlates not with the lengths of their associated transcripts but, instead, with the presence of transcription factor IIF (TFIIF) in transcription reactions. By exploiting adenovirus 2 major late promoter derivatives that contain premelted transcriptional start sites and do not require TFIIF, TFIIE, or TFIIH for transcription initiation, we observe (i) that Elongin is capable of strongly stimulating the rate of synthesis of trinucleotide transcripts by a subcomplex of RNA polymerase II, TBP, and TFIIB and (ii) that the ability of Elongin to stimulate synthesis of these short transcripts is substantially reduced by addition of TFIIF to transcription reactions. Here we present these findings, which are consistent with the model that maximal stimulation of elongation by Elongin requires that early elongation intermediates undergo a structural transition that includes loss of TFIIF.
我们之前通过其在体外刺激RNA聚合酶II延伸速率的能力鉴定并纯化了延伸因子（布拉德舍尔，J. N.，杰克逊，K. W.，康纳韦，R. C.，和康纳韦，J. W.（1993年）《生物化学杂志》268卷，25587 - 25593页）。在本报告中，我们提供证据表明延伸因子对延伸的刺激要求早期RNA聚合酶II延伸复合物转变为可被延伸因子激活的形式。我们观察到：（i）延伸因子不能显著刺激完全组装好的起始前复合物进行启动子特异性转录起始的速率；（ii）早期RNA聚合酶II延伸中间体在合成8 - 9个核苷酸长的转录本后才首次变得易受延伸因子的刺激。此外，我们表明延伸因子相对无法刺激早期延伸中间体的延伸，这与它们相关转录本的长度无关，而是与转录反应中存在转录因子IIF（TFIIF）有关。通过利用腺病毒2主要晚期启动子衍生物，这些衍生物含有预熔解的转录起始位点，转录起始不需要TFIIF、TFIIE或TFIIH，我们观察到：（i）延伸因子能够强烈刺激RNA聚合酶II、TBP和TFIIB的亚复合物合成三核苷酸转录本的速率；（ii）在转录反应中加入TFIIF会大大降低延伸因子刺激这些短转录本合成的能力。在此我们展示这些发现，这些发现与以下模型一致，即延伸因子对延伸的最大刺激要求早期延伸中间体经历包括TFIIF丢失的结构转变。
