THE RNA POLYMERASE-II TERNARY COMPLEX CLEAVES THE NASCENT TRANSCRIPT IN A 3'-]5' DIRECTION IN THE PRESENCE OF ELONGATION FACTOR-SII
THE RNA POLYMERASE-II TERNARY COMPLEX CLEAVES THE NASCENT TRANSCRIPT IN A 3'-]5' DIRECTION IN THE PRESENCE OF ELONGATION FACTOR-SII
复制标题
DOI：
10.1101/gad.6.7.1342
复制
发表时间：
1992-07-01
影响因子：
10.5
通讯作者：
LUSE, DS
中科院分区：
文献类型：
--
作者：
IZBAN, MG;LUSE, DS
RNA 聚合酶 II 延长 RNA 链的过程仍然知之甚少。已知需要延伸因子 SII 才能最大限度地提高体外内在终止位点的通读率。我们发现 SII 具有促进三元复合物转录本裂解的额外且意想不到的特性。我们首先注意到，在单一 NTP 限制的转录反应过程中，SII 的添加导致 RNA 聚合酶 II 在内在终止位点生成的转录本缩短。随后使用一系列在 15、18、20、21 和 35 核苷酸转录物合成后人为暂停的三元复合物观察到新生转录物的截短。 5 分钟反应即可生成短至 9 或 10 个核苷酸的转录本。所有这些缩短的 RNA 都保留在活性三元复合物中，因为它们可以被定量追踪。继续截短反应产生短至 4 个核苷酸的 RNA；然而，一旦切割进行到 5' 端的 8 或 9 个碱基以内，所得转录复合物就无法通过添加 NTP 来延长 RNA。转录本裂解需要二价阳离子，似乎主要以 2 个核苷酸增量进行，并被 α-鹅膏蕈碱抑制。 RNA 聚合酶 II 的催化位点在转录本裂解后重新定位，以便聚合在模板链上的正确位置恢复。转录物截短反应的程度和动力学受到 RNA 聚合酶停止位置和转录物序列的影响。
The process by which RNA polymerase II elongates RNA chains remains poorly understood. Elongation factor SII is known to be required to maximize readthrough at intrinsic termination sites in vitro. We found that SII has the additional and unanticipated property of facilitating transcript cleavage by the ternary complex. We first noticed that the addition of SII caused a shortening of transcripts generated by RNA polymerase II at intrinsic termination sites during transcription reactions in which a single NTP was limiting. Truncation of the nascent transcript was subsequently observed using a series of ternary complexes artificially paused after the synthesis of 15-, 18-, 20-, 21-, and 35-nucleotide transcripts. Transcripts as short as 9 or 10 nucleotides were generated in 5-min reactions. All of these shortened RNAs remained in active ternary complexes because they could be chased quantitatively. Continuation of the truncation reaction produced RNAs as short as 4 nucleotides; however, once cleavage had proceeded to within 8 or 9 bases of the 5' end, the resulting transcription complexes could not elongate the RNAs with NTP addition. Transcript cleavage requires a divalent cation, appears to proceed primarily in 2-nucleotide increments, and is inhibited by alpha-amanitin. The catalytic site of RNA polymerase II is repositioned after transcript cleavage such that polymerization resumes at the proper location on the template strand. The extent and kinetics of the transcript truncation reaction are affected by both the position at which RNA polymerase is halted and the sequence of the transcript.