Buermeyer A B, Strasheim L A, McMahon S L, Farnham P J
McArdle Laboratory for Cancer Research, Medical School, University of Wisconsin, Madison 53706.
J Biol Chem. 1995 Mar 24;270(12):6798-807. doi: 10.1074/jbc.270.12.6798.
We have used an in vitro RNA polymerase II (RNAP II) inhibition-restimulation assay to investigate the inability of a form of RNAP II (RNAP IIB) that lacks the conserved, C-terminal heptapeptide repeat domain (CTD) to transcribe the dihydrofolate reductase (dhfr) promoter. Our previous studies demonstrated promoter-specific responses to RNAP IIB in the inhibition-restimulation assay and suggested the existence of cis-acting elements that alleviate the requirement for the CTD. We have now identified elements from two different classes of promoters that can convert dhfr to a CTD-independent promoter. First, addition of a consensus TATA box to the dhfr promoter resulted in a promoter capable of CTD-independent transcription and increased the promoter's affinity for the general transcription factor TFIID. These results suggest that high affinity for TFIID correlates with an ability to be transcribed by RNAP IIB, supporting a proposed interaction between the CTD and TFIID. Second, transfer of a combination of two elements (located at -25 and +1) from the rep-3b promoter, which does not contain a consensus TATA box but can nonetheless be transcribed by RNAP IIB, into the dhfr promoter also allowed CTD-independent transcription. These elements do not constitute a high affinity binding site for TFIID, indicating that an additional mechanism exists to allow CTD-independent transcription. Thus, elements from two classes of CTD-independent promoters that can obviate a requirement for the CTD appear to function via distinct mechanisms. Our finding that a change in a basal element can affect a requirement for the CTD is consistent with a role for the CTD during the formation of the transcriptional preinitiation complex.
我们使用了一种体外RNA聚合酶II（RNAP II）抑制-再刺激试验，以研究一种缺乏保守的C末端七肽重复结构域（CTD）的RNAP II形式（RNAP IIB）无法转录二氢叶酸还原酶（dhfr）启动子的原因。我们之前的研究在抑制-再刺激试验中证明了对RNAP IIB的启动子特异性反应，并表明存在可减轻对CTD需求的顺式作用元件。我们现在已经从两类不同的启动子中鉴定出了能够将dhfr转化为不依赖CTD的启动子的元件。首先，在dhfr启动子中添加一个共有TATA框，产生了一个能够进行不依赖CTD转录的启动子，并增加了该启动子对通用转录因子TFIID的亲和力。这些结果表明，对TFIID的高亲和力与被RNAP IIB转录的能力相关，支持了CTD与TFIID之间存在相互作用的提议。其次，将来自rep-3b启动子（该启动子不包含共有TATA框，但仍可被RNAP IIB转录）的两个元件（位于-25和+1处）组合转移到dhfr启动子中，也允许进行不依赖CTD的转录。这些元件并不构成TFIID的高亲和力结合位点，表明存在另一种机制允许进行不依赖CTD的转录。因此，两类不依赖CTD的启动子中能够消除对CTD需求的元件似乎通过不同的机制发挥作用。我们发现基础元件的变化会影响对CTD的需求，这与CTD在转录起始前复合物形成过程中的作用是一致的。
