TFB1 or TFB2 is sufficient for Thermococcus kodakaraensis viability and for basal transcription in vitro
TFB1 or TFB2 is sufficient for Thermococcus kodakaraensis viability and for basal transcription in vitro
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DOI：
10.1016/j.jmb.2006.12.069
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发表时间：
2007-03-23
影响因子：
5.6
通讯作者：
Reeve, John N.
中科院分区：
文献类型：
--
作者：
Santangelo, Thomas J.;Cubonova, L'ubomira;Reeve, John N.
古细菌RNA聚合酶（RNAP）与真核生物RNAP II（Pol II）最为相似，但启动基础转录仅需要两种古细菌通用转录因子TBP（TATA盒结合蛋白）和TFB（真核生物通用转录因子TFIIB的古细菌同源物）的支持。然而，许多古细菌基因组编码一种以上的TFB和/或TBP，导致不同的TFB/TBP组合可能被用来指导从不同的启动子在古细菌中的起始的假设。作为该假设的第一个测试，我们已经确定了从Thermococcus kodakaraensis（T.k.）启动多种T. k.当提供T.k. TBP和TFB 1或TFB 2，这两个TFB编码在T.k.基因组在体外每个启动子都有活性的情况下，转录起始发生在TFB 1或TFB 2中，尽管TFB 2的起始最佳盐浓度（类似于250 mM K+）通常高于TFB 1（类似于200 mM K+）。与这种体外功能冗余一致，T.k.已构建了TFB 1-（tfb 1; TK 1280）或TFB 2-（tjb 2; TK 2287）编码基因缺失的菌株。这些突变体在实验室条件下没有表现出可检测的生长缺陷。TFB 1和TFB 2之间的结构域交换已经确定了一个中心区域，该区域有助于TFB活性的盐敏感性，并且删除预测形成TFB 2的B指区尖端的残基对启动子识别或转录起始没有可检测的影响，但确实消除了非常短的（
Archaeal RNA polymerases (RNAPs) are most similar to eukaryotic RNAP II (Pol II) but require the support of only two archaeal general transcription factors, TBP (TATA-box binding protein) and TFB (archaeal homologue of the eukaryotic general transcription factor TFIIB) to initiate basal transcription. However, many archaeal genomes encode more than one TFB and/or TBP leading to the hypothesis that different TFB/TBP combinations may be employed to direct initiation from different promoters in Archaea. As a first test of this hypothesis, we have determined the ability of RNAP purified from Thermococcus kodakaraensis (T.k.) to initiate transcription from a variety of T.k. promoters in vitro when provided with T.k. TBP and either TFB1 or TFB2, the two TFBs encoded in the T.k. genome. With every promoter active in vitro, transcription initiation occurred with either TFB1 or TFB2 although the optimum salt concentration for initiation was generally higher for TFB2 (similar to 250 mM K+) than for TFB1 (similar to 200 mM K+). Consistent with this functional redundancy in vitro, T.k. strains have been constructed with the TFB1- (tfb1; TK1280) or TFB2- (tjb2; TK2287) encoding gene deleted. These mutants exhibit no detectable growth defects under laboratory conditions. Domain swapping between TFB1 and TFB2 has identified a central region that contributes to the salt sensitivity of TFB activity, and deleting residues predicted to form the tip of the B-finger region of TFB2 had no detectable effects on promoter recognition or transcription initiation but did eliminate the production of very short (