MUTANT T7 RNA-POLYMERASE AS A DNA-POLYMERASE
MUTANT T7 RNA-POLYMERASE AS A DNA-POLYMERASE
复制标题
DOI：
10.1002/j.1460-2075.1995.tb00140.x
复制
发表时间：
1995-09-15
期刊：
影响因子：
11.4
通讯作者：
PADILLA, R
中科院分区：
文献类型：
--
作者：
SOUSA, R;PADILLA, R
我们已经确定了T7 RNA聚合酶（RNAP）突变体，有效地利用脱氧核糖核苷三磷酸。在体外，该突变体将合成RNA、DNA或混合dNMP/rNMP组合物的“转录本”，这取决于合成反应中存在的ntp的混合物。该突变是保守的，将活性位点内的Tyr639改变为苯丙氨酸，不影响启动子特异性或整体活性。这种酪氨酸的非保守突变也会减少脱氧核糖和三磷酸核糖核苷之间的区别，但这些突变也会导致活性的大幅降低。在活性位点及其周围的26个其他残基突变中，没有一个对rNTP/dNTP区分有显著影响。DNA聚合酶（DNAP） I中相应的酪氨酸突变增加了错误编码，尽管DNAP I突变对dNTP/rNTP区分的影响尚未报道。因此，这种保守的酪氨酸可能在许多聚合酶中发挥类似的作用，通过感应底物/模板/产物结构中由于底物结构不适当或不匹配而导致的不正确的几何形状。T7 RNAP既可以使用RNA模板，也可以使用DNA模板，具有引物延伸和从头起始的能力。Y639F突变体保留了使用RNA或DNA模板的能力。因此，该突变体可以显示从头启动或引物DNA定向DNA聚合酶、逆转录酶、RNA定向RNA聚合酶或DNA定向RNA聚合酶的活性，这仅仅取决于在合成反应中呈现给它的模板和底物。
We have identified a T7 RNA polymerase (RNAP) mutant that efficiently utilizes deoxyribonucleoside triphosphates. In vitro this mutant will synthesize RNA, DNA or 'transcripts' of mixed dNMP/rNMP composition depending on the mix of NTPs present in the synthesis reaction. The mutation is conservative, changes Tyr639 within the active site to phenylalanine and does not affect promoter specificity or overall activity. Non-conservative mutations of this tyrosine also reduce discrimination between deoxyribo- and ribonucleoside triphosphates, but these mutations also cause large activity reductions. Of 26 mutations of other residues in and around the active site examined none showed marked effects on rNTP/dNTP discrimination. Mutations of the corresponding tyrosine in DNA polymerase (DNAP) I increase miscoding, though effects on dNTP/rNTP discrimination for the DNAP I mutations have not been reported. This conserved tyrosine may therefore play a similar role in many polymerases by sensing incorrect geometry in the structure of the substrate/template/product due to inappropriate substrate structure or mismatches. T7 RNAP can use RNA templates as well as DNA templates and is capable of both primer extension and de novo initiation. The Y639F mutant retains the ability to use RNA or DNA templates. Thus this mutant can display de novo initiated or primed DNA-directed DNA polymerase, reverse transcriptase, RNA-directed RNA polymerase or DNA-directed RNA polymerase activities depending simply on the templates and substrates presented to it in the synthesis reaction.