Izawa M, Sasaki N, Watahiki M, Ohara E, Yoneda Y, Muramatsu M, Okazaki Y, Hayashizaki Y
Genome Science Laboratory, Tsukuba Life Science Center, The Institute of Physical and Chemical Research (RIKEN), Koyadai 3-1-1, Tsukuba-city, Ibaraki 305, Japan.
J Biol Chem. 1998 Jun 5;273(23):14242-6. doi: 10.1074/jbc.273.23.14242.
When analyzing the elongation mechanisms in T7 RNA polymerase (T7 RNAP)by using site-directed mutagenesis and a protein expression system, we identified the recognition sites of the rNTP 3'-OH group in T7 RNAP. On the basis of three-dimensional crystal structure analysis, we selected and analyzed six candidate sites interacting with the 3'-OH group of rNTP in T7 RNAP. We found that the Phe-644 and Phe-667 sites are responsible for the high selectivity of T7 RNAP for rNTPs. Also, we constructed the protein mutations of these residues, F644Y and F667Y, which display a >200-fold higher affinity than the wild type for 3'-dNTPs. These findings indicate that the phenylalanine residues of 644 and 667 specifically interact with the 3'-OH group. Thus, these mutants, F644Y and F667Y, with incorporation of 3'-dNTP terminators, which is similar to native rNTPs, can offer low backgrounds and equal intensities of the sequencing ladders in our method, called "transcriptional sequencing. "
通过定点诱变和蛋白质表达系统分析T7 RNA聚合酶（T7 RNAP）的延伸机制时，我们确定了T7 RNAP中rNTP 3'-OH基团的识别位点。基于三维晶体结构分析，我们选择并分析了T7 RNAP中与rNTP的3'-OH基团相互作用的六个候选位点。我们发现Phe-644和Phe-667位点决定了T7 RNAP对rNTPs的高选择性。此外，我们构建了这些残基的蛋白质突变体F644Y和F667Y，它们对3'-dNTPs的亲和力比野生型高200倍以上。这些发现表明644和667位的苯丙氨酸残基与3'-OH基团特异性相互作用。因此，在我们称为“转录测序”的方法中，这些掺入了与天然rNTPs相似的3'-dNTP终止子的突变体F644Y和F667Y，能够提供低背景和强度相同的测序梯。
