Huang Y, Beaudry A, McSwiggen J, Sousa R
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760, USA.
Biochemistry. 1997 Nov 4;36(44):13718-28. doi: 10.1021/bi971609o.
The catalytic specificity of T7 RNA polymerase (RNAP) for ribonucleoside triphosphates vs deoxynucleoside triphosphates {(kcat/Km)rNTP/(kcat/Km)dNTP} during transcript elongation is approximately 80. Mutation of tyrosine 639 to phenylalanine reduces specificity by a factor of approximately 20 and largely eliminates the Km difference between rNTPs and dNTPs. The remaining specificity factor of approximately 4 is kcat-mediated and is nearly eliminated if Mn2+ is substituted for Mg2+ in the reaction. Mn2+ substitution does not significantly affect the Km difference between rNTPs and dNTPs. Mn2+ substitution also enhances the activity of poorly active mutant enzymes carrying nonconservative substitutions in the active site, and its effects are generally consistent with the Mn2+-catalyzed reaction being less restrictive in its requirements for alignment of the reactive groups. In addition to discrimination occurring at the level of nucleoside monophosphate (NMP) incorporation, it is also found that transcripts containing deoxynucleoside monophosphates (dNMPs) are more poorly extended than transcripts of canonical structure, though a severe barrier to transcript extension is seen only when the 3' region of the transcript is heavily substituted with dNMPs. The barrier to extension of transcripts heavily substituted with dNMPs is reduced for sequences known to be amenable to forming A-like helices and is larger for sequences that resist transformation from B-form DNA.DNA structures. The barrier to extension of dNMP-substituted transcripts is also reduced by solution conditions known to destabilize B-form DNA and to stabilize A-form structures. These observations imply a requirement for a non-B-form, possibly A-like, conformation in the transcript.template hybrid that is disrupted when the transcript is of predominantly deoxyribose structure.
在转录延伸过程中，T7 RNA聚合酶（RNAP）对核糖核苷三磷酸与脱氧核苷三磷酸的催化特异性{（kcat/Km）rNTP/（kcat/Km）dNTP}约为80。酪氨酸639突变为苯丙氨酸会使特异性降低约20倍，并在很大程度上消除了rNTP和dNTP之间的Km差异。剩余约4的特异性因子是由kcat介导的，并且如果在反应中用Mn2+替代Mg2+，该特异性因子几乎会被消除。Mn2+替代不会显著影响rNTP和dNTP之间的Km差异。Mn2+替代还增强了在活性位点携带非保守替代的低活性突变酶的活性，其效果通常与Mn2+催化的反应对反应基团排列要求的限制较少一致。除了在单磷酸核苷（NMP）掺入水平发生的辨别之外，还发现含有脱氧单磷酸核苷（dNMP）的转录本比具有典型结构的转录本延伸得更差，尽管只有当转录本的3'区域被dNMP大量取代时，才会出现严重的转录延伸障碍。对于已知易于形成A样螺旋的序列，被dNMP大量取代的转录本的延伸障碍会降低，而对于抵抗从B型DNA转变的序列，延伸障碍会更大。DNA结构。已知会破坏B型DNA并稳定A型结构的溶液条件也会降低dNMP替代转录本的延伸障碍。这些观察结果表明，转录本-模板杂交体需要一种非B型、可能是A样的构象，当转录本主要是脱氧核糖结构时，这种构象会被破坏。
