Huang Y, Eckstein F, Padilla R, Sousa R
Department of Biochemistry, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, Texas 78284-7760, USA.
Biochemistry. 1997 Jul 8;36(27):8231-42. doi: 10.1021/bi962674l.
The mechanism by which T7 RNA polymerase (RNAP) discriminates between rNTP and dNTP substrates has been characterized. During transcript elongation T7 RNAP uses rNTPs 70-80-fold more efficiently than dNTPs. Discrimination of the hydrogen-bonding character of the ribose 2'-substituent contributes a largely Km-mediated factor of approximately 20 to this preference for rNTPs. Discrimination of 2'-substituent H-bonding character appears to be made through a hydrogen bond to the hydroxyl group of tyrosine 639. This hydrogen bond makes little net contribution to either rNTP ground or transition state binding energy apparently because it is balanced by the energy of desolvation of the tyrosine hydroxyl. This mechanism may reflect a strategy to facilitate translocation by minimizing contributions from polymerase-NMP moiety interactions to NTP binding energy so as to minimize the affinity of the NTP binding site for the 3'-NMP of the product nucleic acid.
T7 RNA聚合酶（RNAP）区分核糖核苷三磷酸（rNTP）和脱氧核糖核苷三磷酸（dNTP）底物的机制已得到表征。在转录延伸过程中，T7 RNAP使用rNTP的效率比dNTP高70至80倍。对核糖2'-取代基氢键特征的区分对这种对rNTP的偏好贡献了一个很大程度上由Km介导的约20的因子。对2'-取代基氢键特征的区分似乎是通过与酪氨酸639的羟基形成氢键来实现的。这种氢键对rNTP基态或过渡态结合能几乎没有净贡献，显然是因为它被酪氨酸羟基去溶剂化的能量所平衡。这种机制可能反映了一种策略，即通过最小化聚合酶-NMP部分相互作用对NTP结合能的贡献来促进转位，从而最小化NTP结合位点对产物核酸3'-NMP的亲和力。
