Brieba L G, Sousa R
Department of Biochemistry, University of Texas Health Sciences Center, 7703 Floyd Curl Drive, San Antonio, Texas 78284-7760, USA.
Biochemistry. 2000 Feb 8;39(5):919-23. doi: 10.1021/bi992324+.
On the basis of their recently described T7 RNA polymerase-T7 promoter crystal structure, Cheetham et al. [(1999) Nature 399, 80] propose that discrimination of the hydrogen bonding character of the elongating NTP ribose 2'-substituent involves a hydrogen bond to histidine 784. This would contradict a previous conclusion, based on the characterization of mutant RNAPs, that discrimination of the hydrogen bonding character of the ribose 2'-substituent depends solely on the hydroxyl group of tyrosine 639. To resolve this point, we prepared and characterized histidine 784 point mutants. We find that while these mutations reduce the activity of the polymerase, they do not significantly reduce the level of ribose discrimination. Furthermore, a mutant with alanine at position 784 preferentially utilizes NTPs with 2'-substituents capable of acting as hydrogen bond donors or acceptors (2'-OH and 2'-NH(2)) over NTPs with substituents that lack such properties (2'-F and 2'-H). In contrast, mutation of tyrosine 639 to phenylalanine eliminates discrimination of ribose 2'-group hydrogen bonding character. The effects on ribose discrimination of mutating tyrosine 639 to phenylalanine are independent of the side chain at position 784. These results indicate that histidine 784 is not involved in discrimination of the ribose 2'-group of the elongating NTP. The ability of T7RNAP tyrosine 639, which is conserved in both RNA and DNA polymerases, to select for rNTPs appears to be due to the fact that in RNAPs this tyrosine is available to hydrogen bond to the ribose 2'-OH, while in DNAPs it is hydrogen bonded to a glutamic acid.
基于他们最近描述的T7 RNA聚合酶-T7启动子晶体结构，Cheetham等人[（1999年）《自然》399卷，80页]提出，对正在延伸的NTP核糖2'-取代基氢键性质的辨别涉及与组氨酸784形成氢键。这与之前基于突变RNA聚合酶的特性得出的结论相矛盾，即对核糖2'-取代基氢键性质的辨别仅取决于酪氨酸639的羟基。为了解决这一问题，我们制备并表征了组氨酸784点突变体。我们发现，虽然这些突变降低了聚合酶的活性，但它们并未显著降低核糖辨别水平。此外，784位为丙氨酸的突变体优先利用具有能够作为氢键供体或受体的2'-取代基（2'-OH和2'-NH₂）的NTP，而不是具有缺乏此类性质的取代基（2'-F和2'-H）的NTP。相比之下，酪氨酸639突变为苯丙氨酸消除了对核糖2'-基团氢键性质的辨别。将酪氨酸639突变为苯丙氨酸对核糖辨别产生的影响与784位的侧链无关。这些结果表明，组氨酸784不参与对正在延伸的NTP核糖2'-基团的辨别。T7 RNA聚合酶的酪氨酸639在RNA和DNA聚合酶中均保守，其选择rNTP的能力似乎是由于在RNA聚合酶中该酪氨酸可与核糖2'-OH形成氢键，而在DNA聚合酶中它与谷氨酸形成氢键。
