Roles of histidine 784 and tyrosine 639 in ribose discrimination by T7 RNApolymerase

On the basis of their recently described T7 RNA polymerase-T7 promoter crys tal structure, Cheetham et al. [(1999) Nature 399, 80] propose that discrim ination of the hydrogen bonding character of the elongating NTP ribose 2'-s ubstituent 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'-substitu ent depends solely on the hydroxyl group of tyrosine 639. To resolve this p oint, we prepared and characterized histidine 784 point mutants. We find th at while these mutations reduce the activity of the polymerase, they do not significantly reduce the level of ribose discrimination. Furthermore, a mu tant with alanine at position 784 preferentially utilizes NTPs with 2'-subs tituents capable of acting as hydrogen bond donors or accepters (2'-OH and 2'-NH2) over NTPs with substituents that lack such properties (2'-F and 2'- H). In contrast, mutation of tyrosine 639 to phenylalanine eliminates discr imination of ribose 2'-group hydrogen bonding character. The effects on rib ose discrimination of mutating tyrosine 639 to phenylalanine are independen t 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 elongat ing 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 tha t 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.