Roles of histidine 784 and tyrosine 639 in ribose discrimination by T7 RNA polymerase.
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.
Brieba
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LG
Department of Biochemistry, University of Texas Health Sciences Center, 7703 Floyd Curl Drive, San Antonio, Texas 78284-7760, USA.
Sousa
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R
D017123:Bacteriophage T7 / Q000201:enzymology* / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000737:chemistry; D004789:Enzyme Activation / Q000235:genetics; D006639:Histidine / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D012266:Ribose / Q000378:metabolism*; D014158:Transcription, Genetic; D014443:Tyrosine / Q000737:chemistry* / Q000235:genetics; D014764:Viral Proteins
D003854:Deoxyribonucleotides; D014764:Viral Proteins; D014443:Tyrosine; D006639:Histidine; D012266:Ribose; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases