Targeted mutagenesis identifies Asp-569 as a catalytically critical residue in T7 RNA polymerase.
In order to look more closely at a well-conserved region in T7 RNA polymerase (T7 RNAP) containing, as shown earlier, the functionally essential residues Pro-563 and Tyr-571, we used targeted mutagenesis to change those residues within this region that are invariant in all single-subunit RNA polymerases, and characterized the mutant enzymes in vitro. The most interesting finding of this study was the crucial importance of the acidic group of Asp-569. In addition, we have shown that the phenolic ring is the most significant functional group of Tyr-571, with the hydroxy group also contributing to promoter binding.
Rechinsky
V O
VO
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow.
Chernov
B K
BK
Dragan
S M
SM
Kostyuk
D A
DA
Tunitskaya
V L
VL
Kochetkov
S N
SN
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000502:physiology*; D017123:Bacteriophage T7 / Q000201:enzymology*; D004268:DNA-Binding Proteins / Q000737:chemistry; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic; D014443:Tyrosine / Q000502:physiology; D014764:Viral Proteins
D004268:DNA-Binding Proteins; D014764:Viral Proteins; D001224:Aspartic Acid; D014443:Tyrosine; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases