Effects of substitutions in a conserved DX(2)GR sequence motif, found in many DNA-dependent nucleotide polymerases, on transcription by T7 RNA polymerase.
The region in bacteriophage T7 RNA polymerase (RNAP) comprising residues 421-425 contains a sequence motif (DX(2)GR) that is conserved among many DNA-dependent nucleotide polymerases. We have found that alterations in this motif result in enzymes that display weaker retention of the RNA product during transcript initiation, a decreased ability to make the transition to a stable elongation complex, and changes in substrate binding and catalytic activity. Many of these defects are coupled with an altered response to the presence or absence of the non-template strand. The observed constellation of defects supports a role for the motif in interacting with and stabilizing the RNA:DNA hybrid during the early stages of transcript initiation. This is consistent with the position of the motif in a T7 RNAP initiation complex. Although a conserved DX(2)GR sequence motif is also observed in multisubunit RNAPs, the structural organization of the motif and the manner in which it interacts with the RNA:DNA hybrid in the latter enzymes is different from that in T7 RNAP. However, another element in the multisubunit RNAPs that contains a highly conserved arginine residue may play the same role as R425 in T7 RNAP. (c) 2002 Elsevier Science Ltd.
Imburgio
Diane
D
Department of Microbiology and Immunology, Morse Institute of Molecular Genetics, SUNY Health Science Center at Brooklyn, 450 Clarkson Avenue, Brooklyn, NY 11203-2098, USA.
Anikin
Michael
M
McAllister
William T
WT
D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D017123:Bacteriophage T7 / Q000201:enzymology*; D001483:Base Sequence; D002384:Catalysis; D017124:Conserved Sequence / Q000235:genetics*; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011068:Poly G / Q000096:biosynthesis / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013698:Templates, Genetic; D024363:Transcription Initiation Site; D014158:Transcription, Genetic; D014764:Viral Proteins
D014764:Viral Proteins; D011068:Poly G; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases