Initiation, elongation, and processivity of carboxyl-terminal mutants of T7 RNA polymerase.
Bacteriophage T7 RNA polymerase is a single-subunit enzyme which has a C-terminal amino acid sequence of Phe-Ala-Phe-Ala883 (FAFA883). Closely related hydrophobic sequences are present at the C termini of seven other single-subunit RNA polymerases, including the mitochondrial RNA polymerase. Mutations at any of the four C-terminal residues depress initiation rates of T7 RNA polymerase from 50 to 95%, accompanied by large increases in the K(m) values for the initiating nucleotide, GTP, as well as the K(m)'s for promoter DNA. The dramatic drops in initiation rates shown by the mutant enzymes remain after correcting for any alteration in saturation of the enzyme by the initiating nucleotide or the promoter DNA resulting from the changes in K(m). In contrast, the high processivity of the enzyme is not altered by mutations in the last four residues. However, the propensity for the enzyme to add an untemplated nucleotide at the 3'-ends of transcripts is abolished by the A880AFA883 mutation. The C-terminal FAFA sequence or foot appears to interact both with the initiating NTP and with the most downstream nucleotides of the promoter, possibly through hydrophobic interactions with the minor groove, in the region where free radical footprinting of the polymerase-promoter DNA complex suggests that the enzyme binds across the minor groove.
Gardner
L P
LP
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520-8114, USA.
Mookhtiar
K A
KA
Coleman
J E
JE
D017123:Bacteriophage T7 / Q000201:enzymology* / Q000235:genetics; D017931:DNA Primers; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D011401:Promoter Regions, Genetic / Q000235:genetics; D011487:Protein Conformation; D012333:RNA, Messenger / Q000378:metabolism; D014158:Transcription, Genetic; D014764:Viral Proteins
D017931:DNA Primers; D012333:RNA, Messenger; D014764:Viral Proteins; D006160:Guanosine Triphosphate; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases