Processivity of T7 RNA polymerase requires the C-terminal Phe882-Ala883-COO- or "foot".
The role of the C-terminal Phe882-Ala883 residues of bacteriophage T7 RNA polymerase in specific transcription has been investigated by means of site-directed mutagenesis. A mutant enzyme that lacks the C-terminal Phe882-Ala883 residues, denoted the "foot" mutant, has been cloned and overproduced, and the effects of the deletion on promoter recognition, initiation, and elongation have been determined. Gel retardation assays and DNase I footprinting show that the foot mutant specifically recognizes and binds to T7 promoters, although this binding appears to be approximately 30-fold weaker than that of the wild-type enzyme. Transcription assays using oligonucleotide templates that contain the consensus T7 promoter show a dramatic decrease in transcriptional activity for the foot mutant. With templates whose coding region begins CCC..., the mutant synthesizes poly(G) products even in the presence of all four nucleotides. The synthesis of poly(G) products from such templates has previously been observed for the wild-type enzyme when GTP is the sole nucleotide present in the reaction and is thought to occur by a novel mechanism involving slippage of the RNA chain 3' to 5' relative to the template [Martin, C.T., Muller, D.K., & Coleman, J.E. (1988) Biochemistry 27, 3966-3974]. These data suggest that the loss in transcriptional activity by the foot mutant results from a severe decrease in processivity as well as catalytic efficiency of the enzyme. Removal of the C-terminal Phe and Ala residues from the wild-type enzyme with carboxypeptidase A generates the phenotype of the mutant precisely, proving that all of the properties of the foot mutant derive from the loss of the Phe-Ala-COOH moiety.(ABSTRACT TRUNCATED AT 250 WORDS)
Mookhtiar
K A
KA
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06510.
Peluso
P S
PS
Muller
D K
DK
Dunn
J J
JJ
Coleman
J E
JE
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002268:Carboxypeptidases / Q000378:metabolism; D043422:Carboxypeptidases A; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics*; D003850:Deoxyribonuclease I; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000235:genetics; D012323:RNA Processing, Post-Transcriptional; D013604:T-Phages / Q000201:enzymology / Q000235:genetics*; D013816:Thermodynamics
D010649:Phenylalanine; D012321:DNA-Directed RNA Polymerases; D003850:Deoxyribonuclease I; D002268:Carboxypeptidases; D043422:Carboxypeptidases A; D000409:Alanine