Initiation, elongation, and processivity of carboxyl-terminal mutants of T7 RNA polymerase

Lisa P. Gardner, Kasim A. Mookhtiar, Joseph E. Coleman

Research output: Contribution to journalArticle

27 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)2908-2918
Number of pages11
JournalBiochemistry
Volume36
Issue number10
DOIs
StatePublished - Mar 11 1997

Fingerprint

Elongation
Enzymes
Nucleotides
DNA-Directed RNA Polymerases
Mutation
DNA
DNA-Directed DNA Polymerase
Guanosine Triphosphate
Hydrophobic and Hydrophilic Interactions
Free Radicals
bacteriophage T7 RNA polymerase
Foot
Amino Acid Sequence
Amino Acids

ASJC Scopus subject areas

  • Biochemistry

Cite this

Initiation, elongation, and processivity of carboxyl-terminal mutants of T7 RNA polymerase. / Gardner, Lisa P.; Mookhtiar, Kasim A.; Coleman, Joseph E.

In: Biochemistry, Vol. 36, No. 10, 11.03.1997, p. 2908-2918.

Research output: Contribution to journalArticle

Gardner, Lisa P. ; Mookhtiar, Kasim A. ; Coleman, Joseph E. / Initiation, elongation, and processivity of carboxyl-terminal mutants of T7 RNA polymerase. In: Biochemistry. 1997 ; Vol. 36, No. 10. pp. 2908-2918.
@article{5efa1466754a41c98e9f384697b5f9e3,
title = "Initiation, elongation, and processivity of carboxyl-terminal mutants of T7 RNA polymerase",
abstract = "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.",
author = "Gardner, {Lisa P.} and Mookhtiar, {Kasim A.} and Coleman, {Joseph E.}",
year = "1997",
month = "3",
day = "11",
doi = "10.1021/bi962397i",
language = "English (US)",
volume = "36",
pages = "2908--2918",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "10",

}

TY - JOUR

T1 - Initiation, elongation, and processivity of carboxyl-terminal mutants of T7 RNA polymerase

AU - Gardner, Lisa P.

AU - Mookhtiar, Kasim A.

AU - Coleman, Joseph E.

PY - 1997/3/11

Y1 - 1997/3/11

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0030995087&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0030995087&partnerID=8YFLogxK

U2 - 10.1021/bi962397i

DO - 10.1021/bi962397i

M3 - Article

C2 - 9062120

AN - SCOPUS:0030995087

VL - 36

SP - 2908

EP - 2918

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 10

ER -