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Information on EC 2.7.7.6 - DNA-directed RNA polymerase and Organism(s) Escherichia phage T7 and UniProt Accession P00573
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2.7.7.6 DNA-directed RNA polymeraseIUBMB Comments
Catalyses DNA-template-directed extension of the 3'- end of an RNA strand by one nucleotide at a time. Can initiate a chain de novo. In eukaryotes, three forms of the enzyme have been distinguished on the basis of sensitivity to alpha-amanitin, and the type of RNA synthesized. See also EC 2.7.7.19 (polynucleotide adenylyltransferase) and EC 2.7.7.48 (RNA-directed RNA polymerase).
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Word Map
- 2.7.7.6
- chromatin
- histone
- rrna
- strand
-
rnaps
- nucleosome
- tata
- drosophila
-
pausing
- pre-mrnas
- tbp
-
nucleolar
- bacteriophage
-
polyadenylation
-
single-stranded
-
cyclin-dependent
-
holoenzyme
- cyclin
-
spacers
- helicase
-
fidelity
- polya
-
coactivator
-
protein-coding
-
transactivation
- adenovirus
- xenopus
- ribonucleoproteins
-
sequence-specific
-
stall
-
pause
-
chip-seq
- vaccinia
-
multisubunit
-
ribozyme
- heptapeptide
- duplex
-
h3k4me3
-
spliceosome
- nucleoplasm
-
run-on
-
supercoiled
-
trimethylation
-
subcomplexes
-
exonuclease
- heterochromatin
-
translesion
- medicine
- primase
- molecular biology
- analysis
- diagnostics
- drug development
-
misincorporation
-
proofread
- synthesis
The taxonomic range for the selected organisms is: Escherichia phage T7
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Synonyms
rna polymerase ii, pol ii, t7 rna polymerase, rna polymerase i, pol iii, rna polymerase iii, pol i, rnapii, rnap ii, dna-dependent rna polymerase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
C RNA formation factors
-
-
-
-
chloroplast soluble RNA polymerase
-
-
-
-
deoxyribonucleic acid-dependent ribonucleic acid polymerase
-
-
-
-
DNA-dependent ribonucleate nucleotidyltransferase
-
-
-
-
DNA-dependent RNA nucleotidyltransferase
-
-
-
-
DNA-dependent RNA polymerase
nucleotidyltransferase, ribonucleate
-
-
-
-
Pol II
-
-
-
-
ribonucleate nucleotidyltransferase
-
-
-
-
ribonucleate polymerase
-
-
-
-
ribonucleic acid formation factors, C
-
-
-
-
ribonucleic acid nucleotidyltransferase
-
-
-
-
ribonucleic acid polymerase
-
-
-
-
ribonucleic acid transcriptase
-
-
-
-
ribonucleic polymerase
-
-
-
-
ribonucleic transcriptase
-
-
-
-
RNA formation factors, C
-
-
-
-
RNA nucleotidyltransferase
-
-
-
-
RNA nucleotidyltransferase (DNA-directed)
-
-
-
-
RNA polymerase
-
-
-
-
RNA polymerase I
-
-
-
-
RNA polymerase II
-
-
-
-
RNA polymerase III
-
-
-
-
RNA transcriptase
-
-
-
-
RNAP
RNAP I
-
-
-
-
RNAP II
-
-
-
-
RNAP III
-
-
-
-
transcriptase
-
-
-
-
DNA-dependent RNA polymerase
-
-
-
-
RNAP
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
nucleotidyl group transfer
-
-
-
-
SYSTEMATIC NAME
IUBMB Comments
nucleoside-triphosphate:RNA nucleotidyltransferase (DNA-directed)
Catalyses DNA-template-directed extension of the 3'- end of an RNA strand by one nucleotide at a time. Can initiate a chain de novo. In eukaryotes, three forms of the enzyme have been distinguished on the basis of sensitivity to alpha-amanitin, and the type of RNA synthesized. See also EC 2.7.7.19 (polynucleotide adenylyltransferase) and EC 2.7.7.48 (RNA-directed RNA polymerase).
CAS REGISTRY NUMBER
COMMENTARY
9014-24-8
-
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
DNA + 5-[[[2-(1H-indol-3-yl)ethyl]amino]carbonyl]-1-deazaUTP
?
-
-
-
-
?
nucleoside triphosphate + A10G2A2C2C
?
-
oligonucleotide extension
-
-
?
nucleoside triphosphate + T10G2T2C2C
?
-
oligonucleotide extension
-
-
?
nucleoside triphosphate + RNAn
diphosphate + RNAn+1
substrate specifically binds to the enzyme in the open conformation, where it is base paired with the acceptor template base, while Tyr639 provides discrimination of ribose versus deoxyribose substrates. Substrate selection occurs prior to the isomerization to the catalytically active conformation
-
-
?
nucleoside triphosphate + RNAn
diphosphate + RNAn+1
mechanism for de novo RNA synthesis, transcription begins with a marked preference for GTP at the +1 and +2 positions
-
-
?
nucleoside triphosphate + RNAn
diphosphate + RNAn+1
-
the catalytic specificity for ribonucleoside triphosphates vs. deoxynucleoside triphosphates during transcript elongation is 80
-
-
?
nucleoside triphosphate + RNAn
diphosphate + RNAn+1
-
the enzyme requires DNA as template
-
-
?
nucleoside triphosphate + RNAn
diphosphate + RNAn+1
-
mediates fast promoter-independent extension of unstable nucleic acid complexes
-
-
?
nucleoside triphosphate + RNAn
diphosphate + RNAn+1
-
short DNA or RNA substrates are good substrates for the enzyme
-
-
?
nucleoside triphosphate + RNAn
diphosphate + RNAn+1
-
the single subunit DNA-dependent RNA polymerase from bacteriophage T7 catalyzes both promoterdependent transcription initiation and promoter-independent elongation
-
-
?
nucleoside triphosphate + RNAn
diphosphate + RNAn+1
-
T7 RNAP undergoes a slow conformational change to form an elongation competent complex with the promoter-free substrate. The complex binds to a correct NTP and incorporates the nucleoside monophosphate into RNA primer very efficiently. In the presence of inorganic pyrophosphate, the elongation complex catalyzes the reverse pyrophosphorolysis reaction at a maximum rate of 0.8 per s
-
-
?
nucleoside triphosphate + RNAn
diphosphate + RNAn+1
-
two proton transfer occurs in the transition state for nucleotidyl-transfer reaction. Associative-like transition-state structure
-
-
?
nucleoside triphosphate + RNAn
diphosphate + RNAn+1
-
template is DNA
-
-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
nucleoside triphosphate + RNAn
diphosphate + RNAn+1
-
mediates fast promoter-independent extension of unstable nucleic acid complexes
-
-
?
nucleoside triphosphate + RNAn
diphosphate + RNAn+1
-
the single subunit DNA-dependent RNA polymerase from bacteriophage T7 catalyzes both promoterdependent transcription initiation and promoter-independent elongation
-
-
?
nucleoside triphosphate + RNAn
diphosphate + RNAn+1
-
template is DNA
-
-
?
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
cisplatin
-
a single cisplatin 1,2-d(CG) intrastrand cross-link or a single cisplatin 1,3-d(GTG) intrastrand cross-link is a strong block to the enzyme. The efficiency of the block at a cisplatin 1,2-d(GG) intrastrand cross-link is similar in several different nucleotide sequence contexts. Some blockage is also observed when the single cisplatin 1,3-d(GTG) intrastrand cross-link is located in the non-transcribed strand. Cisplatin-induced lesions in the transcribed DNA strand constitute a strong physical barrier to RNA polymerase progression
additional information
-
no inhibition by ent-16-ketobeyeran-19-oic acid, i.e. isosteviol, and related compounds
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.015
dGTP
-
pH 8.0, 37°C, dGTP as elongation substrate during dinucleotide synthesis, activation by Mg2+, mutant enzyme Y639F
0.025
dGTP
-
pH 8.0, 37°C, dGTP as elongation substrate during dinucleotide synthesis, activation by Mg2+, mutant enzyme Y639F
0.387
dGTP
-
pH 8.0, 37°C, dGTP as elongation substrate during dinucleotide synthesis, activation with Mn2+
0.75
dGTP
-
pH 8.0, 37°C, initiating nucleotide in dGrA synthesis, mutant enzyme Y639F
0.85
dGTP
-
pH 8.0, 37°C, dGTP as elongation substrate during dinucleotide synthesis, activation by Mg2+, wild-type enzyme
0.88
dGTP
-
pH 8.0, 37°C, initiating nucleotide in rGrA synthesis, wild-type enzyme
1.1
dGTP
-
pH 8.0, 37°C, initiating nucleotide in dGrA synthesis, mutant enzyme Y639F/S641A
1.4
dGTP
-
pH 8.0, 37°C, initiating nucleotide in dGrA synthesis, wild-type enzyme
0.0103
rGTP
-
pH 8.0, 37°C, rGTP as elongation substrate during dinucleotide synthesis, activation by Mn2+, wild-type enzyme
0.0104
rGTP
-
pH 8.0, 37°C, rGTP as elongation substrate during dinucleotide synthesis, activation by Mg2+, mutant enzyme Y639F
0.0153
rGTP
-
pH 8.0, 37°C, rGTP as elongation substrate during dinucleotide synthesis, activation by Mn2+, mutant enzyme Y639F
0.0175
rGTP
-
pH 8.0, 37°C, rGTP as elongation substrate during dinucleotide synthesis, activation by Mg2+, wild-type enzyme
0.21
rGTP
-
pH 8.0, 37°C, initiating nucleotide in rGrA synthesis, mutant enzyme S641A
0.22
rGTP
-
pH 8.0, 37°C, initiating nucleotide in rGrA synthesis, mutant enzyme Y639F/S641A
0.25
rGTP
-
pH 8.0, 37°C, initiating nucleotide in rGrA synthesis, wild-type enzyme
0.32
rGTP
-
pH 8.0, 37°C, initiating nucleotide in rGrA synthesis, mutant enzyme Y639F
additional information
additional information
-
relative KM-values for UTP derivatives
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.1
dGTP
-
pH 8.0, 37°C, initiating nucleotide in dGrA synthesis, mutant enzyme Y639F/S641A
0.22
dGTP
-
pH 8.0, 37°C, initiating nucleotide in dGrA synthesis, wild-type enzyme
0.32
dGTP
-
pH 8.0, 37°C, initiating nucleotide in dGrA synthesis, mutant enzyme S641A
0.34
dGTP
-
pH 8.0, 37°C, initiating nucleotide in dGrA synthesis, mutant enzyme Y639F
0.25
rGTP
-
pH 8.0, 37°C, initiating nucleotide in rGrA synthesis, mutant enzyme Y639F/S641A
0.26
rGTP
-
pH 8.0, 37°C, initiating nucleotide in rGrA synthesis, wild-type enzyme
0.28
rGTP
-
pH 8.0, 37°C, initiating nucleotide in rGrA synthesis, mutant enzyme Y639F
0.38
rGTP
-
pH 8.0, 37°C, initiating nucleotide in rGrA synthesis, mutant enzyme S641A
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). RPOL_BPT7
883
0
98855
Swiss-Prot
other Location (Reliability: 1)
PDB
SCOP
CATH
UNIPROT
ORGANISM
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
-
existence of two possible conformers: E and E that are in rapid equilibrium. Both forms can form the quarternary complex, but only the E form is capable of catalyzing phosphodiester bond formation
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
D421A
-
mutation results in an enzyme with reduced activity and altered patterns of transcription
D421T
-
mutation results in an enzyme with reduced activity and altered patterns of transcription
K631R
-
the fraction of catalytically active E form is 38% compared to 100% for the wild-type enzyme. The synthesis of long transcripts is markedly diminished for the mutant due to decreasing processivity
R423A
-
mutation results in an enzyme with reduced activity and altered patterns of transcription
R423K
-
mutation results in an enzyme with reduced activity and altered patterns of transcription
R425K
-
mutation results in an enzyme with reduced activity and altered patterns of transcription
S641A
-
mutation reduces activity in presence of Mg2+ to 93% of the activity of the wild-type enzyme
W422A
-
mutation results in an enzyme that has nearly normal levels of activity and exhibits patterns of transcription that are similar to that of the wild-type enzyme
W422F
-
mutation results in an enzyme that has nearly normal levels of activity and exhibits patterns of transcription that are similar to that of the wild-type enzyme
W422R
-
mutation results in an enzyme that has nearly normal levels of activity and exhibits patterns of transcription that are similar to that of the wild-type enzyme
W422S
-
mutation results in an enzyme that has nearly normal levels of activity and exhibitspatterns of transcription that arew similar to that of the wild-type enzyme
Y639/S641A
-
mutation reduces activity in presence of Mg2+ to 89% of the activity of the wild-type enzyme
Y639C
-
mutation reduces activity in presence of Mg2+ to 7.5% of the activity of the wild-type enzyme. The mutation reduces the catalytic specificity for ribonucleoside triphosphates versus deoxynucleoside triphosphates during transcript elongation, which is about 80 for the wild-type enzyme. The remaining specificity factor is 11
Y639F
Y639H
-
mutation reduces activity in presence of Mg2+ to 3.7% of the activity of the wild-type enzyme
Y639L
-
mutation reduces activity in presence of Mg2+ to 43% of the activity of the wild-type enzyme. The mutation reduces the catalytic specificity for ribonucleoside triphosphates versus deoxynucleoside triphosphates during transcript elongation, which is about 80 for the wild-type enzyme. The remaining specificity factor is 11
Y639M
-
mutation reduces activity in presence of Mg2+ to 50% of the activity of the wild-type enzyme. The mutation reduces the catalytic specificity for ribonucleoside triphosphates versus deoxynucleoside triphosphates during transcript elongation, which is about 80 for the wild-type enzyme. The remaining specificity factor is 5.5
Y639Q
-
mutation reduces activity in presence of Mg2+ to 1% of the activity of the wild-type enzyme. The mutation reduces the catalytic specificity for ribonucleoside triphosphates vs deoxynucleoside triphosphates during transcript elongation, which is about 80 for the wild-type enzyme. The remaining specificity factor is 4.5
Y639T
-
mutation reduces activity in presence of Mg2+ to 1.3% of the activity of the wild-type enzyme. The mutation reduces the catalytic specificity for ribonucleoside triphosphates versus deoxynucleoside triphosphates during transcript elongation, which is about 80 for the wild-type enzyme. The remaining specificity factor is 6.5
Y639V
-
mutation reduces activity in presence of Mg2+ to 4.3% of the activity of the wild-type enzyme. The mutation reduces the catalytic specificity for ribonucleoside triphosphates versus deoxynucleoside triphosphates during transcript elongation, which is about 80 for the wild-type enzyme. The remaining specificity factor is 19
additional information
-
RNAP mutants: the N-terminal region of T7 RNAP contains a nascent RNA binding site that functions to retain the nascent chain within the ternary complex. The region surrounding residue 240 is involved in binding the initiating NTP. Residues at the very C terminus of T7 RNAP are involved in binding the elongating NTP
Y639F
-
mutation reduces the catalytic specificity for ribonucleoside triphosphates vs deoxynucleoside triphosphates during transcript elongation, which is about 80 for the wild-type enzyme by a factor of 20 and largely eliminates the KM-difference between rNTPs and dNTPs. The remaining specificity factor of 4 is turnover-number-mediated and is nearly eliminated if Mn2+ is substituted for Mg2+ in the reaction. Mn2+ substitution does not significantly affect the Km difference between rNTPs and dNTPs
Y639F
-
the fraction of catalytically active E form is 32% compared to 100% for the wild-type enzyme
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
construction of plasmids with polylinker cloning sites adjacent to the RNA polymerase promoter
-
expression of the T7 RNA polymerase tagged with yellow fluorescent protein and His6 in Rhodobacter capsulatus strain B10S establishing an additional expression system for the enzyme, method optimization and system evaluation, comparison to the Escherichia coli strain BL21(DE3) expression system, both using the expression vector pRhotHi-2-yfp-His6, overview. T7 RNA polymerase subcloning in Escherichia coli strain DH5alpha. Quantitative expression analysis, overview
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
synthesis
-
the enzyme is useful for in vitro transcription reactions to produce preparative quantities of transcribed RNA and labeled RNA probes, transcripts thousands of nucleotides in length, as are the major applications of these reactions, method evaluation, overview. Phage T7 RNA polymerase is an extremely processive enzyme in high-yield transcription of DNA sequences inserted downstream from the corresponding T7 promoter, applications overview
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Patra, D.; Lafer, E.M.; Sousa, R.
Isolation and characterization of mutant bacteriophage T7 RNA polymerases
J. Mol. Biol.
224
307-318
1992
Escherichia phage T7
Chung, Y.J.; Sousa, R.; Rose, J.P.; Lafer, E.; Wang, B.C.
Crystallographic structure of phage T7 RNA polymerase at resolution of 4.0 A
Struct. Funct. Nucleic Acids Proteins (Wu, F. Y. -H. , Wu, C. -W. , Eds. ) Raven
New York
55-59
1990
Escherichia phage T7
-
Huang, Y.; Beaudry, A.; McSwiggen, J.; Sousa, R.
Determinants of ribose specificity in RNA polymerization: effects of Mn2+ and deoxynucleoside monophosphate incorporation into transcripts
Biochemistry
36
13718-13728
1997
Escherichia phage T7
Tornaletti, S.; Patrick, S.M.; Turchi, J.J.; Hanawalt, P.C.
Behavior of T7 RNA polymerase and Mammalian RNA polymerase II at site-specific cisplatin adducts in the template DNA
J. Biol. Chem.
278
35791-35797
2003
Escherichia phage T7, Rattus norvegicus
Imburgio, D.; Anikin, M.; McAllister, W.T.
Effects of substitutions in a conserved DX2GR sequence motif, found in many DNA-dependent nucleotide polymerases, on transcription by T7 RNA polymerase
J. Mol. Biol.
319
37-51
2002
Escherichia phage T7, Escherichia coli
Woody, A.Y.M.; Osumi-Davis, P.A.; Hiremath, M.M.; Woody, R.W.
Pre-steady-state and steady-state kinetic studies on transcription initiation catalyzed by T7 RNA polymerase and its active-site mutants K631R and Y639F
Biochemistry
37
15958-15964
1998
Escherichia phage T7
Mizushina, Y.; Saito, A.; Tanaka, A.; Nakajima, N.; Kuriyama, I.; Takemura, M.; Takeuchi, T.; Sugawara, F.; Yoshida, H.
Structural analysis of catechin derivatives as mammalian DNA polymerase inhibitors
Biochem. Biophys. Res. Commun.
333
101-109
2005
Escherichia phage T7
Zaher, H.S.; Unrau, P.J.
T7 RNA polymerase mediates fast promoter-independent extension of unstable nucleic acid complexes
Biochemistry
43
7873-7880
2004
Escherichia phage T7
Temiakov, D.; Patlan, V.; Anikin, M.; McAllister, W.T.; Yokoyama, S.; Vassylyev, D.G.
Structural basis for substrate selection by T7 RNA polymerase
Cell
116
381-391
2004
Escherichia phage T7 (P00573)
Vaught, J.D.; Dewey, T.; Eaton, B.E.
T7 RNA polymerase transcription with 5-position modified UTP derivatives
J. Am. Chem. Soc.
126
11231-11237
2004
Escherichia phage T7
Mizushina, Y.; Akihisa, T.; Ukiya, M.; Hamasaki, Y.; Murakami-Nakai, C.; Kuriyama, I.; Takeuchi, T.; Sugawara, F.; Yoshida, H.
Structural analysis of isosteviol and related compounds as DNA polymerase and DNA topoisomerase inhibitors
Life Sci.
77
2127-2140
2005
Escherichia phage T7
Anand, V.S.; Patel, S.S.
Transient state kinetics of transcription elongation by T7 RNA polymerase
J. Biol. Chem.
281
35677-35685
2006
Escherichia phage T7
Kennedy, W.P.; Momand, J.R.; Yin, Y.W.
Mechanism for de novo RNA synthesis and initiating nucleotide specificity by T7 RNA polymerase
J. Mol. Biol.
370
256-268
2007
Escherichia phage T7 (P00573), Escherichia phage T7
Castro, C.; Smidansky, E.; Maksimchuk, K.R.; Arnold, J.J.; Korneeva, V.S.; Goette, M.; Konigsberg, W.; Cameron, C.E.
Two proton transfers in the transition state for nucleotidyl transfer catalyzed by RNA- and DNA-dependent RNA and DNA polymerases
Proc. Natl. Acad. Sci. USA
104
4267-4272
2007
Escherichia phage T7
Paschal, B.M.; McReynolds, L.A.; Noren, C.J.; Nichols, N.M.
RNA polymerases
Curr. Protoc. Mol. Biol.
Chapter 3
Unit3.8
2008
Enterobacteria phage T3, Escherichia phage T7, Escherichia coli, Zindervirus SP6
Katzke, N.; Arvani, S.; Bergmann, R.; Circolone, F.; Markert, A.; Svensson, V.; Jaeger, K.E.; Heck, A.; Drepper, T.
A novel T7 RNA polymerase dependent expression system for high-level protein production in the phototrophic bacterium Rhodobacter capsulatus
Protein Expr. Purif.
69
137-146
2010
Escherichia phage T7
EXTERNAL LINKS (specific for EC-Number 2.7.7.6)
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