Asp537, Asp812 are essential and Lys631, His811 are catalytically significant in bacteriophage T7 RNA polymerase activity

Osumi-Davis, Patricia A.; Aguilera, Marcela C. de; Woody, Robert W.; Woody, A-Young Moon

doi:10.1016/0022-2836(92)90122-z

Cited by 79 publications

(53 citation statements)

References 38 publications

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“…Motif D has been in search of a function other than structural scaffolding since solution of the first RT structures (37). In all cases in which this putative general acid has been changed, the observed rate of nucleotide incorporation has been diminished substantially (38)(39)(40). Particularly noteworthy is the observation that chemistry appears to become more rate-limiting by changing the putative general acid of RB69 DNA polymerase, Lys-560, to alanine (39).…”

Section: Discussionmentioning

confidence: 99%

Two proton transfers in the transition state for nucleotidyl transfer catalyzed by RNA- and DNA-dependent RNA and DNA polymerases

Castro

Smidansky

Maksimchuk

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

146

190

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The rate-limiting step for nucleotide incorporation in the presteady state for most nucleic acid polymerases is thought to be a conformational change. As a result, very little information is available on the role of active-site residues in the chemistry of nucleotidyl transfer. For the poliovirus RNA-dependent RNA polymerase (3D pol ), chemistry is partially (Mg 2؉ ) or completely (Mn 2؉ ) rate limiting. Here we show that nucleotidyl transfer depends on two ionizable groups with pK a values of 7.0 or 8.2 and 10.5, depending upon the divalent cation used in the reaction. A solvent deuterium isotope effect of three to seven was observed on the rate constant for nucleotide incorporation in the pre-steady state; none was observed in the steady state. Proton-inventory experiments were consistent with two protons being transferred during the rate-limiting transition state of the reaction, suggesting that both deprotonation of the 3 -hydroxyl nucleophile and protonation of the pyrophosphate leaving group occur in the transition state for phosphodiester bond formation. Importantly, two proton transfers occur in the transition state for nucleotidyl-transfer reactions catalyzed by RB69 DNA-dependent DNA polymerase, T7 DNA-dependent RNA polymerase and HIV reverse transcriptase. Interpretation of these data in the context of known polymerase structures suggests the existence of a general base for deprotonation of the 3 -OH nucleophile, although use of a water molecule cannot be ruled out conclusively, and a general acid for protonation of the pyrophosphate leaving group in all nucleic acid polymerases. These data imply an associative-like transition-state structure.general-acid-base catalysis ͉ phosphoryl transfer ͉ two-metal-ion mechanism N ucleic acid polymerases are essential for the maintenance and expression of the genomes of all organisms. All classes of polymerases use the same five-step kinetic scheme for nucleotide incorporation (1-6). The kinetic mechanism for the RNAdependent RNA polymerase (RdRp 1 ) from poliovirus (3D pol ) is shown in Scheme 1. One of the advantages of this system is that once 3D pol assembles onto the primer-template substrate, this complex has a half-life of Ͼ2 h (7), greatly simplifying kinetic analysis. In step one, the enzyme-nucleic acid complex (ER n ) binds the nucleoside triphosphate forming a ternary complex (ER n NTP).Step two involves a conformational change (*ER n NTP) that orients the triphosphate for catalysis. In step three, nucleotidyl transfer occurs (*ER nϩ1 PP i ), followed by a second conformational-change step (ER nϩ1 PP i ) and pyrophosphate release (ER nϩ1 ).Although the sequence of events occurring during the nucleotide-addition cycle is identical for all polymerases, the ratelimiting step appears to be different. In most cases, the first conformational-change step (step two) is rate-limiting (2, 8, 9). In one, chemistry (step three) is rate-limiting (10), and in some (e.g., T4 and RB69 DNA polymerases), the rate-limiting step has not been established. For 3D pol , bot...

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Section: Discussionmentioning

confidence: 99%

Two proton transfers in the transition state for nucleotidyl transfer catalyzed by RNA- and DNA-dependent RNA and DNA polymerases

Castro

Smidansky

Maksimchuk

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

146

190

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“…Not only do the conserved A, B, and C motifs occur in similar spatial relationships, with conserved residues similarly located, but nearly all the secondary structure elements within the polymerase domain are equivalently located. Mutational studies have demonstrated the importance of Asp537 and Asp8I2, corresponding to the invariant carbox ylates of motifs A and C respectively, and Lys63 1, an invariant residue of motif B in DNA-directed polymerases (48,49). Like the two structures described above, T7 RNA polymerase also seems to be built in a modular fashion, having an N-terminal domain that is structurally unrelated to any of the domains of Klenow fragment or reverse transcriptase, and that appears to carry out functions specific to RNA synthesis.…”

Section: Comparison Of Polymerase Domain Structures In Reverse Transcmentioning

confidence: 99%

Function and Structure Relationships in DNA Polymerases

Joyce¹

1994

Annual Review of Biochemistry

134

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“…[41][42][43] Mutations at the K631 position have been shown to inhibit transcription. 44 Thus, we hypothesized that a sterically demanding caging group installed on K631 will block incoming NTPs from the active site and inhibit T7RNAp activity. In addition, the carbamate linker changes the electronic nature of the ε-amino group, effectively preventing protonation and removing the positive charge.…”

Section: Introductionmentioning

confidence: 99%

Genetically Encoded Light-Activated Transcription for Spatiotemporal Control of Gene Expression and Gene Silencing in Mammalian Cells

et al. 2013

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Photocaging provides a method to spatially and temporally control biological function and gene expression with high resolution. Proteins can be photochemically controlled through the sitespecific installation of caging groups on amino acid side chains that are essential for protein function. The photocaging of a synthetic gene network using unnatural amino acid mutagenesis in mammalian cells was demonstrated with an engineered bacteriophage RNA polymerase. A caged T7 RNA polymerase was expressed in cells with an expanded genetic code and used in the photochemical activation of genes under control of an orthogonal T7 promoter, demonstrating tight spatial and temporal control. The synthetic gene expression system was validated with two reporter genes (luciferase and EGFP) and applied to the light-triggered transcription of short hairpin RNA constructs for the induction of RNA interference.

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Asp537, Asp812 are essential and Lys631, His811 are catalytically significant in bacteriophage T7 RNA polymerase activity

Cited by 79 publications

References 38 publications

Two proton transfers in the transition state for nucleotidyl transfer catalyzed by RNA- and DNA-dependent RNA and DNA polymerases

Two proton transfers in the transition state for nucleotidyl transfer catalyzed by RNA- and DNA-dependent RNA and DNA polymerases

Function and Structure Relationships in DNA Polymerases

Genetically Encoded Light-Activated Transcription for Spatiotemporal Control of Gene Expression and Gene Silencing in Mammalian Cells

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