Biomedical subjects

S Shuman

Publications and source records attributed to S Shuman.

At least 145 records · Page 8Linked to original sources

PubMed1995

Characterization of ts 16, a temperature-sensitive mutant of vaccinia virus.

We have characterized a temperature-sensitive mutant of vaccinia virus, ts16, originally isolated by Condit et al. (Virology 128:429-443, 1983), at the permissive and nonpermissive temperatures. In a previous study by Kane and Shuman (J. Virol 67:2689-2698, 1993), the mutation of ts16 was mapped to the I7 gene, encoding a 47-kDa protein that shows partial homology to the type II topoisomerase of Saccharomyces cerevisiae. The present study extends previous electron microscopy analysis, showing that in BSC40 cells infected with ts16 at the restrictive temperature (40 degrees C), the assembly was arrested at a stage between the spherical immature virus and the intracellular mature virus (IMV). In thawed cryosections, a number of the major proteins normally found in the IMV were subsequently localized to these mutant particles. By using sucrose density gradients, the ts16 particles were purified from cells infected at the permissive and nonpermissive temperatures. These were analyzed by immunogold labelling and negative-staining electron microscopy, and their protein composition was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. While the ts16 virus particles made at the permissive temperature appeared to have a protein pattern identical to that of wild-type IMV, in the mutant particles the three core proteins, p4a, p4b, and 28K, were not proteolytically processed. Consistent with previous data the sucrose-purified particles could be labelled with [3H]thymidine. In addition, anti-DNA labelling on thawed cryosections suggested that most of the mutant particles had taken up DNA. On thawed cryosections of cells infected at the permissive temperature, antibodies to I7 labelled the virus factories, the immature viruses, and the IMVs, while under restrictive conditions these structures were labelled much less, if at all. Surprisingly, however, by Western blotting (immunoblotting) the I7 protein was present in similar amounts in the defective particles and in the IMVs isolated at the permissive temperature. Finally, our data suggest that at the nonpermissive temperature the assembly of ts16 is irreversibly arrested in a stage at which the DNA is in the process of entering but before the particle has completely sealed, as monitored by protease experiments.

Animals↗

PubMed1995

The D1 and D12 subunits are both essential for the transcription termination factor activity of vaccinia virus capping enzyme.

Transcription termination by vaccinia virus RNA polymerase during synthesis of early mRNAs requires a virus-encoded termination factor (VTF). VTF is but one of many activities associated with the vaccinia virus mRNA capping enzyme, a heterodimer of 95- and 33-kDa subunits encoded by the D1 and D12 genes, respectively. Although the three catalytic domains involved in cap formation have been assigned to individual subunits or portions thereof, the structural requirements for VTF activity are unknown. We now report that both full-length subunits are required for transcription termination. The 844-amino acid D1 subunit by itself, which is fully active in triphosphatase and guanylyltransferase functions, has no demonstrable VTF activity in vitro. Neither does the D12 subunit by itself. The heterodimeric methyltransferase domain of D1 (residues 498 to 844) and D12 subunits also has no VTF activity. VTF is not affected by a K-to-M mutation of the guanylyltransferase active site at position 260 (K260M) that abolishes enzyme-GMP complex formation or by a H682A/Y683A double mutation of the D1 subunit, which abrogates methyltransferase activity. Thus, the structural requirements for termination are distinct from those for nucleotidyl transfer and methyl transfer.

Base Sequence↗

PubMed1995

Mutational analysis of vaccinia virus nucleoside triphosphate phosphohydrolase II, a DExH box RNA helicase.

Vaccinia virus nucleoside triphosphate phosphohydrolase II (NPH-II), a 3'-to-5' RNA helicase, displays sequence similarity to members of the DExH family of nucleic acid-dependent nucleoside triphosphatases (NTPases). The contributions of the conserved GxGKT and DExH motifs to enzyme activity were assessed by alanine scanning mutagenesis. Histidine-tagged versions of NPH-II were expressed in vaccinia virus-infected BSC40 cells and purified by nickel affinity and conventional fractionation steps. Wild-type His-NPH-II was indistinguishable from native NPH-II with respect to RNA helicase, RNA binding, and nucleic acid-stimulated NTPase activities. The K-191-->A (K191A), D296A, and E297A mutant proteins bound RNA as well as wild-type His-NPH-II did, but they were severely defective in NTPase and helicase functions. The H299A mutant was active in RNA binding and NTP hydrolysis but was defective in duplex unwinding. Whereas the NTPase of wild-type NPH-II was stimulated > 10-fold by polynucleotide cofactors, the NTPase of the H299A mutant was nucleic acid independent. Because the specific NTPase activity of the H299A mutant in the absence of nucleic acid was near that of wild-type enzyme in the presence of DNA or RNA and because the Km for ATP was unaltered by the H299A substitution, we regard this mutation as a "gain-of-function" mutation and suggest that the histidine residue in the DExH box is required to couple the NTPase and helicase activities.

Acid Anhydride Hydrolases↗

PubMed1995

Mutational analysis of mRNA capping enzyme identifies amino acids involved in GTP binding, enzyme-guanylate formation, and GMP transfer to RNA.

Vaccinia virus mRNA capping enzyme is a multifunctional protein with RNA triphosphatase, RNA guanylyltransferase, RNA (guanine-7) methyltransferase, and transcription termination factor activities. The protein is a heterodimer of 95- and 33-kDa subunits encoded by the vaccinia virus D1 and D12 genes, respectively. The capping reaction entails transfer of GMP from GTP to the 5'-diphosphate end of mRNA via a covalent enzyme-(lysyl-GMP) intermediate. The active site is situated at Lys-260 of the D1 subunit within a sequence element, KxDG (motif I), that is conserved in the capping enzymes from yeasts and other DNA viruses and at the active sites of covalent adenylylation of RNA and DNA ligases. Four additional sequence motifs (II to V) are conserved in the same order and with similar spacing among the capping enzymes and several ATP-dependent ligases. The relevance of these common sequence elements to the RNA capping reaction was addressed by mutational analysis of the vaccinia virus D1 protein. Nine alanine substitution mutations were targeted to motifs II to V. Histidine-tagged versions of the mutated D1 polypeptide were coexpressed in bacteria with the D12 subunit, and the His-tagged heterodimers were purified by Ni affinity and phosphocellulose chromatography steps. Whereas each of the mutated enzymes retained triphosphatase, methyltransferase, and termination factor activities, six of nine mutant enzymes were defective in some aspect of transguanylylation. Individual mutations in motifs III, IV, and V had distinctive effects on the affinity of enzyme for GTP, the rate of covalent catalysis (EpG formation), or the transfer of GMP from enzyme to RNA. These results are concordant with mutational studies of yeast RNA capping enzyme and suggest a conserved structural basis for covalent nucleotidyl transfer.

Amino Acid Sequence↗

PubMed1995

Yeast mRNA cap methyltransferase is a 50-kilodalton protein encoded by an essential gene.

RNA (guanine-7-)methyltransferase, the enzyme responsible for methylating the 5' cap structure of eukaryotic mRNA, was isolated from extracts of Saccharomyces cerevisiae. The yeast enzyme catalyzed methyl group transfer from S-adenosyl-L-methionine to the guanosine base of capped, unmethylated poly(A). Cap methylation was stimulated by low concentrations of salt and was inhibited by S-adenosyl-L-homocysteine, a presumptive product of the reaction, but not by S-adenosyl-D-homocysteine. The methyltransferase sedimented in a glycerol gradient as a single discrete component of 3.2S. A likely candidate for the gene encoding yeast cap methyltransferase was singled out on phylogenetic grounds. The ABD1 gene, located on yeast chromosome II, encodes a 436-amino-acid (50-kDa) polypeptide that displays regional similarity to the catalytic domain of the vaccinia virus cap methyltransferase. That the ABD1 gene product is indeed RNA (guanine-7-)methyltransferase was established by expressing the ABD1 protein in bacteria, purifying the protein to homogeneity, and characterizing the cap methyltransferase activity intrinsic to recombinant ABD1. The physical and biochemical properties of recombinant ABD1 methyltransferase were indistinguishable from those of the cap methyltransferase isolated and partially purified from whole-cell yeast extracts. Our finding that the ABD1 gene is required for yeast growth provides the first genetic evidence that a cap methyltransferase (and, by inference, the cap methyl group) plays an essential role in cellular function in vivo.

Amino Acid Sequence↗

PubMed1994

Vaccinia DNA topoisomerase I: kinetic evidence for general acid-base catalysis and a conformational step.

The pH dependences of the internal equilibrium (Kcl) and rate constants for site-specific DNA strand cleavage (kcl) and resealing (kr) catalyzed by Vaccinia DNA topoisomerase I have been investigated using single-turnover conditions in the pH range 4.6-9.8 at 20 degrees C. The pH dependence of the rate constant for strand cleavage (kcl) shows a bell-shaped profile with apparent pKa values of 6.3 +/- 0.2 and 8.4 +/- 0.2, suggesting base catalysis of the attack of the active site Tyr-274 on the phosphodiester phosphorus, and acid catalysis of the expulsion of the 5'-deoxyribose oxygen. A low pKa (i.e., 6.3) for Tyr-274 in the free enzyme is ruled out by NMR titration from pH 5.1 to 8.8 monitoring the C-zeta chemical shift of [zeta-13C]-tyrosine-enriched topoisomerase. The dependence of the internal equilibrium constant (Kcl) on pH reveals very similar pKa values as kcl (5.8 +/- 0.2 and 8.6 +/- 0.2). However, kr is found to be independent of pH. The differing response of kcl and kr to pH rules out a simple two-state internal cleavage equilibrium and suggests that a conformational change occurs following formation of the covalent complex which retains the correct protonation state for strand religation. A conformation step is further indicated by a 4.6-fold "thio effect" on kcl upon substitution of the nonbridging oxygen atom of the attacked phosphoryl group by sulfur [Stivers, J. T., Shuman, S., & Mildvan, A. S. (1994) Biochemistry 33, 327], and the absence of such an effect on kr, (krphos/krthio = 0.9 +/- 0.2), indicating the rates of cleavage and religation to be limited by covalent chemistry and a conformational step, respectively. The rate constant of this conformational change in the direction of religation agrees with the average rate constant for supercoil release from plasmid substrates, suggesting this conformational change to be a part of the topoisomerization step. Although the general acid and general base catalysts have not yet been identified, the quantitative roles of these and other residues in catalysis are discussed.

Base Sequence↗

PubMed1994

Novel approach to molecular cloning and polynucleotide synthesis using vaccinia DNA topoisomerase.

Construction of chimaeric DNA molecules in vitro relies traditionally on two enzymatic steps catalyzed by separate protein components. Site-specific restriction endonucleases are used to generate linear DNAs with defined termini that can then be joined covalently at their ends via the action of DNA ligase. A novel approach to the synthesis of recombinant DNAs exploits the ability of a single enzyme, vaccinia DNA topoisomerase, to both cleave and rejoin DNA strands with extreme specificity at each step. Placement of the CCCTT cleavage motif for vaccinia topoisomerase near the end of a duplex DNA permits efficient generation of a stable, highly recombinogenic protein-DNA adduct that can religate only to acceptor DNAs that contain complementary single-strand extensions. Linear DNAs containing CCCTT cleavage sites at both ends (bivalent substrates) can be activated by topoisomerase and inserted into a plasmid vector in a simple and rapid in vitro procedure that is especially well suited to the molecular cloning of polymerase chain reaction-amplified DNAs. Activation of polyvalent (e.g. branched) DNA substrates by topoisomerase offers a potentially powerful method for the synthesis of two- and three-dimensional polynucleotide networks.

Base Sequence↗

PubMed1994

Vaccinia topoisomerase binds circumferentially to DNA.

Vaccinia DNA topoisomerase, a member of the eukaryotic type I enzyme family, binds duplex DNA and forms a covalent adduct at sites containing a conserved sequence element 5'-CCCTT decreases in the scissile strand. The protein-DNA interface entails essential contacts with four phosphate moieties within the CCCTT motif, including the scissile phosphate, and three phosphates within the GGGAA sequence on the noncleaved strand. Critical protein-phosphate contacts are arrayed across the minor groove of the DNA helix. Base-specific contacts with the pentamer element are within the major groove and are situated on the opposite face of the helix. Thus, vaccinia topoisomerase binds circumferentially to its target site in duplex DNA. This binding mode suggests that the eukaryotic enzyme adopts a toroidal shape in the DNA-bound state. Conformational isomerization of the bound protein provides a plausible mechanism for DNA relaxation.

Base Sequence↗

PubMed1994

Requirements for noncovalent binding of vaccinia topoisomerase I to duplex DNA.

Vaccinia DNA topoisomerase binds duplex DNA and forms a covalent adduct at sites containing a conserved sequence element 5'(C/T)CCTT decreases in the scissile strand. Distinctive aspects of noncovalent versus covalent interaction emerge from analysis of the binding properties of Topo(Phe-274), a mutated protein which is unable to cleave DNA, but which binds DNA noncovalently. Whereas DNA cleavage by wild type enzyme is most efficient with 'suicide' substrates containing fewer than 10 base pairs distal to the scissile bond, optimal noncovalent binding by Topo(Phe-274) requires at least 10-bp of DNA 3' of the cleavage site. Thus, the region of DNA flanking the pentamer motif serves to stabilize the noncovalent topoisomerase-DNA complex. This result is consistent with the downstream dimensions of the DNA binding site deduced from nuclease footprinting. Topo(Phe-274) binds to duplex DNA lacking the consensus pentamer with 7-10-fold lower affinity than to CCCTT-containing DNA.

Base Sequence↗

PubMed1994

Covalent catalysis in nucleotidyl transfer reactions: essential motifs in Saccharomyces cerevisiae RNA capping enzyme are conserved in Schizosaccharomyces pombe and viral capping enzymes and among polynucleotide ligases.

Formation of the 5' cap structure of eukaryotic mRNAs occurs via transfer of GMP from GTP to the 5' terminus of the primary transcript. RNA guanylyltransferase, the enzyme that catalyzes this reaction, has been isolated from many viral and cellular sources. Though differing in molecular weight and subunit structure, the various guanylyltransferases employ a common catalytic mechanism involving a covalent enzyme-(Lys-GMP) intermediate. Saccharomyces cerevisiae CEG1 is the sole example of a cellular capping enzyme gene. In this report, we describe the identification and characterization of the PCE1 gene encoding the capping enzyme from Schizosaccharomyces pombe. PCE1 was isolated from a cDNA library by functional complementation in Sa. cerevisiae. Induced expression of PCE1 in bacteria and in yeast confirmed that the 47-kDa Sc. pombe protein was enzymatically active. The amino acid sequence of PCE1 is 38% identical (152 of 402 residues) to the 52-kDa capping enzyme from Sa. cerevisiae. Comparison of the two cellular capping enzymes with guanylyltransferases encoded by DNA viruses revealed local sequence similarity at the enzyme's active site and at four additional collinear motifs. Mutational analysis of yeast CEG1 demonstrated that four of the five conserved motifs are essential for capping enzyme function in vivo. Remarkably, the same motifs are conserved in the polynucleotide ligase family of enzymes that employ an enzyme-(Lys-AMP) intermediate. These findings illuminate a shared structural basis for covalent catalysis in nucleotidyl transfer and suggest a common evolutionary origin for capping enzymes and ligases.

Amino Acid Sequence↗

PubMed1994

Stimulation of vaccinia topoisomerase I by nucleoside triphosphates.

The rate of relaxation of supercoiled DNA by purified vaccinia topoisomerase I is stimulated 20-fold by 5 mM ATP. A similar effect is elicited by GTP, CTP, UTP, dATP, and adenosine 5'-(beta, gamma-imido)triphosphate. ATP-mediated rate enhancement requires salt as a coactivator. ADP and inorganic pyrophosphate also stimulate relaxation 10-20-fold, whereas AMP and inorganic phosphate have little effect. A model for allosteric activation of topoisomerase by nucleotides is suggested.

Binding Sites↗

PubMed1994

Mutational analysis of vaccinia DNA topoisomerase defines amino acid residues essential for covalent catalysis.

The eukaryotic family of type I DNA topoisomerases includes the nuclear type I enzymes and the enzymes encoded by vaccinia and other poxviruses. The small size of the vaccinia topoisomerase (314 amino acids as compared to 765-972 amino acids for the cellular enzymes) makes it likely that this protein constitutes the minimal functional unit of a eukaryotic type I enzyme and provides an opportunity for a comprehensive structure-function analysis through mutagenesis. Two protein subregions were targeted for mutagenesis in the present study. The role of the Ser-Lys-X-X-Tyr sequence present at the active site of all family members was examined by replacing each conserved residue with alanine. Alanine substitution at the active site Tyr abrogated topoisomerase activity. In contrast, mutations at Ser-270 and Lys-271 had no effect on enzyme activity. The region of the vaccinia topoisomerase from amino acids 126-142 (MFFIRFGKMKYLKENET) is highly conserved and contains a residue, Gly-132, shown previously to be essential. Twenty-nine different mutations were generated in this region, with at least one substitution at each position. Point mutations were identified at three positions, Arg-130, Tyr-136, and Leu-137, which either abrogated or severely reduced DNA relaxation. The effects on activity could be attributed to a defect in formation of the covalent intermediate. Alterations of 13 other amino acids, including conserved residues, had little or no effect on topoisomerase activity.

Alanine↗

PubMed1994

Intrinsic RNA (guanine-7) methyltransferase activity of the vaccinia virus capping enzyme D1 subunit is stimulated by the D12 subunit. Identification of amino acid residues in the D1 protein required for subunit association and methyl group transfer.

Vaccinia virus mRNA capping enzyme, a heterodimer of virus-encoded D1 and D12 subunits, catalyzes three steps in the synthesis of the m7GpppN cap. By expressing portions of the subunits in bacteria, singly and together, we have localized the RNA (guanine-7) methyltransferase domain to a 305-amino acid carboxyl-terminal segment of the D1 polypeptide (residues 540-844) complexed with the D12 protein. We find that the purified carboxyl D1 protein has a weak intrinsic methyltransferase activity, indicating that the catalytic center resides within this subunit. The basal level of activity can be stimulated 100-fold by addition of purified D12 protein, which is itself catalytically inert. The carboxyl region of D1 forms a heterodimer with the D12 subunit in vivo and in vitro. Analysis of alanine substitution mutants of the D1 protein identifies amino acid residues important for subunit interaction. Our results suggest that subunit heterodimerization is necessary, but not sufficient, for full methyltransferase activity. A mutation of vicinal positions His-682-Tyr-683 that specifically affects catalytic activity but not subunit interaction implicates these residues as constituents of the active site.

Amino Acid Sequence↗

PubMed1994

A role for the H4 subunit of vaccinia RNA polymerase in transcription initiation at a viral early promoter.

The vaccinia virus H4 gene encodes an essential subunit of the DNA-dependent RNA polymerase holoenzyme encapsidated within virus particles (Ahn, B., and Moss, B. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 3536-3540; Kane, E. M., and Shuman, S. (1992) J. Virol. 66, 5752-5762). The role of this protein in transcription of viral early genes was revealed by the effects of affinity-purified anti-H4 antibody on discrete phases of the early transcription reaction in vitro. Anti-H4 specifically prevented the synthesis of a 21-nucleotide nascent RNA chain but had no impact on elongation of the 21-mer RNA by preassembled ternary complexes. Inhibition of initiation but not elongation was also observed with affinity-purified anti-D6 antibody directed against the 70-kDa subunit of the vaccinia early transcription initiation factor (ETF). Native gel mobility-shift assays showed that anti-H4 prevented the NTP-dependent recruitment of RNA polymerase to the preinitiation complex of ETF bound at the early promoter. Two species of ternary complexes could be resolved by native gel electrophoresis. Addition of anti-H4 to preformed complexes elicited a supershift of both ternary species but not of the preinitiation complex. Supeshift by anti-D6 revealed that the more rapidly migrating species of ternary complex did not contain immunoreactive ETF. Loss of ETF from the ternary complex was time-dependent. Thus, whereas the H4 protein was a stable constituent of the elongation complex, ETF was dissociable. We suggest that H4 functions as a molecular bridge to ETF and thereby allows specific recognition of early promoters by the core RNA polymerase. H4 is unlike bacterial sigma factor in that it remains bound to polymerase after the elongation complex is established.

Amino Acid Sequence↗

PubMed1994

Mutational analysis of yeast mRNA capping enzyme.

RNA guanylyltransferase (capping enzyme) catalyzes the transfer of GMP from GTP to the 5'-diphosphate end of mRNA. The capping reaction proceeds via an enzyme-guanylate intermediate in which GMP is linked covalently to a lysine residue of the enzyme. In the capping enzyme of Saccharomyces cerevisiae, GMP is attached to a 52-kDa polypeptide, identified as the product of the essential CEG1 gene. The amino acid sequence of the CEG1 protein includes a motif, Lys70-Thr-Asp-Gly, that is conserved at the active site of vaccinia virus RNA guanylyltransferase and which is similar to the KXDG sequence found at the active sites of RNA and DNA ligases. To evaluate the role of this motif in the function of the yeast enzyme, we have expressed the CEG1 protein in active form in Escherichia coli. Replacement of Lys70 or Gly73 with alanine abrogated enzyme-guanylate formation in vitro; in contrast, alanine substitutions at Thr71 or Asp72 merely reduced activity relative to wild-type enzyme. The K70A and G73A mutations were lethal to yeast, whereas yeast carrying the T71A and D72A alleles of CEG1 were viable. These results implicate Lys70 as the active site of yeast guanylyltransferase and provide evidence that cap formation per se is an essential function in eukaryotic cells.

Amino Acid Sequence↗

PubMed1994

Factor-dependent transcription termination by vaccinia RNA polymerase. Kinetic coupling and requirement for ATP hydrolysis.

Transcription termination during synthesis of vaccinia early mRNAs occurs downstream of a UUUUUNU signal in the nascent transcript and requires a virus-encoded termination factor (VTF), which is identical with the vaccinia mRNA capping enzyme. Using purified transcription complexes halted at defined sites on linear DNA templates, we have examined the order and timing of events during a single round of elongation and termination. We find that although cap synthesis occurs by the time the nascent RNA is 31 nucleotides long, capping enzyme is not stably associated with the elongation complex at this stage. Stable interaction, defined by the formation of a termination-competent complex, requires a longer nascent RNA, e.g. 51 nucleotides, but does not depend on prior transcription of the termination signal. The acquisition of termination competence correlates temporally with the physical association of capping enzyme/VTF with the elongation complex, as revealed by UV cross-linking of the capping enzyme large subunit to the nascent RNA chain. Subsequent induction of termination and transcript release by capping enzyme requires energy, specifically the hydrolysis of ATP. The choice of termination site is flexible and is determined by a kinetic balance between the rate of polymerase elongation and the rate of signaling. Signaling rate is related directly to the concentration of hydrolyzable ATP. An apparent lower limit of 33 nucleotides between the 5' boundary of the termination signal and the most proximal termination site implies that the UUUUUNU signal must be extruded from the RNA polymerase before it can be acted upon by VTF. Similarities between VTF-dependent termination and rho-dependent termination underscore an evolutionarily conserved mechanism for RNA signal transduction to the elongating RNA polymerase.

Adenosine Triphosphate↗

PubMed1994

Vaccinia DNA topoisomerase I: single-turnover and steady-state kinetic analysis of the DNA strand cleavage and ligation reactions.

Vaccinia DNA topoisomerase I catalyzes a reversible, site-specific strand cleavage and resealing reaction with duplex DNA involving a transient 3'-phosphotyrosyl linkage between the DNA and Tyr-274 of the enzyme. Single-turnover and steady-state kinetic measurements, as well as DNA binding studies with DNA duplexes containing the preferred cleavage sequence (5'-CCCTT decreases -3') in 50 mM Tris-HCl, pH 7.5, at 20 degrees C, have permitted the evaluation of the individual rate constants for strand cleavage (kcl) and religation (kr) and for duplex DNA binding and dissociation. The values of kcl = 0.07 s-1 and kr = 0.66 s-1 indicate that the internal cleavage equilibrium (Kcl = kcl/kr = 0.1) favors the uncleaved E.DNA complex. The apparent second-order rate constant kcl/KmDNA + = 8 x 10(5) M-1 s-1 for the single-turnover cleavage reaction is 10(2)-10(3)-fold less than the rate of diffusional encounter and provides an estimate of k(on) (DNA). Single-turnover cleavage experiments using a duplex substrate with a nonbridging racemic phosphorothioate nucleotide substitution at the cleavage site showed biphasic cleavage kinetics with equal amplitudes for each phase, which was fit to a double exponential: kfast = 0.01 s-1 and kslow = 0.0004 s-1. These "thio effects" (= kclphos/kclthiol) of 4.6- and 115-fold indicate that cleavage is at least partially rate-limiting in the single-turnover reaction; the two kinetic phases indicate a strong preference for cleavage of one thio isomer. Multiple-turnover cleavage-religation reactions showed an initial pre-steady-state burst proportional to enzyme, followed by a slower steady-state rate with a value of kcat = 0.006 s-1. The phosphorothioate substrate showed a smaller burst and no significant thio effect on kcat. These results indicate fast chemical steps and largely rate-limiting dissociation of the religated product (KD = 54 nM) in the steady-state, a conclusion confirmed by direct measurement of the rate constant for product dissociation as 0.01 s-1. MgCl2 (5 mM) increases this rate constant by an order of magnitude, thus explaining the divalent cation induced acceleration of DNA supercoil relaxation by this enzyme. No divalent cation binding by the enzyme was detected indicating this effect to result from metal binding to DNA. The rate constants kcl and kr for the nucleophilic attack of Tyr-274 and deoxyribose 5'-OH at phosphorus represent approximately 10(9)- and 10(12)-fold enhancements over the analogous attack of phenolate and alcoholate anions at the phosphorus backbone of DNA in solution at pH 7.5. Consistent with this estimate, the Y274F mutant is at least 10(6)-fold less active in cleavage than the wild-type enzyme.

Base Sequence↗

PubMed1993

RNA binding properties of vaccinia virus capping enzyme.

Vaccinia virus capping enzyme, a heterodimer of 95-kDa and 33-kDa subunits, modifies the 5' RNA end and also acts as a transcription termination factor during synthesis of viral early mRNAs. Termination occurs in response to a specific signal, UUUUUNU, in the nascent RNA chain. We now report that purified capping enzyme binds to defined RNAs in solution to form complexes that are stable during native gel electrophoresis. Multiple enzyme molecules can bind to a single RNA. No particular 5' end structure is required for RNA binding, suggesting that the observed protein-RNA interaction is unrelated to the triphosphatase, guanylyltransferase, or methyltransferase functions of capping enzyme. Although binding does not require a UUUUUNU element in the RNA, complex formation is competed preferentially by poly(U) compared to poly(C). Capping enzyme binds to the synthetic 30-mer homopolymers to form a single protein-RNA complex; affinity for U-30 is 10-fold higher than for A-30. The sites of protein-RNA contact, as detected by UV cross-linking, are located predominantly within the 95-kDa capping enzyme subunit, which is itself sufficient to bind and cross-link to RNA in the absence of the small subunit.

Base Sequence↗