A-Young Moon Woody

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

To define catalytically essential residues of bacteriophage T7 RNA polymerase, we have generated five mutants of the polymerase, D537N, K631M, Y639F, H811Q and D812N, by site-directed mutagenesis and purified them to homogeneity. The choice of specific amino acids for mutagenesis was based upon photoaffinity-labeling studies with 8-azido-ATP and homology comparisons with the Klenow fragment and other DNA/RNA polymerases. Secondary structural analysis by circular dichroism indicates that the protein folding is intact in these mutants. The mutants D537N and D812N are totally inactive. The mutant K631M has 1% activity, confined to short oligonucleotide synthesis. The mutant H811Q has 25% activity for synthesis of both short and long oligonucleotides. The mutant Y639F retains full enzymatic activity although individual kinetic parameters are somewhat different. Kinetic parameters, (kcat)app and (Km)app for the nucleotides, reveal that the mutation of Lys to Met has a much more drastic effect on (kcat)app than on (Km)app, indicating the involvement of K631 primarily in phosphodiester bond formation. The mutation of His to Gln has effects on both (kcat)app and (Km)app; namely, three- to fivefold reduction in (kcat)app and two- to threefold increase in (Km)app, implying that His811 may be involved in both nucleotide binding and phosphodiester bond formation. The ability of the mutant T7 RNA polymerases to bind template has not been greatly impaired. We have shown that amino acids D537 and D812 are essential, that amino acids K631 and H811 play significant roles in catalysis, and that the active site of T7 RNA polymerase is composed of different regions of the polypeptide chain. Possible roles for these catalytically significant residues in the polymerase mechanism are discussed.

Thermal and urea-induced unfolding in T7 RNA polymerase: calorimetry, circular dichroism and fluorescence study.

Structural changes in T7 RNA polymerase (T7RNAP) induced by temperature and urea have been studied over a wide range of conditions to obtain information about the structural organization and the stability of the enzyme. T7RNAP is a large monomeric enzyme (99 kD). Calorimetric studies of the thermal transitions in T7RNAP show that the enzyme consists of three cooperative units that may be regarded as structural domains. Interactions between these structural domains and their stability strongly depend on solvent conditions. The unfolding of T7RNAP under different solvent conditions induces a highly stable intermediate state that lacks specific tertiary interactions, contains a significant amount of residual secondary structure, and undergoes further cooperative unfolding at high urea concentrations. Circular dichroism (CD) studies show that thermal unfolding leads to an intermediate state that has increased β‐sheet and reduced α‐helix content relative to the native state. Urea‐induced unfolding at 25°C reveals a two‐step process. The first transition centered near 3 M urea leads to a plateau from 3.5 to 5.0 M urea, followed by a second transition centered near 6.5 M urea. The CD spectrum of the enzyme in the plateau region, which is similar to that of the enzyme thermally unfolded in the absence of urea, shows little temperature dependence from 15° to 60°C. The second transition leads to a mixture of poly(Pro)II and unordered conformations. As the temperature increases, the ellipticity at 222 nm becomes more negative because of conversion of poly(Pro)II to the unordered conformation. Near‐ultraviolet CD spectra at 25°C at varying concentrations of urea are consistent with this picture. Both thermal and urea denaturation are irreversible, presumably because of processes that follow unfolding.

Spectroscopic studies of Congo red binding to RNA polymerase

The azo dye Congo Red has a high affinity for nucleotide-binding enzymes. We have studied the binding of Congo Red to RNA polymerase by circular dichroism (CD) and difference absorption spectroscopy, steady-state kinetics, and nitrocellulose filter-binding. Induced CD shows that a large number of Congo Red molecules bind to the holoenzyme. CD also demonstrates that the core enzyme at low ionic strengths has a distinctive Congo Red binding site which is not present in the holoenzyme, nor in the core enzyme at higher ionic strengths or in the presence of poly(dT). CD studies indicate that Congo Red can readily displace double-stranded polynucleotides (T7 DNA or poly[d(A-T)] from RNA polymerase. Single-stranded DNA (poly(dT) and T7 DNA in open complexes) is not displaced from RNA polymerase except at high Congo Red concentrations. Both kinetics and nitrocellulose filter-binding measurements support this conclusion. Difference spectra indicate that the bound Congo Red molecules undergo stacking. We postulate that RNA polymerase binds Congo Red in a region with which a segment of DNA normally interacts, and that Congo Red is a potent inhibitor because the stacked dye has a polyanionic character.

Mapping of the active site of T7 RNA polymerase with 8-azidoATP.

The photoaffinity analog of ATP, 8-azidoATP, labels T7 RNA polymerase. Photoincorporation exhibits saturation behavior and is protected against by the substrate ATP. 8-AzidoATP is a competitive inhibitor of ATP incorporation with Ki approximately 40 microM. The photolabeled T7 RNA polymerase, following cyanogen bromide digestion, was analyzed by phenylboronate agarose column chromatography followed by reverse-phase high pressure liquid chromatography. Sequencing of the peptides labeled with radioactive photoprobe allowed the identification of three peptides, P314-M362 (I), L550-M666 (II), and F751-M861 (III). These peptides are in the proximity of the photoprobe 8-azidoATP and, therefore, expected to contain functionally significant residues and define an active site domain. These peptides (I and II) contain residues previously implicated in T7 RNA polymerase activity or show homology to active site regions of the Klenow fragment of DNA polymerase I (II and III).

Effects of ppGpp on transcription by DNA-dependent RNA polymerase from Escherichia coli: circular dichroism, absorption and specific transcription studies

Concrete evidence is presented for conformational changes elicited in RNA polymerase upon binding ppGpp by circular dichroism measurements. In the presence of 100 microM ppGpp, the molar ellipticity of RNA polymerase at 220 nm is reduced by 14% from the initial value of - 11,100 deg X cm2 X dmol-1 at 25 degrees C. In vitro transcription on templates containing the beta-lactamase promoter and colicin E1 promoter on poly[d(A-T)] is inhibited by ppGpp. None of these templates had GC-rich nucleotide sequence near the transcription initiation site, and yet they were influenced by ppGpp. Comparison of the effect on the synthesis of mRNAs for beta-lactamase and colicin E1 and the synthesis of the proteins themselves indicates that the effect of ppGpp is at the level of transcription for the former case and involves coupled transcription-translation for the latter case. Difference absorption, polyacrylamide gel electrophoresis, and nitrocellulose filter-binding studies show that the binding of ppGpp to RNA polymerase does not impair the extent of the interaction between enzyme and DNA. Kinetic studies suggest that ppGpp affects transcription initiation on beta-lactamase promoter. On poly[d(A-T)], ppGpp affects the rate of open complex formation and is a mixed inhibitor with respect to the incorporation of nucleotides. Our results are consistent with the idea that ppGpp acts as a regulator by binding at a site different from the active site and changes the RNA polymerase conformation, causing altered transcriptional behavior on different DNA templates.

Spectroscopic analysis of DNA base-pair opening by Escherichia coli RNA polymerase. Temperature and ionic strength effects

The interaction of Escherichia coli RNA polymerase with poly[d(A-T)] and poly[d-(I-C)] was studied by difference absorption spectroscopy at temperatures, from 5 to 45 degrees C in the absence and presence of Mg2+. The effect of KCl concentration, at a fixed temperature, was studied from 12.5 to 400 mM. Difference absorption experiments permitted calculation of the extent of DNA opening induced by RNA polymerase and estimation of the equilibrium constant associated with the isomerization from a closed to an open RNA polymerase-DNA complex. delta H0 and delta S0 for the closed-to-open transition with poly[d(A-T)] or poly[d(I-C)] complexed with RNA polymerase are significantly lower than the values associated with the helix-to-coil transition for the free polynucleotides. For the RNA polymerase complexes with poly[d(A-T)] and poly[d(I-C)] in 50 mM KCl, delta H0 approximately 15-16 kcal/mol (63-67 kJ/mol) and delta S0 approximately 50-57 cal/K per mol (209-239 J/K per mol). The presence of Mg2+ does not change these parameters appreciably for the RNA polymerase-poly[d(A-T)] complex, but for the RNA polymerase-poly[d(I-C)] complex in the presence of Mg2+, the delta H0 and delta S0 values are larger and temperature-dependent, with delta H0 approximately 22 kcal/mol (92 kJ/mol) and delta S0 approximately 72 cal/K per mol (approx. 300 J/K per mol) at 25 degrees C, and delta Cp0 approximately 2 kcal/K per mol (approx. 8.3 kJ/K per mol). The circular dichroism (CD) changes observed for helix opening induced by RNA polymerase are qualitatively consistent with the thermally induced changes observed for the free polynucleotides, supporting the difference absorption method. The salt-dependent studies indicate that two monovalent cations are released upon helix opening. For poly[d(A-T)], the temperature-dependence of enzyme activity correlates well with the helix opening, implying this step to be the rate-determining step. In the case of poly[d(I-C)], the same is not true, and so the rate-determining step must be a process subsequent to helix opening.

The interaction of RNA polymerase and DNA Effects on the helix-coil transition and light scattering

To characterize the interactions of RNA polymerase with DNA, we have investigated the thermal transition of poly[d(A-T] bound to RNA polymerase from Escherichia coli and the aggregation properties of the enzyme with DNA. The melting curve of the DNA-enzyme complex demonstrates a sharply lowered melting temperature for part of the DNA, whereas for another fraction the double helix is stabilized. This indicates that the DNA-binding site of RNA polymerase serves two functions: (1) to disrupt the double helix at one point, and (2) to maintain the duplex form at other points. The aggregation of DNA and RNA polymerase has been monitored by turbidity measurements, and conditions have been delineated under which aggregation is minimized. Holoenzyme added to double-stranded DNA or single-stranded DNA has little or no tendency to aggregate under most conditions. Core enzyme, on the other hand, aggregate extensively with double-stranded DNA, the only under conditions of low salt (10 mM KCl), without Mg2+, or at high salt (300 mM KCl), with or without Mg2+, can this aggregation be eliminated. Core enzyme also does not aggregate in the presence of single-stranded DNA. These aggregation properties are interpreted as evidence for more than one DNA-binding site on RNA polymerase.