Synthesis and properties of fluorescent nucleotide substrates for DNA-dependent RNA polymerases.

A new class of fluorescent nucleotide analogs which contain the fluorophore 1-aminonaphthalene-5-sulfonate attached via a gamma-phosphoamidate bond has been synthesized. Both the purine and pyrimidine analogs have fluorescence emission maxima at 460 nm. Cleavage of the alpha-beta-phosphoryl bond produces change in both the absorption and fluorescence emission spectra. The fluorescence of the pyrimidine analogs is quenched; cleavage of the alpha-beta-phosphoryl bond of the UTP analog produces about a 14-fold increase in fluorescence intensity at 500 nm. Under the same conditions the fluorescence of the CTP analog increases about 8-fold, whereas the fluorescence of the purine analogs shows only a slight change. These derivatives are good substrates for Escherichia coli RNA polymerase with only slightly increased Km values and with Vmax values about 50 to 70% that of the normal nucleotides. They are used less efficiently by wheat germ RNA polymerase II. The ATP analog can be used by E. coli RNA polymerase to initiate RNA chains.     

Synthesis and characterization of fluorescent dinucleotide substrate for the DNA-dependent RNA polymerase from Escherichia coli

New fluorescent derivatives of dinucleoside monophosphates, (5'-AmNS)UpA/ApU/GpU/CpA, with a fluorophore, 1-aminonaphthalene-5-sulfonic acid (AmNS), attached to the first nucleotide of the dinucleoside monophosphates via a 5'-secondary amine linkage were synthesized in good yield. The chemical structure of (5'-AmNS)ApU was proved by the phosphodiesterase digestion followed by Whatman No. 3MM paper chromatographic and spectroscopic analysis of the digested products. The ability of these analogs to be incorporated into the 5' terminus of RNA chain forming fluorescent oligonucleotides by Escherichia coli RNA polymerase was studied in the presence of a synthetic DNA template. The enzymatic reaction of (5'-AmNS)UpA and [3H]UTP in the presence of poly(dA-dT) yielded (5'-AmNS)UpAp[3H]U in greater than 30% yield with the Km values of 5 and 2.5 microM and Vmax values of 17 and 25 nmol/min/mg of enzyme for (5'-AmNS)UpA and UpA, respectively. The structure of this fluorescent trinucleotide was identified by RNase A digestion and paper chromatographic analysis of the digested products. (5'-AmNS)UpA or (5'-AmNS)ApU exhibits two absorption maxima around 270 and 340-350 nm and a fluorescent emission maximum at 445 nm when excited at 340 nm. These spectral characteristics permit their use as energy donors for the transfer of energy to the intrinsic cobalt of the cobalt-substituted RNA polymerases. Upon hydrolysis of the phosphodiester bond of these analogs by venom phosphodiesterase, the absorption at 340 and 270 nm increased by 5 and 20%, respectively, while their fluorescence at 445 nm was enhanced by 25%. Thus, these analogs can be used for studying the dynamics of initiation and elongation reactions catalyzed by DNA-dependent RNA polymerases by absorption and fluorescence spectroscopies.     

Fluorescence resonance energy transfer studies on the proximity relationship between the intrinsic metal ion and substrate binding sites of Escherichia coli RNA polymerase

DNA-dependent RNA polymerase from Escherichia coli contains 2 mol of zinc/mol of holoenzyme (alpha 2 beta beta' sigma) with one zinc each in the beta and beta' subunits. A new method to substitute selectively the zinc in the beta subunit was developed by the inactivation of RNA polymerase with 0.25 M NaNO3, 1 M NaCl, 1 mM diaminocyclohexane tetraacetic acid, and 0.1 mM dithiothreitol followed by reconstitution with Co(II), Cd(II), or Cu(II). The hybrid Co-Zn, Cd-Zn, or Cu-Zn RNA polymerase thus obtained retains, respectively, 91, 88, and 50% enzyme activity of the reconstituted Zn-Zn RNA polymerase. Co-Zn RNA polymerase exhibits absorption maxima at 395 and 465 nm, and Cu-Zn RNA polymerase at 637 nm (epsilon = 815 M-1 cm-1). 1-Aminonaphthalene-5-sulfonic acid (AmNS) derivatives of ATP, UTP, and dinucleoside monophosphates (diNMPs), UpA or ApU, were synthesized with AmNS attached to NTP via a gamma-phosphoamidate bond or to diNMPs via a 5'-secondary amine linkage. Since the fluorescence emission maxima of (5'-AmNS)UpA, (gamma-AmNS)ATP, and (gamma-AmNS)UTP at 445, 464, and 464 nm, respectively, when excited at 340 nm, overlap the 465-nm absorption band of Co-Zn RNA polymerase, the spatial relationship between fluorescence substrate analogs and the intrinsic Co(II) in Co-Zn RNA polymerase was studied by fluorescence resonance energy transfer technique. The fluorescence of the initiator, (5'-AmNS)UpA, and elongator, (gamma-AmNS)UTP, of the RNA chain, was quenched 20.3 and 7.1%, by the addition of saturation concentration of Zn-Zn RNA polymerase, and 21.3 and 14.7%, respectively, by the addition of template, poly(dA-dT). The fluorescence of (5'-AmNS)UpA and (gamma-AmNS)UTP was quenched 81.8 and 80.6%, respectively, by the addition of the saturation concentration of Co-Zn RNA polymerase in the absence of template, and 82.7 and 82.9% in the presence of template. On the basis of respective Ro values of 21.3 and 21.9 A for the (5'-AmNS)UpA-Co and (gamma-AmNS)UTP-Co pairs, the distances from Co(II) to the initiation site and to the elongation site were calculated to be 17.4 and 17.5 A, respectively, in the absence and 17.2 and 17.4 A in the presence of template.     

Mechanism of inhibition of Escherichia coli RNA polymerase by captan.

Captan (N-trichloromethylthiocyclohex-4-ene-1,2-dicarboximide) was shown to inhibit RNA synthesis in vitro catalysed by Escherichia coli RNA polymerase. Incorporation of [gamma-32P]ATP and [gamma-32P]GTP was inhibited by captan to the same extent as overall RNA synthesis. The ratio of [3H]UTP incorporation to that of [gamma-32P]ATP or of [gamma-32P]GTP in control and captan-treated samples indicated that initiation was inhibited, but the length of RNA chains being synthesized was not altered by captan treatment. Limited-substrate assays in which re-initiation of RNA chains did not occur also showed that captan had no effect on the elongation reaction. Studies which measured the interaction of RNA polymerase with template DNA revealed that the binding of enzyme to DNA was inhibited by captan. Glycerol-gradient sedimentation of the captan-treated RNA polymerase indicated that the inhibition of the enzyme was irreversible and did not result in dissociation of its subunits. These data are consistent with a mechanism in which RNA polymerase activity was irreversibly altered by captan, resulting in an inability of the enzyme to bind to the template. This interaction was probably at the DNA-binding site on the polymerase and did not involve reaction of captan with the DNA template.     

Anomalous asymmetric kinetics of human red cell hexose transfer: role of cytosolic adenosine 5'-triphosphate

Cytosolic adenosine 5'-triphosphate (ATP) modifies the properties of human red cell sugar transport. This interaction has been examined by analysis of substrate-induced sugar transporter intrinsic fluorescence quenching and by determination of Michaelis and velocity constants for D-glucose transport in red cell ghosts and inside-out vesicles lacking and containing ATP. When excited at 295 nm, human erythrocyte ghosts stripped of peripheral proteins display an emission spectrum characterized by a scattering peak and a single emission peak centered at about 333 nm. Addition of sugar transport substrate or cytochalasin B and phloretin (sugar transport inhibitors) reduces emission peak height by 10% and 5%, respectively. Cytochalasin B induced quenching is a simple saturable phenomenon with an apparent Kd (app Kd) of 60 nM and a capacity of 1.4 nmol of sites/mg of membrane protein. Quenching by D-glucose (and other transported sugars) is characterized by at least two (high and low) app Kd parameters. Inhibitor studies indicate that these sites correspond to sugar efflux and influx sites, respectively, and that both sites can exist simultaneously. ATP induces quenching of stripped ghost fluorescence with half-maximal effects at 20-30 microM ATP. ATP reduces the low app Kd and increases the high app Kd for sugar-induced fluorescence quenching. D-Glucose transport in intact red cells is asymmetric (Km and Vmax for influx less than Km and Vmax for efflux). In addition, two operational Km parameters for efflux are detected in zero- and infinite-trans efflux conditions. Protein-mediated sugar transport in ghosts and inside-out vesicles (IOVs) is symmetric with respect to Km and Vmax for entry and exit, and only one Km for exit is detected. Addition of millimolar levels of ATP to the interior of ghosts or to the exterior of IOVs restores both transport asymmetry and two operational Km parameters for native efflux. A model for red cell hexose transport is proposed in which ATP modifies the catalytic properties of the transport system. This model mimics the behavior of the sugar transport systems of intact cells, ghosts, and inside-out vesicles.     

Kinetics of the interaction of methylene diphosphonic acid and inorganic pyrophosphate with DNA-dependent RNA-polymerase from calf thymus

The kinetics of interaction of PPi and its diphosphonic analog, methylenediphosphonic acid (MDPA), with nucleoside triphosphates, DNA and Mg2+ binding sites of DNA-dependent RNA polymerase II from calf thymus was investigated. The values of apparent Km in the NTP polymerization reaction for ATP and CTP equal to 2.7 X 10(-4) and 1.8 X 10(-4) M, respectively, were determined. It was shown that MDPA and PPi competitively inhibited the RNA polymerase reaction with respect to nucleoside triphosphate. The inhibition constants (Ki) of ATP and CTP incorporation for MDPA were 2.2 X 10(-4) and 3.3 X 10(-4) M, respectively, while those of the nucleoside triphosphate incorporation for PPi were equal to 1.4 X 10(-4) and 2.0 X 10(-4) M, respectively. MDPA and PPi were incompetitive inhibitors of template (DNA) and Mn2+. A possible mechanism of inhibition of the RNA polymerase reaction by MDPA is proposed.     

Synthesis and properties of a new fluorescent analog of ATP: Adenosine-5′-triphosphoro-γ-1-(5-sulfonic acid) napthylamidate

An analog of ATP has been synthesized which contains the fluorophore, 1-aminonapthalene-5-sulfonate attached via a γ-phosphoamidate bond. This analog is strongly fluorescent (quantum yield = 0.63) with an emission maximum at 460 nm; the excited state lifetime is 20 nsec. It is a substrate for DNA-dependent RNA polymerase of $\text{E. coli}$ and wheat germ RNA polymerase II. It is also a substrate for $\text{E. coli}$ valyl t-RNA synthetase, venom phosphodiesterase, and potato apyrase. Cleavage of the α-β phosphoryl bond as a result of RNA synthesis or by venom phosphodiesterase produces a 15 nm red shift in the fluorescence emission spectrum. This property should make this nucleotide useful for studies of the mechanisms of enzymatic reactions involving cleavage of the α-β phosphoryl bond.     

Murine DNA polymerase.alpha-primase initiates RNA-primed DNA synthesis preferentially upstream of a 3'-CC(C/A)-5' motif

We find that the purified murine DNA polymerase.alpha-primase complex exhibits the greatest affinity for DNA templates in which CCC occurs 10 nucleotides downstream of a DNA primase initiation site (Km = 6.6 +/- 0.3 pM). Templates with 3'-CCA-5' at this position are less efficiently utilized (Km = 16 +/- 4 pM). Point mutations that disrupt the 3'-CC(C/A)-5' motif further decrease the affinity for DNA approximately 7-fold (Km = 105 +/- 58 pM). Mutations at the primase start site reduce Vmax 2-fold. Template pyrimidines are required for priming, and initiation with ATP is preferred to initiation with GTP. We conclude that a component of the DNA polymerase.alpha-primase complex recognizes a 3'-CC(C/A)-5' motif in the DNA template, downstream of a primase start site, and that this interaction controls site selection and frequency of initiation by DNA primase.     

Interactions of calf thymus DNA polymerase alpha with primer/templates.

The interactions of calf thymus DNA polymerase alpha (pol alpha) with primer/templates were examined. Simply changing the primer from DNA to RNA had little effect on primer/template binding or dNTP polymerization (Km, Vmax and processivity). Surprisingly, however, adding a 5'-triphosphate to the primer greatly changed its interactions with pol alpha (binding, Vmax and Km and processivity). While changing the primer from DNA to RNA greatly altered the abilit of pol alpha to discriminate against nucleotide analogs, it did not compromise the ability of pol alpha to discriminate against non-cognate dNTPs. Thus the nature of the primer appears to affect 'sugar fidelity', without altering 'base fidelity'. DNase protection assays showed that pol alpha strongly protected 9 nt of the primer strand, 13 nt of the duplex template strand and 14 nt of the single-stranded template from hydrolysis by DNase I and weakly protected several bases outside this core region. This large DNA binding domain may account for the ability of a 5'-triphosphate on RNA primers to alter the catalytic properties of pol alpha.     

Inhibitory effects of 9-beta-D-xylofuranosyladenine 5'-triphosphate on DNA-dependent RNA polymerase I and II from cherry salmon (Oncorhynchus masou)

In order to determine the mode of action of cytostatic 9-beta-D-xylofuranosyladenine (xylo-A), the inhibitory effects of 9-beta-D-xylofuranosyladenine 5'-triphosphate (xylo-ATP) on DNA-dependent RNA polymerases I and II purified from cherry salmon (Oncorhynchus masou) liver nuclei were studied. This nucleotide showed strong inhibitory action on both RNA polymerases I and II. The K1 values are 14 microM for polymerase I and 5 microM for polymerase II (Km values of ATP are 37 microM for polymerase I and 40 microM for polymerase II). The mode of xylo-ATP was competitive with respect to the incorporation of AMP into RNA and non-competitive to UTP and CTP.     

Kinetics and template-dependency of ribonucleic acid synthesis by bacterial ribonucleic acid polymerase.

The rate of RNA synthesis catalysed by DNA-dependent RNA polymerase shows a Michealis-Menten-type saturation curve with increasing template concentration. However, the apparent Km is proportional to enzyme concentration, indicating that the reaction does not obey a simple kinetic scheme. The action of inhibitors also indicates a more complex interaction between the enzyme and the DNA template; many inhibitors of RNA synthesis either decrease Vmax. without affecting Km, or increase Km without affecting Vmax. All of these observations can be accounted for quantitatively by a reaction pathway in which the non-specific binding sites of the viral DNA template inhibit competitively the binding of the enzyme to the initiation sites. In terms of this pathway the two classes of inhibitors of RNA synthesis must then act predominantly either on the rate of elongation or on the availability of the binding sites respectively.     

Spatial relationship between the intrinsic metal in the beta subunit and cysteine-132 in the sigma subunit of Escherichia coli RNA polymerase: a resonance energy transfer study

Fluorescence excited-state energy transfer measurements were carried out between the N-(1-pyrene)maleimide (PM)-labeled sigma subunit and Co in the beta subunit of Co-Zn RNA polymerase (RPase). sigma subunit with or without PM labeling was cleaved with 2-nitro-5-thiocyanobenzoic acid, and the reaction products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One molecule of the fluorescent probe (PM) was found to be attached to the cysteine-132 residue of the sigma subunit. When excited at 340 nm, the fluorescence emission bands from 380 to 420 nm of PM-labeled sigma overlap with the charge transfer absorption band of Co-Zn RPase around 400 nm. Based on F?rster's equation, the R0 values for the donor-acceptor pair were calculated to be 21.5 and 22 A in the absence and presence of template analog (dA-dT)60, respectively. Using these R0 values and the observed energy transfer efficiencies, the distance between the cysteine-132 of the sigma subunit and Co located at the initiation site of the beta subunit was calculated to be 22 A with or without the template present, indicating that no major conformational change of the enzyme was induced upon template binding. However, a small but significant change in the above distance was observed upon the addition of ATP to RPase in the presence (dA-dT)60 but not in the absence of (dA-dT)60 template. The biological implications of these observations are discussed.     

DNA synthesis in vitro with an endoplasmic-reticulum-DNA-polymerase complex from unfertilized sea urchin eggs

An endoplasmic-reticulum-DNA-polymerase complex was prepared from unfertilized sea urchin eggs and its DNA-synthesizing activity was examined using single-stranded DNA of bacteriophage fd as a template. The complex catalyzed the ribonucleotide-dependent DNA synthesis which required dNTPs, NTPs, Mg2+ and single-stranded DNA. The DNA synthesis was sensitive to aphidicolin and N-ethylmaleimide but was resistant to 2',3'-dideoxyribosylthymine 5'-triphosphate (ddTTP) and alpha-amanitin, suggesting the involvement of DNA polymerase alpha. In parallel with the DNA synthesis, a small amount of RNA was synthesized in the presence of 100 micrograms/ml alpha-amanitin. The Km value of ribonucleotides for the RNA synthesis coincided with that for the DNA synthesis, suggesting a correlation between the DNA and RNA syntheses. Labelling of the products with [gamma-32P]ATP followed by DNA digestion with pancreatic DNase I revealed the attachment of an oligoribonucleotide (7-11 bases in length) at the 5' ends of the DNA products. These observations suggest that in DNA synthesis, primer RNA synthesis occurs first, followed by DNA chain elongation. During 1-90-min incubation, the amount of the DNA synthesized increased but the length was not significantly increased. Over 80% of the number of synthesized DNA molecules comprised a single population of short DNA fragments (60-200 bases, on average 120 bases in length) and the number of fragments increased, depending on the incubation time. However, DNA fragments of various sizes (about 100-6000 bases) were synthesized with DNA polymerase alpha solubilized from the endoplasmic-reticulum-DNA-polymerase complex. All this evidence suggests that in vitro, the complex preferentially synthesizes a particular size of short DNA fragments. The significance of the fragments is discussed.