Synthesis and characterization of 5-[(4-Azidophenacyl)thio]uridine 5'-triphosphate, a cleavable photo-cross-linking nucleotide analogue.

The synthesis, isolation, and characterization of a new photo-cross-linking uridine 5'-triphosphate analogue are described. This nucleotide analogue, 5-[(4-azidophenacyl)thio]uridine 5'-triphosphate (5-APAS-UTP), contains an aryl azide group approximately 10 A from the uridine ring. The azide is photoactivated by irradiation at 300 nm, resulting in covalent attachment of the nucleotide to adjacent molecules. The nucleotide can be desulfurated with Raney nickel to cause molecular cleavage between the base and the aryl azide. Desulfuration yields uridine 5'-triphosphate and p-azidoacetophenone. If the analogue is cross-linked to another molecule, desulfuration leaves the analogue's acetophenone group attached to that molecule. This effectively leaves behind a molecular tag on molecules that interact with the uridine analogue either as monomeric nucleotide or as part of an RNA molecule. This nucleotide analogue can be incorporated into internal positions in RNA by transcription in vitro with Escherichia coli RNA polymerase. It can therefore be used to examine interactions between RNA and other molecules (e.g., proteins or nucleic acids). Because the sulfur atom can be selectively removed, the covalent bonds formed between analogue-containing RNA and other molecules can be cleaved, when desired, to facilitate identification of the cross-linked molecules and RNA nucleotides in the cross-linked complex.

Structure determination of the UDP-disaccharide fragment of cytoplasmic cofactor isolated from Methanobacterium thermoautotrophicum.

The methylcoenzyme M methylreductase reaction has an absolute requirement for 7-mercaptoheptanoylthreonine phosphate or component B, which is the active component of the intact molecule previously referred to as cytoplasmic cofactor. A hydrolytic fragment of cytoplasmic cofactor has been purified and identified as uridine 5'-(O-2-acetamido-2-deoxy-beta-manno-pyranuronosyl acid (1----4)-2-acetamido-2-deoxy-alpha-glucopyranosyl diphosphate) by high resolution NMR and fast atom bombardment mass spectro-metry. It is postulated that UDP-disaccharide may function to anchor 7-mercaptoheptanoyl threonine phosphate at the active site of the methyl-reductase enzyme complex.

Binding interactions between yeast tRNA ligase and a precursor transfer ribonucleic acid containing two photoreactive uridine analogues.

Yeast tRNA ligase, from Saccharomyces cerevisiae, is one of the protein components that is involved in the splicing reaction of intron-containing yeast precursor tRNAs. It is an unusual protein because it has three distinct catalytic activities. It functions as a polynucleotide kinase, as a cyclic phosphodiesterase, and as an RNA ligase. We have studied the binding interactions between ligase and precursor tRNAs containing two photoreactive uridine analogues, 4-thiouridine and 5-bromouridine. When irradiated with long ultraviolet light, RNA containing these analogues can form specific covalent bonds with associated proteins. In this paper, we show that 4-thiouridine triphosphate and 5-bromouridine triphosphate were readily incorporated into a precursor tRNA(Phe) that was synthesized, in vitro, with bacteriophage T7 RNA polymerase. The analogue-containing precursor tRNAs were authentic substrates for the two splicing enzymes that were tested (endonuclease and ligase), and they formed specific covalent bonds with ligase when they were irradiated with long-wavelength ultraviolet light. We have determined the position of three major cross-links and one minor cross-link on precursor tRNA(Phe) that were located within the intron and near the 3' splice site. On the basis of these data, we present a model for the in vivo splicing reaction of yeast precursor tRNAs.

AraUTP-Affi-Gel 10: a novel affinity absorbent for the specific purification of DNA polymerase alpha-primase.

For the specific purification of eukaryotic DNA-dependent DNA polymerase alpha, we prepared two novel affinity resins bearing 5-(E)-(4-aminostyryl) araUTP as a ligand. One of them was araUTP-Sepharose 4B which was coupled directly with the ligand and the other was araUTP-Affi-Gel 10 which was coupled with the ligand through a spacer. No DNA polymerase alpha-primase activity from cherry salmon (Oncorhynchus masou) testes was bound on the araUTP-Sepharose 4B in all cases examined. On the other hand, the araUTP-Affi-Gel 10 retains this enzyme activity when poly(dA) or poly(dA)-oligo(dT)12-18 is present. The retained enzyme activity was sharply eluted around 100-mM KCl concentrations as a single peak, and this fraction showed a specific activity of about 170,000 units/mg as alpha-polymerase activity. The highly purified DNA polymerase alpha-primase isolated using the araUTP-Affi-Gel 10 contained only three polypeptides, which showed Mr values of 120,000, 62,000, and 58,000, respectively, as judged using sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

p-(Bromoacetamido)phenyl uridyl pyrophosphate: an active-site-directed irreversible inhibitor for uridine diphosphate galactose 4-epimerase.

The synthesis of p-(bromoacetamido)phenyl uridyl pyrophosphate (BUP) is described. This compound is an active-site-directed irreversible inhibitor of Escherichia coli UDP-galactose 4-epimerase. The inactivation follows pseudo-first-order kinetics at pH 8.5 in nonnucleophilic buffers, and a saturation effect is seen in the pseudo-first-order rate constant as the concentration of BUP is increased. The half-saturation parameter for BUP in the inactivation is 0.21 +/- 0.02 mM, which compares favorably with the inhibition constant of 0.3 +/- 0.05 mM for BUP acting as a competitive reversible inhibitor of the enzyme. The inactivation rate is slow, however, with a minimum half-time of 12 h at pH 8.5 and 27 degrees C. Both specific alkylation and nonspecific alkylation by BUP occur, but nonspecific alkylation is faster than the inactivation and the rate of inactivation correlates well with the rate of covalent incorporation of one molecule of [14C]BUP at the active site.

Recognition of structure of 5-halogenated derivatives of ara-UTP by DNA polymerase gamma and reverse transcriptase.

This report deals with the test of a series of 5-halogenated derivatives of ara-UTP for the inhibition of DNA polymerase gamma and viral reverse transcriptase. The compounds newly synthesized and tested were; ara5-FUTP, ara5-C1UTP, ara5-BrUTP and ara5-IUTP. The results were: 1) All these compounds were inhibitory to DNA polymerase gamma and reverse transcriptase. The mode of inhibitions was, in all cases, competitive against dTTP. 2) Ki values for these inhibitors with DNA polymerase gamma were lower than those with reverse transcriptase. 3) Ara5-ClUTP was less inhibitory to reverse transcriptase than other derivatives.

RNA synthesis in isolated nuclei: the use of mercurated nucleotides.

We have used uridine 5' triphosphate-5-mercury (Hg-UTP) in place of UTP to study RNA synthesis in a previously described isolated nuclei system (1). Employing isopycnic density gradient centrifugation to separate RNAs based upon their relative content of Hg-U, several conclusions can be drawn. In vitro RNA synthesis consists of end addition onto pre-initiated HnRNA molecules as well as apparent initiation of new HnRNA molecules de novo. Synthesis in our system continues linearly for greater than two hours. The chain elongation rate has been measured to be about 500 nucleotides per minute. The methods used to make these measurements are generally applicable to other in vitro systems.

Steric and charge factors in the resistance of uridylyl(3' - 5')N6-(N-threonylcarbonyl)adenosine to venom phosphodiesterase hydrolysis.

The contribution of steric and negative charge factors to the resistance of uridylyl(3' - 5')N6-(N-threonylcarbonyl)adenosine to venom phosphodiesterase was investigated. The hydrolysis rates of uridylyl(3'-5')N6-(N-threonylcarbonyl)-adenosine, its model derivatives, methyl ester and O-benzyl ester, together with unmodified uridyly (3'-5')adenosine, were studied. It was found that the contribution of both factors is of the same order. The steric inhibition of digestion is distinctly higher than that confirmed by N6-(delta2-isopentenyl)adenosine [1], which is ascribed to the rigid conformation of the threonylcarbonyladenosine side chain.

[Addition of the fluorescent label to the 3'-OH end of DNA and the 3'-OH end of nascent RNA]. / Vvedenie fluorestsentnoi metki na 3'OH-konets DNK i 3'-OH-konets rastushchei tsepi RNK.

3'(2')-O-acyl derivatives of the uridine triphosphate were synthesized. Acyl residues contained fluorescent dye; fluoresceine or rodamine C. Optical properties and stability of UTP analogues were studied. Their ability to serve as the substrates for calf thymus terminal deoxyribonucleotidyl transferase and E. coli RNA polymerase was also examined. It was shown that both enzymes were able to use tested analogues as substrates. Incorporation of the analogues into nascent RNA and DNA chains inhibited the synthetic reaction because of primer inactivation. The rate of the incorporation of the analogues showed an exponential time dependence