adenosine-5--o-(3-thiotriphosphate) and sarkosyl

Transcription initiation by RNA polymerase II is a complex, multistep process that minimally involves transcription complex assembly, open complex formation, and promoter clearance. Hydrolysis of the beta--gamma phosphoanhydride bond of ATP has previously been shown to be required for open complex formation, as well as for the phosphorylation of the carboxy-terminal domain of the largest subunit of RNA polymerase II. The observation that ATP-dependent activation of transcription complexes can be blocked by ATP analogues that contain nonhydrolyzable beta--gamma phosphoanhydride bonds (such as ATPgammaS) was exploited to develop a functional kinetic assay for ATP-activated transcription complexes. Activated complexes on the promoter present in the long terminal repeat of the proviral DNA of mouse mammary tumor virus were defined as those that could productively initiate transcription in the presence of excess ATPgammaS. Activation is dependent on treatment of assembled preinitiation complexes with ATP (or dATP) prior to addition of ATPgammaS. At least 15-35% of the total number of preinitiation complexes present become activated within 2 min in the presence of (d)ATP, and activation appears to be rapidly reversible. The time course of formation of activated complexes in the presence of dATP is characterized by two kinetic phases: a rapid formation followed by a relatively slow decay. Activated complexes were estimated to form with a half-time of less than 1 min.

Topics: Adenosine Triphosphate; Cell Nucleus; Deoxyadenine Nucleotides; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Enzyme Stability; Half-Life; HeLa Cells; Humans; Kinetics; Mammary Tumor Virus, Mouse; Promoter Regions, Genetic; RNA Polymerase II; Sarcosine; Transcription, Genetic; Transcriptional Activation

We have investigated the role that ATP plays in the synthesis of accurately initiated transcripts from the adenovirus 2 major late and mouse interleukin-3 promoters by a purified RNA polymerase II transcription system prepared from rat liver. The synthesis of 250-330 nucleotide run-off transcripts and 4-9 nucleotide Sarkosyl-resistant transcription intermediates requires ATP both for RNA synthesis and for activation of the system prior to RNA synthesis. Activation specifically requires an adenine nucleoside triphosphate containing a hydrolyzable beta, gamma-phosphoanhydride bond. ATP, adenine-9-beta-D-arabinofuranoside (araATP), and dATP are potent activators of transcription; they activate transcription to 50% of maximum at 2 microM. ATP analogs containing nonhydrolyzable beta, gamma-phosphoanhydride bonds such as adenyl-5'-yl imidodephosphate, adenosine 5'-(beta, gamma-methylene)triphosphate, and adenosine 5'-O-(thio)triphosphate (ATP gamma S) function efficiently in chain elongation, but do not activate transcription. Furthermore, ATP gamma S is a potent, reversible inhibitor of ATP activation. 20 microM ATP gamma S inhibits the synthesis of both full-length run-off transcripts and sarkosyl-resistant intermediates by 50% when the concentration of ATP is 10 microM. ATP gamma S inhibition can be overcome by high concentrations of ATP, dATP, araATP, or ddATP. Inhibition of the synthesis of Sarkosyl-resistant transcription intermediates by ATP gamma S is prevented by preincubation of the transcription enzymes and DNA template with ATP and magnesium prior to the addition of ATP gamma S and the remaining ribonucleoside triphosphates. Thus we argue that ATP activates the transcription system in a step prior to RNA synthesis.

Topics: Adenosine Triphosphate; Animals; Liver; Mice; Nucleotides; Promoter Regions, Genetic; Rats; RNA; RNA Polymerase II; Sarcosine; Transcription, Genetic; Vidarabine Phosphate