DNA-activated ATPase activity associated with Xenopus transcription factor A.
Highly purified preparations of Xenopus transcription factor A exhibit DNA-activated ATPase (ATP phosphorylase, EC 3.6.1.3) activity, which is inhibited by affinity-purified anti-factor A antibodies but not by nonspecific gamma-globulins. This enzymatic activity copurifies with both factor A and the 7 S particle, a ribonucleoprotein complex composed of factor A and 5 S RNA in a one-to-one stoichiometric ratio. At equal concentrations of protein, factor A and the 7 S particle catalyze the hydrolysis of ATP to ADP and Pi at similar rates. Kinetic analysis demonstrates that factor A is a fairly typical ATPase with a Vmax of 1.7 nmol/min/mg of protein and a KM of 5.0 X 10(-5) M, whereas the corresponding values for the 7 S particle are 2.7 nmol/min/mg of protein and 1.4 X 10(-4) M, respectively. Besides ATP, dATP is also an effective substrate for the enzyme with a Vmax of 0.7 nmol/min/mg of protein and a KM of 3.3 X 10(-5) M in reactions catalyzed by the 7 S particle. The ATPase activity of free factor A, but not the 7 S particle, can be stimulated approximately 3-fold by the addition of pBR322 plasmid DNA. Proteolytic fragments of factor A generated by treatment of the 7 S particle with papain and trypsin retain a portion of their catalytic activity, 50 and 10%, respectively, in concert with their relative size. Radioactive ATP and dATP can be photocross-linked to factor A by UV irradiation. These radioactive substrates are also cross-linked to the papain- and trypsin-generated fragments with markedly decreased efficiencies. UV photocross-linking of non-substrate nucleotides to factor A was not detectable. These results provide evidence that the ATPase activity is intrinsic to the factor A protein which is essential for the specific initiation of 5 S RNA gene transcription.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000528:radiation effects; D000818:Animals; D004247:DNA / Q000378:metabolism*; D004789:Enzyme Activation; D007700:Kinetics; D008970:Molecular Weight; D010777:Photochemistry; D011487:Protein Conformation; D015534:Trans-Activators; D014157:Transcription Factors / Q000378:metabolism*; D014466:Ultraviolet Rays; D029867:Xenopus Proteins; D014982:Xenopus laevis
C044544:TFAM protein, Xenopus; D015534:Trans-Activators; D014157:Transcription Factors; C097078:XL-MTTFA protein, Xenopus; D029867:Xenopus Proteins; D000255:Adenosine Triphosphate; D004247:DNA; D000251:Adenosine Triphosphatases