TFIIF, a basal eukaryotic transcription factor, is a substrate for poly(ADP-ribosyl)ation.
We have examined the susceptibility of some of the basal eukaryotic transcription factors as covalent targets for poly(ADP-ribosyl)ation. Human recombinant TATA-binding protein, transcription factor (TF)IIB and TFIIF (made up of the 30 and 74 kDa RNA polymerase II-associated proteins RAP30 and RAP74) were incubated with calf thymus poly(ADP-ribose) polymerase and [32P]NAD+ at 37 degrees C. On lithium dodecyl sulphate/PAGE and autoradiography, two bands of radioactivity, coincident with RAP30 and RAP74, were observed. No radioactivity co-migrated with TATA-binding protein or TFIIB. The phenomenon was dependent on the presence of nicked DNA, which is essential for poly(ADP-ribose) polymerase activity. Covalent modification of TFIIF increased with time of incubation, with increasing TFIIF concentration and with increasing NAD+ concentration. High-resolution PAGE confirmed that the radioactive species associated with RAP30 and RAP74 were ADP-ribose polymers. From these observations, we conclude that both TFIIF subunits are highly specific substrates for covalent poly(ADP-ribosyl)ation.
Rawling
J M
JM
Department of Microbiology and Immunology, University of North Texas Health Science Center, 3500 Camp Bowie Blvd., Fort Worth, TX 76107-2699, USA.
Alvarez-Gonzalez
R
R
D000818:Animals; D002417:Cattle; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D011064:Poly Adenosine Diphosphate Ribose / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000302:isolation & purification / Q000378:metabolism*; D012319:RNA Polymerase II / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D016385:TATA Box; D035181:TATA-Box Binding Protein; D013950:Thymus Gland / Q000201:enzymology; D035581:Transcription Factor TFIIB; D014157:Transcription Factors / Q000302:isolation & purification / Q000378:metabolism*
D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D035181:TATA-Box Binding Protein; D035581:Transcription Factor TFIIB; D014157:Transcription Factors; D009243:NAD; D011064:Poly Adenosine Diphosphate Ribose; D011065:Poly(ADP-ribose) Polymerases; D012319:RNA Polymerase II