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...

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Bibliographic Details
Published in:Biochemical journal 1997-05, Vol.324 ( Pt 1) (1), p.249-253
Main Authors: Rawling, J M, Alvarez-Gonzalez, R
Format: Article
Language:English
Subjects:
Animals
Cattle
DNA-Binding Proteins - isolation & purification
DNA-Binding Proteins - metabolism
Electrophoresis, Polyacrylamide Gel
Humans
Kinetics
Molecular Weight
NAD - metabolism
Poly Adenosine Diphosphate Ribose - metabolism
Poly(ADP-ribose) Polymerases - isolation & purification
Poly(ADP-ribose) Polymerases - metabolism
Recombinant Proteins - metabolism
RNA Polymerase II - isolation & purification
RNA Polymerase II - metabolism
Substrate Specificity
TATA Box
TATA-Box Binding Protein
Thymus Gland - enzymology
Transcription Factor TFIIB
Transcription Factors - isolation & purification
Transcription Factors - metabolism
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Summary: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.
ISSN:0264-6021
1470-8728
DOI:10.1042/bj3240249