I propose to develop general procedures for the identification and purification of specific DNA binding proteins, in particular those involved in the transcription of the SV40 genome by human RNA polymerase II by the use of the photoaffinity labelling techniques which employ specifically modified DNA duplexes that represent appropriate protein binding domains. I will first develop chemical procedures for the unambiguous introduction of a photo-reactive phenyl azide group anywhere in a DNA template by using novel reagents, compatible with automated DNA synthesizer, for the synthesis of modified oligonucleotides. The chemical linker employed to attach radioactive phenyl azide group would be constructeed (incorporating a disulfide or ester linkage) to be rapidly cleaved after photochemical crosslinking thus transferring the radiolabel from DNA to protein. A systematic study of the placement of photo-reactive group and the length of the linker arm by which the azide group is attached to the duplex will be carried out to establish optimum labelling strategy. Such radiolabelled photo-reactive DNA probes, representing various protein binding domains of the SV40 enhancer region or the TATA box promoter region, would be synthesized and then photochemically crosslinked to the specific binding proteins in extracts from HeLa cells. These transcription proteins, after radiolabel transfer from the DNA, would then be identified by gel electrophoresis, isoelectric focusing and by tryptic mapping analysis. Affinity chromatographic procedures, for the purification of specific transcription proteins, will be developed by attaching synthetic DNA duplexes (representing individual protein binding domains) to cellulose, either by direct chemical linkage or via a cellulose-biotin-avidin-biotinylated DNA complex. The transcription proteins specifically retained on these affinity columns will be eluted under appropriate conditions. The transcription factor associated with the DNA dependent ATPase activity will also be identified by photochemical crosslinking with dATP or AMP-PNP analogs and purified by AMP-PNP based affinity chromatography. Finally, the purified transcription proteins will be used to reconstruct an in vitro transcription system in which the transcription of genes under the control of SV40 or other viral promoter would be studied. We anticipate that our studies will also help in further defining the general mechanism(s) of eucaryotic RNA transcription.
NIH ExPorter
NIH ExPorter