Analysis of promoter-transcription factor interactions by site-specific photo-cross-linking

Abstract

Cells depend upon the proper functioning of proteins for cell cycle regulation and controlled cell growth, factors whose malfunction contributes to many human diseases. Transcription of RNA is carried out by DNA-dependent RNA polymerases I, II, and III. Transcription of ribosomal RNA (rRNA) by RNA polymerase (pol) I from DNA is a key point of regulation in the synthesis of ribosomes, the translational machinery for the production of proteins. Ribosomes are composed of rRNA and proteins, the structural and the functional components, respectively. rRNA comprises 75% of the entire RNA in the cell; its transcription is highly active. Our experimental organism is Acanthamoeba castellanii, a free-living amoeba. In A. castellanii, Pol I transcribes the genes which form the 35S rRNA preribosomal transcript. Pol I is recruited to the DNA by protein-protein interactions with Transcription Initiation Factor IB (TIF-IB) to form the initiation complex. TIF-IB has been shown by MPE footprinting to protect the DNA from -17 to -67 relative to the transcription initiation site, or tis (+1). The A. castellanii subunits have been identified as TATA-binding protein (TBP), which is 32kDa in size, and four TBP-associated factors (TAFIS), having relative molecular masses of 145kDa, 99kDa, 96kDa, 91kDa. Proteins can be photo-cross-linked to DNA by site-specific derivatization with an aryl-azido group, which is activated to a nitrene upon exposure to UV light. This results in a covalent cross-link of promoter DNA to the proximal protein subunit. TIF-IB subunits have been partially mapped by photo-cross-linking in the initiation complex before promoter clearance by Pol I by Xiaoliang Gong, a former member of the Paule lab. Using a new, site-specific method, we have completed the cross-linking of TIF-IB in the regions where it had not been done, as well as cross-link Pol I on the promoter in the preinitiation and elongation complexes. The results are similar to cross-linking results with RNA polymerase III, but differ from cross-linking done with RNA polymerase II. We have also worked in collaboration with Mary Robinson in Laurie Stargell's lab. Mary found enhanced DNA-protein interactions with certain combinations of factors, including TBP, TFIIA and TAFII40. We have set out to map these factors, as well as TFIIB and miniTFIIA, on a yeast promoter. TFIIA and TFIIB cross-link specifically on the promoter, whereas TBP and miniTFIIA do not. We have also observed that the addition of the TAFII40/TAFII19 heterodimer enhances cross-linking of TFIIB. Also, the TAFII40/TAFII19 heterodimer has a strong affinity for DNA, while TAFII40 does not cross-link to the DNA.