B. Franklin Pugh
Biochemistry of Eukaryotic Transcription Initiation
Our research is aimed at understanding how mammalian transcriptional regulators communicate with RNA polymerase to turn genes on. Our approach is to biochemically dismantle and reconstruct distinct types of transcription complexes, with an eye toward identifying common mechanistic themes.
Our working hypothesis regarding transcription control of a typical mRNA promoter is illustrated in the accompanying figure. RNA polymerase II requires a set of basal initiation factors in order to function at the promoters of mRNA-coding genes. These factors are called TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH. TFIID is a multisubunit complex which appears to interface transcriptional activators with the basal transcription machinery. An important component of TFIID is the TATA binding protein called TBP. The TATA box is a DNA element located 30 nucleotides upstream from the transcriptional start site. It helps assemble and position the transcription complex by binding TFIID, which then recruits the other basal factors and pol II. By itself, the TATA box is poorly suited to recruit TFIID, and so it relies on sequence-specific transcription factors, like Sp1, for help. In this way, Sp1 controls the expression of the gene.
To identify common mechanistic themes, we are exploring different types of transcription complexes. One such system is the RNA polymerase III transcription complex. Pol III transcribes tRNA genes, which utilize downstream promoter elements. TFIIIC binds to the promoter and recruits TFIIIB 30 nucleotide upstream of the transcriptional start site. TFIIIB then recruits pol III. This ordered assembly process is reminiscent of the pol II system. Indeed, TFIIIB appears to be the pol III counterpart of TFIID; it is a multisubunit complex which also contains TBP.
Interestingly, most pol III and pol II promoters lack a TATA box but nevertheless require TBP. How can TBP function without its cognate binding site? Mechanistic studies on TBP reveal that it binds to DNA with extremely high affinity regardless of the underlying sequence. Moreover, TBP can rapidly slide along the DNA like a train on a railroad track. TBP, in effect, appears to anchor transcription complexes to DNA. We envision that promoter elements like TATA, initiators, and GC boxes directly and indirectly help to properly position TBP at the promoter. Indirect positioning occurs through TBP-associated factors (TAFs) which either bind to a specific sequence or interact with a sequence-specific transcription factor like Sp1.
Our rapidly evolving understanding of the eukaryotic transcription machinery now puts us in position to ask basic enzymological questions on the mechanism of transcription initiation and gene regulation. Questions we wish to answer include the following: 1) What are the elementary kinetic steps in transcription initiation? 2) Which step is the rate-limiting? 3) What exactly do activators do when they speed up the slow step? 4) Do activators target more than one step in the initiation process? Answers to these questions and many more, lead us to a greater understanding of how our bodies control our genes, how our genes get out of control as in the case of oncogenesis, and how they are controlled by outside forces such as viruses.