Transcription is the first step and the key control point in the pathway of gene expression. Transcriptional regulation underlies oncogenesis, development, and other fundamental processes. For example many oncoproteins, including the most common in a wide range of cancers, are transcriptional activator proteins, and many morphogens whose gradients of concentration in early embryos define body form, are transcriptional activator proteins as well. The purpose of the proposed research is to provide a structural basis for understanding the mechanism of transcription, regulation of the process, and altered regulation as occurs in tumor cells.
Regulation of transcription occurs by way of regulatory proteins, which may number in the tens to hundreds for a single promoter. They exert their effects on RNA polymerase and general factors, assembled in a giant "initiation complex" at the promoter. Although most regulation of transcription is thought to occur at the initiation stage of the process, recent evidence suggests that paused elongation complexes are a key and major regulation site as well. It is this paused complex that is substrate for various elongation factors such as TFIIS that can regulate the amount of read through. Indeed, an additional elongation factor, SIII, has recently been shown to be a target of the VHL (Von Hippel-Lindau) tumor-suppressor protein and able to directly regulate its function.
The goal of the proposed research is to determine the x-ray structure at atomic resolution of RNA polymerase II in the midst of transcribing DNA to RNA (ternary complex). The ternary complex is paused at a single base due to starvation of a single nucleotide (UTP) or paused intrinsically due to structural elements inherent in the DNA templates' primary sequence. In both cases, a DNA/RNA/polymerase II complex is formed. The problem is challenging, since the polymerase alone comprises 15 polypeptides with a total mass of nearly 600,000 Daltons, and the generation and purification of homogenous transcribing complex adds an additional factor of complexity. Solution of the problem will require a combination of biochemical studies and electron and x-ray crystallography. Progress has recently been made in the lab of Roger Kornberg in the development of two-dimensional (2-D) protein crystallography and its application to the structure determination of yeast RNA polymerase II by electron microscopy at 16 resolution; growth of 3-D crystals of RNA polymerase II giving x-ray diffraction to 3 resolution; and finally, the applicant's crystallization of a functional paused elongation complex and collection of a complete data set to low resolution. The proposed research will provide a basis for the basic transcription mechanism, pausing of polymerase II on DNA, as well as an understanding of the interactions of polymerase II with DNA and RNA.