RNA Polymerase Unveiled

Multisubunit RNA polymerases (RNAPs), whose core subunits are conserved throughout evolution, synthesize essentially all cellular RNA molecules. Understanding the mechanism and regulation of RNAP has been a key goal of molecular biologists since the discovery of the enzyme by Samuel Weiss in the late 1950s. Three papers in this issue of Cell from Seth Darst's and Roger Kornberg's groups mark an epochal transition in the field from conceptual modeling based on biochemical probes, to analysis at three-dimensional, atomic resolution. This long-sought view is the culmination of efforts begun nearly two decades ago by Kornberg to overcome the daunting challenges posed by the size and complexity of RNAPs (the similar to 400 kDa core RNAP is beta ', beta , and two smaller alpha subunits in bacteria; the even larger eukaryotic RNAPs contain eight or more additional small subunits). Kornberg developed methods to form 2-D crystals of soluble proteins on lipid bilayers and then, joined by Darst, first produced low-resolution electron crystallographic structures of RNAP around 1990. Working independently, their groups now have delivered three new structures of multisubunit RNAPs. Using S. cerevisiae RNA polymerase II (yRNAPII), Kornberg's group has generated a 6 Ae X-ray diffraction map of the enzyme alone and a lower resolution EM structure of its active form complexed with DNA and nascent RNA transcript. This required finding ways to crystallize yRNAPII (by deleting the genes that encode two small subunits, Rpb4 and Rpb7, and excluding oxygen) and finding a way to surpass the typical 10 Ae phasing limit of the only heavy metal compound (W sub(18)) that did not eliminate high-resolution diffraction from yRNAPII crystals. Using the core RNAP from the bacterium Thermus aquaticus (Taq), Darst's group has solved the first high-resolution structure (3.3 Ae) of a multisubunit RNAP. This impressive achievement reveals the positions of the alpha carbon backbone and most key amino acid side chains in a protein of complexity beyond that of any high-resolution crystal structure heretofore reported.