December 4-7, 2001
Friiberghs Manor, Örsundsbro and Stockholm University
by Roger D. Kornberg
Department of Structural Biology,
Stanford University School of Medicine, Stanford CA 94305,
USA
A complete RNA polymerase II transcription system has been derived from the yeast Saccharomyces cerevisiae. Fractionation and sequence analysis have identified nearly 60 required polypeptides, as well as components of chromatin-remodeling (SWI/SNF, RSC), complexes. The required polypeptides comprise the 12-subunit RNA polymerase II, multiple "general transcription factors", and a 20-subunit "Mediator". The general transcription factors are responsible for promoter recognition and for melting the DNA template for the initiation of transcription. Mediator makes the key connection between enhancers and promoters. It transduces regulatory information from activator and repressor proteins to RNA polymerase II. There is a perfect one-to-one correspondence and high degree of sequence homology between all subunits of yeast and human polymerases and general transcription factors. Mediator exhibits a high degree of evolutionary conservation as well.
Structural studies of the RNA polymerase II transcription machinery began with electron microscope analysis of two-dimensional protein crystals formed on lipid layers. This led to the derivation of a 10-subunit form of RNA polymerase II especially conducive to crystallization, and to the use of two-dimensional crystals as seeds for the growth of large single crystals for X-ray analysis. The large size of the polymerase, over half a million Daltons, presented unusual technical difficulties, that were overcome by crystal growth and maintenance in anaerobic conditions, crystal shrinkage, and the use of nonstandard heavy atom derivatives. Structure determination at 2.8 Angstroms resolution from multiple crystal forms revealed a division of the polymerase in massive mobile elements, surrounding a nucleic-acid-binding cleft.
Structural studies of the RNA polymerase II transcription machinery began with electron microscope analysis of two-dimensional protein crystals formed on lipid layers. This led to the derivation of a 10-subunit form of RNA polymerase II especially conducive to crystallization, and to the use of two-dimensional crystals as seeds for the growth of large single crystals for X-ray analysis. The large size of the polymerase, over half a million Daltons, presented unusual technical difficulties, that were overcome by crystal growth and maintenance in anaerobic conditions, crystal shrinkage, and the use of nonstandard heavy atom derivatives. Structure determination at 2.8 Angstroms resolution from multiple crystal forms revealed a division of the polymerase in massive mobile elements, surrounding a nucleic-acid-binding cleft.
RNA polymerase II was also crystallized in the form of an actively transcribing complex, containing template DNA and product RNA. The structure of this complex was solved by molecular replacement at 3.3 Angstroms resolution, revealing the DNA entering and unwinding in the active center cleft. Nine base pairs of DNA-RNA hybrid could be seen extending from the active center at nearly right angles to the entering DNA. Changes in protein structure between the transcribing complex and free enzyme include closure of a mobile element termed the "clamp" over the DNA and RNA, and ordering of a series of "switches" at the base of the clamp to create a binding site complementary to the DNA-RNA hybrid. Protein-nucleic acid contacts help explain DNA and RNA strand separation, the specificity of RNA synthesis, "abortive cycling" during transcription initiation, and RNA and DNA translocation during transcription elongation.
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