ABSTRACT
The crystal structure of RNA polymerase II in the act of transcription was determined at 3.3 A resolution. Duplex DNA is seen entering the main cleft of the enzyme and unwinding before the active site. Nine base pairs of DNA-RNA hybrid extend from the active center at nearly right angles to the entering DNA, with the 3' end of the RNA in the nucleotide addition site. The 3' end is positioned above a pore, through which nucleotides may enter and through which RNA may be extruded during back-tracking. The 5'-most residue of the RNA is close to the point of entry to an exit groove. Changes in protein structure between the transcribing complex and free enzyme include closure of a 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.
Subject(s)
DNA, Fungal/chemistry , RNA Polymerase II/chemistry , RNA Polymerase II/metabolism , RNA, Fungal/chemistry , RNA, Messenger/chemistry , Saccharomyces cerevisiae/enzymology , Transcription, Genetic , Base Pairing , Base Sequence , Binding Sites , Crystallography, X-Ray , DNA, Fungal/metabolism , Metals/metabolism , Models, Genetic , Models, Molecular , Molecular Sequence Data , Nucleic Acid Conformation , Protein Conformation , Protein Structure, Quaternary , Protein Structure, Secondary , Protein Structure, Tertiary , RNA, Fungal/biosynthesis , RNA, Fungal/metabolism , RNA, Messenger/biosynthesis , RNA, Messenger/metabolism , Saccharomyces cerevisiae/geneticsABSTRACT
A backbone model of a 10-subunit yeast RNA polymerase II has been derived from x-ray diffraction data extending to 3 angstroms resolution. All 10 subunits exhibit a high degree of identity with the corresponding human proteins, and 9 of the 10 subunits are conserved among the three eukaryotic RNA polymerases I, II, and III. Notable features of the model include a pair of jaws, formed by subunits Rpb1, Rpb5, and Rpb9, that appear to grip DNA downstream of the active center. A clamp on the DNA nearer the active center, formed by Rpb1, Rpb2, and Rpb6, may be locked in the closed position by RNA, accounting for the great stability of transcribing complexes. A pore in the protein complex beneath the active center may allow entry of substrates for polymerization and exit of the transcript during proofreading and passage through pause sites in the DNA.
Subject(s)
Models, Molecular , RNA Polymerase II/chemistry , Transcription Factors, General , Transcription, Genetic , Transcriptional Elongation Factors , Amino Acid Motifs , Binding Sites , Catalytic Domain , Crystallization , Crystallography, X-Ray , DNA, Fungal/chemistry , DNA, Fungal/metabolism , Enzyme Stability , Escherichia coli/enzymology , Humans , Protein Binding , Protein Structure, Quaternary , Protein Structure, Secondary , RNA Polymerase II/genetics , RNA Polymerase II/metabolism , RNA, Fungal/chemistry , RNA, Fungal/metabolism , RNA, Messenger/chemistry , RNA, Messenger/metabolism , Thermus/enzymology , Transcription Factors/chemistry , Transcription Factors/metabolismABSTRACT
X-ray diffraction data from two forms of yeast RNA polymerase II crystals indicate that the two largest subunits of the polymerase, Rpb1 and Rpb2, may have similar folds, as is suggested by secondary structure predictions. DNA may bind between the two subunits with its 2-fold axis aligned to a pseudo 2-fold axis of the protein.
Subject(s)
DNA/metabolism , Fungal Proteins/chemistry , Protein Structure, Tertiary , RNA Polymerase II/chemistry , Amino Acid Sequence , Binding Sites , Fungal Proteins/metabolism , Molecular Sequence Data , Protein Folding , Protein Structure, Secondary , RNA Polymerase II/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , X-Ray Diffraction , Yeasts/enzymologyABSTRACT
The structure of an actively transcribing complex, containing yeast RNA polymerase II with associated template DNA and product RNA, was determined by electron crystallography. Nucleic acid, in all likelihood the "transcription bubble" at the active center of the enzyme, occupies a previously noted 25 A channel in the protein structure. Details are indicative of a roughly 90 degrees bend of the DNA between upstream and downstream regions. The DNA apparently lies entirely on one face of the polymerase, rather than passing through a hole to the opposite side, as previously suggested.
Subject(s)
DNA, Fungal/chemistry , RNA Polymerase II/chemistry , RNA, Fungal/chemistry , RNA, Messenger/chemistry , Saccharomyces cerevisiae/enzymology , Crystallography , DNA, Fungal/ultrastructure , Image Processing, Computer-Assisted , Microscopy, Electron , Models, Molecular , RNA Polymerase II/ultrastructure , RNA, Fungal/ultrastructure , RNA, Messenger/ultrastructure , Streptavidin/chemistry , Streptavidin/ultrastructure , Transcription, GeneticABSTRACT
Appropriate treatment of X-ray diffraction from an unoriented 18-heavy atom cluster derivative of a yeast RNA polymerase II crystal gave significant phase information to 5 A resolution. The validity of the phases was shown by close similarity of a 6 A electron density map to a 16 A molecular envelope of the polymerase from electron crystallography. Comparison of the 6 A X-ray map with results of electron crystallography of a paused transcription elongation complex suggests functional roles for two mobile protein domains: the tip of a flexible arm forms a downstream DNA clamp; and a hinged domain may serve as an RNA clamp, enclosing the transcript from about 8-18 residues upstream of the 3'-end in a tunnel.