Yeast RNA Polymerase II at 5 Å Resolution

Abstract

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.

The molecular basis of eukaryotic transcription

“…Jianhua Fu, who discovered the best diffracting form of pol II crystals, addressed the phase problem by data collection from a large number of crystals and with the use of heavy atom clusters developed by others. He found matched pairs of native and derivative crystals from which phases to 5-Å resolution could be derived (25). The resulting electron density map corresponded closely to the structure of pol II at 16-Å resolution determined from 2D crystals by electron microscopy and 3D reconstruction.…”

Conformational flexibility of bacterial RNA polymerase

“…The very high conservation of sequence, structure, and function between E. coli and Taq (Fig. 1), and the fact that related conformational changes of a similar scale have been observed in different crystal forms and different functional complexes of yeast RNAP II (24,(27)(28)(29)(30), leads to the conclusion that the observed conformational change reflects the normal flexibility of the RNAP rather than differences between E. coli and Taq RNAPs or crystallization artifacts.…”

“…From a comparison of two crystal forms of yeast RNAP II, a highly mobile domain termed the ''clamp'' was identified (24,28,29), comprising primarily the N terminus of Rpb1, and the C terminus of Rpb2 (the homologous structure in Taq RNAP corresponds to residues 1-624 of ␤Ј and 1054-1115 of ␤). In the different structures of RNAP II, the clamp undergoes a swinging motion, resulting in opening or closing of the main DNA͞RNA cleft, and this motion was proposed to be important for allowing entry of DNA into the cleft for the initiation of transcription, and for closing on the DNA and the DNA͞RNA hybrid to provide processivity during transcription elongation.…”

Fcp1 directly recognizes the C-terminal domain (CTD) and interacts with a site on RNA polymerase II distinct from the CTD

“…There has been no known mechanism to date that could explain this noncatalytic activity of Fcp1. But the sharply biased surface-charge distribution of Pol II (positive in the DNA͞RNA cleft and negative elsewhere), which indicates that the polymerase binds heparin polymers in the cleft, prompted us to think that the loss of heparin binding may result from three possible mechanisms: (i) an occlusion caused by direct Fcp1 binding in the DNA͞RNA site; (ii) a blockage of the cleft by Fcp1 binding at its opening; and (iii) a closure of the cleft by the clamp domain (55)(56)(57). The first mechanism might be discounted by the considerations that (i) the DNA͞RNA site is Ϸ75 Å deep but only 25 Å wide, a potential topological problem for a protein as large as pombe Fcp1 (82 kDa) and that (ii) the nucleic acid cleft is occupied by the DNA template and RNA transcript, which would exclude Fcp1 from interacting with Pol II elongation complex, however Fcp1 was observed to stimulate a purified elongation complex (40).…”