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References
Have Your PIC!
Science 8 November 2013: Vol. 342 no. 6159 pp. 706-707 DOI: 10.1126/science.1246170
http://www.sciencemag.org/content/342...

Architecture of an RNA Polymerase II Transcription Pre-Initiation Complex
Science 8 November 2013: Vol. 342 no. 6159 DOI: 10.1126/science.1238724
http://www.sciencemag.org/content/342...

Structured Abstract
Introduction: RNA polymerase II (pol II) is capable of RNA synthesis but is unable to recognize a promoter or to initiate transcription. For these essential functions, a set of general transcription factors (GTFs)—termed TFIIB, -D, -E, -F, and -H—is required. The GTFs escort promoter DNA through the stages of recruitment to pol II, unwinding to create a transcription bubble, descent into the pol II cleft, and RNA synthesis to a length of 25 residues and transition to a stable elongating complex. The structural basis for these transactions is largely unknown. Only TFIIB has been solved by means of x-ray diffraction, in a complex with pol II. We report on the structure of a complete set of GTFs, assembled with pol II and promoter DNA in a 32-protein, 1.5 megaDalton "pre-initiation complex" (PIC), as revealed with cryo-electron microscopy (cryo-EM) and chemical cross-linking. Methods: Three technical advances enabled the structural analysis of the PIC. First, a procedure was established for the preparation of a stable, abundant PIC. Both the homogeneity and functional activity of the purified PIC were demonstrated. Second, an algorithm was developed for alignment of cryo-EM images that requires no prior information (no "search model") and that can distinguish multiple conformational states. Last, a computational method was devised for determining the arrangement of protein subunits and domains within a cryo-EM density map from a pattern of chemical cross-linking. Results: The density map of the PIC showed a pronounced division in two parts, one pol II and the other the GTFs. Promoter DNA followed a straight path, in contact with the GTFs but well separated from pol II, suspended above the active center cleft. Cross-linking and computational analysis led to a most probable arrangement of the GTFs, with IIB at the upstream end of the pol II cleft, followed by IIF, IIE, and IIH. The Ssl2 helicase subunit of IIH was located at the downstream end of the cleft. Discussion: A principle of the PIC revealed by this work is the interaction of promoter DNA with the GTFs and not with pol II. The GTFs position the DNA above the pol II cleft, but interaction with pol II can only occur after melting of the DNA to enable bending for entry in the cleft. Contact of the DNA with the Ssl2 helicase in the PIC leads to melting (in the presence of adenosine triphosphatase). Cryo-EM by others, based on sequential assembly and analysis of partial complexes rather than of the complete PIC, did not show a separation between pol II and GTFs and revealed direct DNA--pol II interaction. The discrepancy calls attention to a role of the GTFs in preventing direct DNA-polymerase interaction.

Abstract
The protein density and arrangement of subunits of a complete, 32-protein, RNA polymerase II (pol II) transcription pre-initiation complex (PIC) were determined by means of cryogenic electron microscopy and a combination of chemical cross-linking and mass spectrometry. The PIC showed a marked division in two parts, one containing all the general transcription factors (GTFs) and the other pol II. Promoter DNA was associated only with the GTFs, suspended above the pol II cleft and not in contact with pol II. This structural principle of the PIC underlies its conversion to a transcriptionally active state; the PIC is poised for the formation of a transcription bubble and descent of the DNA into the pol II cleft.