You are currently viewing the abstract.View Full Text
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.
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.
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.
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.
Pre-Initiation Complex in 3D
The regulation of gene expression is critical for almost every aspect of biology. Transcription—generating an RNA copy of a gene—requires the assembly of a large pre-initiation complex (PIC) at every RNA polymerase II (pol II) promoter. Roughly 32 proteins—the subunits of pol II and the general transcription factors—form a PIC that can recognize a minimal TATA-box promoter, select a transcription start site, and synthesize a nascent transcript. Murakami et al. (p. 10.1126/science.1238724, published online 26 September; see the Perspective by Malik and Roeder) determined the three-dimensional map of the Saccharomyces cerevisiae 30-subunit PIC using cryo-electron microscopy. The saddle-shaped TATA binding protein, the boot-shaped transcription factor IIA (TFIIA), and promoter DNA ∼27 bp downstream of the TATA-box could all be seen. Cross-linking and mass spectrometry was used to determine the spatial proximity of the 30 subunits, revealing that the PIC forms two lobes with TFIIF forming a bridge between them.
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.