The non-natural photoreactive amino acid p-Benzoyl-L-Phenylalanine (Bpa) was incorporated into the RNA polymerase (Pol) II surface surrounding the central cleft formed by the Rpb1 and Rpb2 subunits. Photocrosslinking of Preinitiation Complexes (PICs) with these Pol II derivatives and hydroxyl radical cleavage assays revealed that the TFIIF dimerization domain interacts with the Rpb2 lobe and protrusion domains adjacent to Rpb9 while TFIIE crosslinks to the Rpb1 clamp domain on the opposite side of the Pol II central cleft. Mutations in the Rpb2 lobe and protrusion domains were found to alter both Pol II-TFIIF binding and the transcription start site, a phenotype associated with mutations in TFIIF, Rpb9, and TFIIB. In combination with previous biochemical and structural studies, these new findings illuminate the structural organization of the PIC and reveal a network of protein-protein interactions involved in transcription start site selection.The earliest step in transcription initiation is recruitment of Pol II and the general transcription factors to form the Preinitiation Complex (PIC) 1 . Upon addition of ATP, the PIC transitions to the Open Complex state, resulting in separation of the DNA strands surrounding the transcription start site and insertion of the template strand into the active center of Pol II. Next, Pol II must locate the transcription start site, which in S. cerevisiae, can be over 60 base pairs distant from the initial site of DNA strand separation 2 . Mutations in the general factor TFIIF and in the B-finger domain of the general factor TFIIB can alter the transcription start site, suggesting these two factors are involved in start site recognition 3-7 . Consistent with this role, crystallography and protein-protein crosslinking in the PIC have positioned both of these factors within the Pol II active site cleft 8-10 . Additionally, mutations in the Pol II subunit Rpb9 have been found to alter the transcription start site, but it remains unclear how this subunit, which is located distant from the Pol II active site, participates in start site selection 11,12 .Initiation of RNA synthesis begins with DNA-NTP base pairing, phosphodiester bond formation, and translocation of the DNA-RNA hybrid within the active center of Pol II. After synthesis of 8−12 bases of RNA, Pol II escapes from the promoter into a stable elongation state 13-15 . The nucleation of transcription factors at the promoter and the structural transition into the open and elongation complexes involves a complex set of protein-protein and protein-DNA interactions. Elucidating these molecular interactions within the transcription machinery Phone: 206 667 5261 Fax 206 667 6497 shahn@fhcrc.org. AUTHOR CONTRIBUTIONS L.W. modified the non-natural amino acid incorporation system and performed the experiment in Figure 1. H-T.C. performed and designed the remaining experiments. S.H. supervised the study. H-T.C. and S.H. wrote the manuscript.Publisher's Disclaimer: This PDF receipt will only be used as the basis for generating ...
ternary elongation complex, where it likely functions in accurate termination. Our work explains how the Rpc37/53 dimer is anchored on the Pol III core and acts as a hub to integrate a protein network for initiation and termination.RNA polymerase III (Pol III) catalyzes the RNA synthesis of diverse untranslated transcripts, including precursor tRNAs, 5S rRNA, and certain small nuclear RNAs, such as U6 and 7SL RNAs. The other two nuclear RNA polymerases, Pol I and Pol II, transcribe pre-rRNA and mRNA, respectively. In humans, Pol III also participates in the transcription of a number of microRNAs (19) and plays a role in tumorigenesis (41,58). Among the nuclear RNA polymerases, Pol III is the largest enzyme, comprising 17 subunits with a total molecular mass of approximately 0.7 MDa; its core structure of 12 subunits is conserved among all nuclear Pols (18). The remaining five subunits are specific to Pol III and form two subcomplexes with distinct functions in transcription, Rpc82/34/31 and Rpc37/53. The former subcomplex interacts with the initiation transcription factor TFIIIB for the recruitment of polymerase and also participates in promoter opening and transcription initiation (6,50,55,56), whereas the latter participates in promoter opening and transcription termination and reinitiation (34,38). Previous yeast two-hybrid screenings provided an overview of protein-protein interactions among Pol III subunits (26), and electron microscopy analysis of Pol III suggested the location of the subcomplexes on the Pol III core structure (24,25,32,52). However, much more information on proteinprotein interactions between these subcomplexes and the initiation factors is necessary to understand the mechanisms of initiation, elongation, and termination and to determine how the mechanisms used by the Pol III machinery differ from those used by Pol I and II.A conserved dimerization module has recently been detected in the Pol I, II, and III machineries as part of Rpa49/ 34.5 of Pol I, the general transcription factor TFIIF of Pol II, and Rpc37/53 of Pol III (8,13,18,36). The presence of the TFIIF-like complexes is unique to the eukaryotic transcription systems. However, other than the conserved dimerization module and a winged-helix (WH) fold that is present as a single copy in each of the Tfg1 and Tfg2 subunits of TFIIF and as tandem repeats in Rpa49, the overall structural characteristics of the three TFIIF-like complexes differ; thus, their functions in transcription may also differ. For example, although all three of these TFIIF-like complexes are involved in transcription and elongation, they exert opposite effects on elongation, with Rpa49/34.5 and TFIIF increasing and Rpc37/53 reducing the elongation rate (36,38,59).Structural and biochemical analyses have suggested how individual TFIIF-like complexes establish protein interactions for their respective transcriptional activities. The TFIIF dimerization module, which resides in subunits Tfg1 and Tfg2, has been mapped to the Pol II lobe domain above the active site c...
The RNA polymerase (pol) II general transcription factor TFIIF functions at several steps in transcription initiation including preinitiation complex (PIC) formation and start site selection. We find that two structured TFIIF domains bind Pol II at separate locations far from the active site with the TFIIF dimerization domain on the Pol II lobe and the winged helix domain of the TFIIF small subunit Tfg2 above the Pol II protrusion where it may interact with upstream promoter DNA. Binding of the winged helix to the protrusion is PIC specific. Anchoring of these two structured TFIIF domains at separate sites locates an essential and unstructured region of Tfg2 near the Pol II active site cleft where it may interact with flexible regions of Pol II and the general factor TFIIB to promote initiation and start site selection. Consistent with this mechanism, mutations far from the enzyme active site, which alter the binding of either structured TFIIF domains to Pol II, have similar defects in transcription start site usage.
Biochemical probes positioned on the surface of the general transcription factor TFIIB were used to probe the architecture of the RNA polymerase II (Pol II) transcription preinitiation complex (PIC). In PICs, the TFIIB linker and core domains are positioned over the central cleft and wall of Pol II. This positioning is not observed in the smaller Pol II-TFIIB complex. These results lead to a new model for the structure of the PIC, which agrees with most previously documented protein-DNA interactions within Pol II and archaea PICs. Specific interaction of the TFIIB core domain with Pol II positions and orients the promoter DNA over the Pol II central cleft, and TBP-DNA bending leads to bending of the promoter around the surface of Pol II. The TFIIF subunit Tfg1 was found in close proximity to the TFIIB B finger, linker, and core domains, suggesting that these two factors closely cooperate during initiation.
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