Podcasts about ii pol ii

Protein-coding genes in eukaryotes are transcribed by RNA polymerase II (Pol II). This process is tightly regulated and coupled to RNA processing. Many transcription and RNA processing factors are recruited to Pol II via its conserved C-terminal domain (CTD) containing 27 heptapeptide repeats of the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 in Saccharomyces cerevisiae. These repeats can be differentially phosphorylated during the transcription cycle serving as a code for interacting factors. During transcription initiation, Ser5 is phosphorylated at the 5’-end of genes and this phosphorylation is required for RNA capping enzyme binding. During transcription elongation, the Pol II CTD becomes phosphorylated at Tyr1 and Ser2 and binds the elongation factor Spt5. Spt5 also contains a repetitive C-terminal region (CTR) required for cotranscriptional recruitment of proteins. At the 3’-end of genes, Ser2-phosphorylated Pol II associates with the cleavage and polyadenylation factor (CPF) and is dephosphorylated at Tyr1 residues. This work shows that CPF is a Pol II CTD phosphatase and that its subunit Glc7 dephosphorylates Tyr1 in vitro. In vivo, Glc7 activity is required for normal Tyr1 dephosphorylation at the polyadenylation (pA) site, for recruitment of termination factors Pcf11 and Rtt103, and for normal Pol II termination. These results show that transcription termination involves Tyr1 dephosphorylation of the CTD and indicate that pre-mRNA processing and transcription termination are coupled via CPF-dependent Pol II Tyr1 dephosphorylation. Additionally, 19 kinases were tested for activity on Tyr1 in yeast by selective inhibition or knock-out in vivo. However, none of the candidates was identified as the Tyr1 kinase. Possibly this enzyme is an atypical kinase not known to be involved in transcription so far. Furthermore, this work reports a new role of the Spt5 CTR in recruitment of RNA 3’-end processing factors. The results show that the Spt5 CTR as well as RNA is required for normal recruitment of the pre-mRNA cleavage factor (CF) I to the 3’-end of yeast genes. Genome-wide ChIP profiling detects occupancy peaks of CFI subunits around 100 base pairs downstream of the pA site of genes. CFI recruitment to this defined region may result from simultaneous binding to the Spt5 CTR, to nascent RNA containing the pA sequence, and to the elongating Pol II isoform that is phosphorylated at Ser2 of the CTD. Consistent with this model, the CTR interacts with CFI in vitro, but is not required for pA site recognition and transcription termination in vivo. In summary, we characterized two new aspects of transcription and RNA processing regulation by two different C-terminal repetitive protein domains. CTD Tyr1 phosphorylation, which is removed by Glc7, regulates termination factor recruitment by masking their binding site, the Spt5 CTR is involved in recruitment of CFI. Both results greatly contribute to a more detailed understanding of the mechanisms involved in transcription termination and RNA 3’-end processing.

RNA polymerase II (Pol II) is the eukaryotic enzyme responsible for transcribing all protein-coding genes into messenger RNA (mRNA). This thesis describes studies on the molecular mechanisms of Pol II translocation, alpha-amanitin inhibition and DNA lesion recognition by Pol II. To study how Pol II translocates after nucleotide incorporation, we prepared elongation complex (EC) crystals in which pre- and post-translocation states interconvert. Crystal soaking with the inhibitor alpha-amanitin locked the EC in a new state that we identified as a translocation intermediate at 3.4 Å resolution. The DNA base entering the active site occupies a “pre-templating” position above the central bridge helix, which is shifted and occludes the standard templating position. A leucine residue in the trigger loop forms a wedge next to the shifted bridge helix, but moves by 13 Å to close the active site for nucleotide incorporation. Our results support a Brownian ratchet mechanism of elongation that involves swinging of the trigger loop between open, wedged, and closed positions, and suggest that alpha-amanitin impairs nucleotide incorporation and translocation by trapping the trigger loop and bridge helix in a translocation intermediate. Cells use transcription-coupled repair (TCR) to efficiently eliminate DNA lesions such as UV-induced cyclobutane pyrimidine dimers (CPDs). Here we present the structure-based mechanism for the first step in eukaryotic TCR, CPD-induced stalling of Pol II. A CPD in the transcribed strand slowly passes a translocation barrier, and enters the polymerase active site. The CPD 5’-thymine then directs uridine monophosphate (UMP) misincorporation into mRNA, which blocks translocation. Artificial replacement of the UMP by adenosine monophosphate (AMP) enables CPD bypass, thus Pol II stalling requires CPD-directed misincorporation. In the stalled complex, the lesion is inaccessible, and the polymerase conformation is unchanged. This is consistent with non-allosteric recruitment of repair factors and excision of a lesion-containing DNA fragment in the presence of Pol II. CPD recognition is compared with the recognition of a cisplatin-induced guanine-guanine intrastrand crosslink. Similarities and differences in the detailed mechanism of transcriptional stalling at the two different dinucleotide lesions are discussed.

TFIIF is the only general transcription factor that has been implicated in the preinitiation complex assembly, open complex formation, initiation and transcription elongation. In addition, TFIIF stimulates Fcp1, a central phosphatase needed for recycling of RNA polymerase II (Pol II) after transcription by dephosphorylation of the Pol II C-terminal domain (CTD). This thesis reports the X-ray structure of the small CTD phosphatase Scp1, which is homologous to the Fcp1 catalytic domain. The structure shows a core fold and an active center similar to phosphotransferases and –hydrolases that solely share a DXDX(V/T) signature motif with Fcp1/Scp1. It was further demonstrated that the first aspartate in the signature motif undergoes metalassisted phosphorylation during catalysis, resulting in a phosphoaspartate intermediate that was structurally mimicked with the inhibitor beryllofluoride. Specificity may result from CTD binding to a conserved hydrophobic pocket between the active site and an insertion domain that is unique to Fcp1/Scp1. Fcp1 specificity may additionally arise from phosphatase recruitment near the CTD via the Pol II subcomplex Rpb4/7, which is shown to be required for Fcp1 binding to the polymerase in vitro. Until now, the main impediment in the high resolution crystallographic studies of TFIIF in complex with Pol II and other members of transcription machinery was unavailability of soluble, stoichiometric TFIIF complex in sufficient amounts. This thesis reports on the development of the overexpression system in yeast and a purification protocol that enabled for the first time to isolate milligram amounts of a pure and soluble, 15-subunit (~0,7 MDa) stoichiometric Pol IITFIIF complex. Such complex together with the promoter DNA, RNA, TBP and TFIIB assembles in vitro into the yeast initially transcribing complex, which can now be studied structurally.

The Mediator of transcriptional regulation is the central coactivator that enables a response of RNA polymerase II (Pol II) to activators and repressors. Yeast Mediator has a size of more than one MDa and consists of 25 different polypeptides. Biochemical studies defined three Mediator modules in yeast, the head (MED17) the middle (MED9/MED10) and the tail (MED15) modules. During this work, an E.coli coexpression-copurification system was developed, which allowed to study pairwise interactions of Mediator middle module subunits. With the help of this system I reconstituted a complex of two essential and conserved yeast Mediator middle module proteins, the MED7/MED21 heterodimer, and solved its crystal structure. The heterodimer forms an extended structure, which spans one third of the Mediator length, and almost the diameter of Pol II. It shows a four helix bundle and a coiled-coil protrusion connected by a flexible hinge. Multiple conserved patches can be identified on the surface, which allow for assembly of the middle module. A combination of the coexpression-copurification system and assembly of subcomplexes allowed the reconstitution of a five-subunit Mediator middle module subcomplex. The reconstituted subcomplex is able to bind Pol II in vitro. MED6 associates with the middle module and forms a bridge to the head module. The potential flexibility of this bridge and the MED7/MED21 hinge can account for changes in Mediator structure upon its binding to Pol II or to activators.

RNA polymerase II (Pol II) is the central enzyme, that synthetizes all mRNA in eukaryotic cells. In this work, I solved the structure of the complete, initiation-competent 12-subunit yeast RNA polymerase II at 3.8 Å. I also solved the structure of the Pol II subcomplex of Rpb4/7 alone at 2.3 Å resolution. These structures reveal the details of Pol II assembly from 12 subunits and give important insights into the initiation of transcription. The refined, atomic model of the complete 12-subunit Pol II enabled homology modeling of the two other nuclear RNA polymerases. In Pol I and Pol III, 65 % and 77 % of the Pol II fold are conserved, respectively. Together with a recent structure of a Pol II elongation complex, these results show that the basic mechanism of transcription applies also to the two other nuclear RNA polymerases

This thesis describes crystal structures of complete, 12-subunit yeast RNA polymerase II (Pol II) in complex with a synthetic transcription bubble and product RNA, with an NTP substrate analogue, and in complex with the transcription elongation factor TFIIS. The structure of the Pol II-transcription bubble-RNA complex reveals incoming template and non-template DNA, a seven base-pair DNA-RNA hybrid, and three nucleotides each of separating DNA and RNA. Based on this structure, those parts of Pol II were identified which are involved in separating template DNA from non-template DNA before the active site, and DNA from product RNA at the upstream end of the DNA-RNA hybrid. In both instances, strand separation can be explained by Pol II-induced duplex distortions. Only parts of the complete transcription bubble present in the complexes are ordered in the crystal structure, explaining the way in which high processivity of Pol II is reconciled with rapid translocation along the DNA template. The presence of an NTP substrate analogue in a conserved putative pre-insertion site was unveiled in a Pol II-transcription bubble-RNA complex crystal soaked with the substrate analogue GMPCPP. The structure of the Pol II-TFIIS complex was obtained from Pol II crystals soaked with TFIIS. TFIIS extends from the Pol II surface to the active site and complements the active site with two essential and invariant acidic residues for hydrolytic RNA cleavage. TFIIS also induces extensive structural changes in Pol II that reposition nucleic acids, in particular RNA, near the active centre. These results support the idea that Pol II contains a single tuneable active site for RNA polymerisation and cleavage. The technical obstacles imposed by crystal structure determination of large, transient protein-DNA-RNA complexes were overcome by two novel, fluorescence-based assays to monitor and optimise the composition of the crystals. Both assays are not limited to Pol II complexes, but can serve as a general tool for the crystallographic community.