2,085 research outputs found

    RNA Polymerase II during Transcript Elongation: deaiing with DNA damage and staying phosphorylated

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    This thesis covers two topics related to transcript elongation in Saccharomyces cerevisiae - the regulation of the phosphatase Fcp1 and transcription-coupled DNA damage repair. Formation of RNA polymerase II (RNAPII) complexes throughout the transcription cycle is mediated in part by the phosphorylation state of the C-terminal domain (CTD) of the largest RNAPII subunit. Although a multitude of kinases can phosphorylate the CTD, currently only one CTD-specific phosphatase, Fcp1, has been identified. This work studies the possibility that Fcp1 might be associated with the elongating form of RNAPII. The phosphatase co-fractionates with RNAPII in association with the elongation factor Elongator. Furthermore, genetic studies show that a double mutant that carries a deletion of an Elongator gene as well as a temperature sensitive fcp1 mutation has a synthetic lethal phenotype at the permissive temperature. In vitro assays using crude extracts demonstrate that the CTD of RNAPII becomes dephosphorylated in a Fcp1-dependent manner. In contrast, in a reconstituted DNA-RNA-RNAPII system, the addition of the purified phosphatase does not stimulate such dephosphoryation. These results indicate a close relationship between Fcp1 phosphatase and the elongating form of RNAPII. Transcription-coupled DNA damage repair is a term applied to the preferential repair of DNA damage on the coding strand within active genes. The second half of this thesis describes the characterisation of nucleotide excision repair (NER) of the intrastrand 1,3-(pGpTpG)-cisplatin lesion in Saccharomyces cerevisiae as well as an attempt to reconstitute a transcription-coupled NER reaction (TC-NER) in vitro. Using modified yeast extracts, the excision products of the above lesion by NER were found to be between 23 and 26 nucleotides long, via incisions around the 15th phosphodiester bond 3' and 7th the phosphodiester bond 5' of the damage. The attempt to reconstitute TC-NER in vitro was hindered by difficulties with the transcription substrate and the functional instability of purified NER proteins

    Cryo-EM structure of mammalian RNA polymerase II in complex with human RPAP2

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    Nuclear import of RNA polymerase II (Pol II) involves the conserved factor RPAP2. Here we report the cryo-electron microscopy (cryo-EM) structure of mammalian Pol II in complex with human RPAP2 at 2.8 Å resolution. The structure shows that RPAP2 binds between the jaw domains of the polymerase subunits RPB1 and RPB5. RPAP2 is incompatible with binding of downstream DNA during transcription and is displaced upon formation of a transcription pre-initiation complex

    Structure of the Complete RNA Polymerase II Elongation Complex and its Interaction with the Elongation Factor TFIIS

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    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

    Activation of the human Mediator kinase CDK8 by MED12

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    Investigating the Assembly of Ribonucleoprotein complexes (RNPs)

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    Eukaryotic cells contain numerous small nuclear ribonucleoproteins (RNPs) that function in different RNA-processing events in the nucleus. The spliceosomal U1, U2, U5 and U4/U6 snRNPs (small nuclear RNPs) make up one class of complexes that are catalysing a well defined process called pre-mRNA splicing. Pre-mRNA splicing is the process by which non-coding regions (introns) are removed from pre-mRNA transcripts and the protein coding elements (exons) assembled into mature mRNAs before the RNA leaves the nucleus. Another class comprises the box C/D snoRNPs (small nucleolar RNPs) that are involved in processing of the ribosomal RNA (rRNA) in the nucleolus. These complexes function as guides in the site-specific 2' O-methylation of riboses in the precursor rRNA as well as assisting in rRNA biogenesis. Though the above-named U4/U6 snRNP and the box C/D snoRNP complexes are distinct in both composition and function, they share a similar RNA component and protein composition. Their RNA component can form a so called kink-turn (k-turn) motif (in the U4 5′ stem-loop and box C/D and B/C motif in box C/D snoRNAs), which is bound by a 15.5 kilodalton protein (15.5K). Besides protein 15.5K, the U4/U6 snRNP and the box C/D snoRNP complexes possesses several complex-specific proteins. In addition to seven Sm and seven LSm proteins that bind the Sm site of the U4 snRNA and the 3' end of U6 snRNA respectively, four proteins have been found to be associated with the human U4/U6 snRNP. These include the hPrp31 protein (also called 61K) and three proteins with molecular weights of 20, 60 and 90 kDa that form a biochemically stable, hCypH/hPrp4/hPrp3 (also 20/60/90K) protein complex. As well the box C/D snoRNPs are associated with a number of complex-specific proteins, which practically perform processing of the rRNA. The box C/D snoRNPs, like the U8 and U14 box C/D snoRNA, have been shown to bind proteins NOP56, NOP58, TIP48, TIP49 and fibrillarin. Lastly, the 15.5K protein and the hU3–55K protein belong to the complex assembled on the U3 box B/C motif of the U3 box C/D snoRNA. The assembly of the RNP complexes is a multiple-stage process that is initiated by 15.5K protein binding to the respective k-turn motif. Protein 15.5K is unique in that it is essential for the hierarchical assembly of the above-named three RNP complexes. Protein 15.5K interacts with the cognate RNAs via an induced-fit mechanism, which results in the folding of the surrounding RNA to create binding site(s) for the RNP-specific proteins. It has been shown that protein-RNA interactions are essential for the binding of complex-specific proteins to the U4/U6 snRNP, C/D snoRNP, and the RNP complex assembled on the U3 box B/C motif. However, at the beginning of this work it was unknown whether protein 15.5K also mediates RNP formation through direct protein-protein interactions with the complex-specific proteins. To investigate this possibility, a series of protein 15.5K mutations were created in which the surface properties of the protein had been changed. Within the scope of this work, their ability to support the formation of the three distinct RNP complexes was assessed and the formation of each RNP was found to require a distinct set of regions on the surface of the 15.5K protein. This implies that protein-protein contacts are essential for RNP formation in each complex. Further supporting this idea, direct protein protein interaction was observed between hU3 55K and 15.5K. In conclusion, the data obtained suggest that the formation of each RNP involves the direct recognition of specific elements in both 15.5K protein and the specific RNA. The U4/U6 snRNP-specific protein hPrp31 and the box C/D snoRNP-specific proteins NOP56 and NOP58 have been shown to be homologous, sharing a conserved domain, the so-called Nop domain. At the beginning of this work, it was unclear how protein hPrp31 and proteins NOP56/NOP58 assemble specifically onto the U4/U6 snRNP and box C/D snoRNPs, respectively. To address this question, structural requirements for the association of protein hPrp31 with the U4 snRNP in vitro were analysed. By employing point and deletion mutants of the U4 snRNA, structural features within the U4 snRNA necessary for binding of protein hPrp31 to the U4/U6 snRNP were determined. The findings indicate that the specificity of hPrp31 binding is provided by stems I and II of the U4 snRNA and suggest a way in which protein hPrp31 and proteins NOP56/NOP58 may specifically assemble onto the U4/U6 snRNP and box C/D snoRNPs, respectively. Furthermore, the results presented here provide evidence that the Nop domain on its own is necessary and sufficient for hPrp31 binding to the U4 snRNP, thus suggesting that this domain may be a novel RNA-binding domain. Recently it has been shown that protein hPrp31 is also of clinical interest. Two mutations (A194E, A216P) in the human hPrp31 gene (PRPF31) are correlated with the autosomal dominant form of retinitis pigmentosa, a disorder that leads to degeneration of the photoreceptors in the retina of the eye. It has been shown that hPrp31 plays a key role in U4/U6.U5 tri snRNP formation, which is assumed to take place in Cajal bodies (CBs), subnuclear organelles of animal and plant cells. Nevertheless, it is so far unknown whether the mutations in protein hPrp31 affect the stability of the tri snRNP. Using fluorescence microscopy and biochemical methods it has been shown here how hPrp31 mutations influence the localisation of protein hPrp31 in vivo and how they influence U4/U6.U5 tri snRNP formation

    In Vivo Consequences of Altered Pol II Catalysis

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    Gene transcription by RNA polymerase II (Pol II) is an essential process. Using Saccharomyces cerevisiae as a model system, our lab has previously identified and partially characterized Pol II activity mutants that can alter catalysis rate to faster or slower than wild type in vitro. In my dissertation research I use a set of these Pol II activity mutants to determine consequences of altered catalysis rate on polymerase functions, co-transcriptional pre-mRNA processing and gene expression in vivo. I show that alteration in Pol II catalytic rate, either increase or decrease, leads to a decreased Pol II occupancy and an apparent reduction in elongation rate on a commonly used reporter gene in vivo. Measurement of in vivo elongation rate on this reporter requires transcriptional shutoff followed by ChIP. I discover that some Pol II catalytic mutants can compromise the kinetics of transcription shutoff by glucose, which is generally assumed to be unaffected by transcription mutants. Further, I show that Pol II catalytic mutants affect model gene expression and the effects on gene expression are exacerbated with increased promoter strength and gene length. My results suggest that gene expression defects in the Pol II mutants may in part result from defective mRNA processing. Additionally, I show that mRNA half-lives for that model gene are increased in Pol II mutant strains and the magnitude of half-life changes correlate both with mutants’ growth and the magnitude of reporter gene expression defects. Finally, I test if altered Pol II elongation sensitizes cells to nucleotide depletion and find that Pol II mutants and several elongation factor mutants respond to GTP starvation similarly to wild type and that putative elongation defects are not likely to drive the cellular response to limiting GTP. Altogether my findings reveal wide-ranging in vivo effects of Pol II catalytic mutants, which will be critical for precise use of these Pol II catalytic mutants in gene regulation studies

    Cryo-EM structures of eukaryotic translation termination and ribosome recycling complexes containing eRF1, eRF3 and ABCE1

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    Translation of an mRNA template into a polypeptide chain is terminated on a stop codon. The stop is recognized in the ribosomal A site by release factors, that subsequently release the polypeptide. This event is followed by ribosome recycling leading to a dissociation of the ribosome into subunits. Eukaryotic eRF1 recognizes all three stop codons and is delivered to the ribosomal A site in a ternary complex with GTP-bound eRF3. ABCE1, a highly conserved ATPase, stimulates peptide release and splits the ribosome in concert with eRF1. The first goal of this work was to study the structural rearrangements of eRF1, eRF3 and ABCE1 on the ribosome during translation termination and ribosome recycling. Two cryo-EM structures were obtained at sub-nanometer resolution: the pre-termination complex containing eRF1 and eRF3, and a termination/pre-recycling complex containing eRF1 and ABCE1. The pre-termination stage showed eRF1 packed against eRF3, unable to catalyze peptide release. In the termination/pre-recycling complex, eRF1 assumed an extended conformation which is further stabilized by ABCE1, with the central domain of eRF1 swung out toward the CCA end of the P-site tRNA. ABCE1 adopted a half-closed conformation of its two nucleotide-binding domains in the termination/pre-recycling complex. According to a model based of these results, splitting the ribosome would require the closing of the two nucleotide-binding domains and a rotation of the iron-sulfur cluster domain of ABCE1, which would in turn push eRF1 into the intersubunit space. Supporting this idea, ABCE1 was shown to remain bound to the small ribosomal subunit after in vitro splitting. 40S-bound ABCE1 adopted a fully closed conformation and in which re-association of the large ribosomal subunit is prevented. As a second goal of this work, native S. cerevisiae ABCE1-bound small ribosomal subunits were purified to complement the in vitro studies and explore the supposed involvement of ABCE1 in translation initiation. Cryo-EM of native 40S-ABCE1 complexes indeed confirmed the closed conformation of ABCE1. Moreover, these complexes were associated with initiator tRNA and eIF1A, an initiation factor which binds the ribosomal A site and is involved in multiple processes in initiation including subunit joining. These results are clearly hinting at an active role of ABCE1 during translation initiation. Yet, the exact role of ABCE1 will be subject of further studies

    High-resolution structure of the human translation termination complex

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