Podcasts about rnap ii

Background: A Xist RNA decorated Barr body is the structural hallmark of the compacted inactive X territory in female mammals. Using super resolution three-dimensional structured illumination microscopy (3D-SIM) and quantitative image analysis, we compared its ultrastructure with active chromosome territories (CTs) in human and mouse somatic cells, and explored the spatio-temporal process of Barr body formation at onset of inactivation in early differentiating mouse embryonic stem cells (ESCs). Results: We demonstrate that all CTs are composed of structurally linked chromatin domain clusters (CDCs). In active CTs the periphery of CDCs harbors low-density chromatin enriched with transcriptionally competent markers, called the perichromatin region (PR). The PR borders on a contiguous channel system, the interchromatin compartment (IC), which starts at nuclear pores and pervades CTs. We propose that the PR and macromolecular complexes in IC channels together form the transcriptionally permissive active nuclear compartment (ANC). The Barr body differs from active CTs by a partially collapsed ANC with CDCs coming significantly closer together, although a rudimentary IC channel system connected to nuclear pores is maintained. Distinct Xist RNA foci, closely adjacent to the nuclear matrix scaffold attachment factor-A (SAF-A) localize throughout Xi along the rudimentary ANC. In early differentiating ESCs initial Xist RNA spreading precedes Barr body formation, which occurs concurrent with the subsequent exclusion of RNA polymerase II (RNAP II). Induction of a transgenic autosomal Xist RNA in a male ESC triggers the formation of an `autosomal Barr body' with less compacted chromatin and incomplete RNAP II exclusion. Conclusions: 3D-SIM provides experimental evidence for profound differences between the functional architecture of transcriptionally active CTs and the Barr body. Basic structural features of CT organization such as CDCs and IC channels are however still recognized, arguing against a uniform compaction of the Barr body at the nucleosome level. The localization of distinct Xist RNA foci at boundaries of the rudimentary ANC may be considered as snap-shots of a dynamic interaction with silenced genes. Enrichment of SAF-A within Xi territories and its close spatial association with Xist RNA suggests their cooperative function for structural organization of Xi.

Replication of the genome is strongly inhibited when high fidelity DNA polymerases encounter unrepaired DNA lesions, which can not be processed. The highly stringent active sites of these polymerases are unable to accommodate damaged bases and therefore DNA lesions block the replication fork progression. In order to overcome this problem, cells have evolved mechanisms for either repairing the damage, or synthesising past it with specially adapted polymerasases. Eukaryotic DNA polymerase eta (Pol eta), belonging to the Y-family of DNA polymerases, is outstanding in its ability to replicate through a variety of highly distorting DNA lesions such as cyclobutane pyrimidine dimers (CPDs), which are the main UV-induced lesions. Also cisplatin induced 1,2-d(GpG) adducts (Pt-GGs), which are formed in a typical cancer therapy with cisplatin can be processed by Pol eta. The bypass of such intrastrand crosslinks by high fidelity DNA polymerases is particularly difficult because two adjacent coding bases are simultaneously damaged. Thus, replication by Pol eta allows organisms to survive exposure to sunlight or, in the case of cisplatin, gives rise to resistances against cisplatin treatment. Mutations in the human POLH gene, encoding Pol eta, causes the variant form of xeroderma pigmentosum (XP V), characterized by the failure to copy through CPDs. This leads to strongly increased UV sensitivity and skin cancer predisposition. This thesis describes mechanistic investigations of the translesion synthesis (TLS) process by S. cerevisiae DNA Pol eta at atomic resolution, which were undertaken in collaboration with the Hopfner group. To study this process, cisplatin lesioned DNA had to be prepared first. Once this technique was established, the catalytic fragment of Pol eta was crystallized as ternary complex with incoming 2',3'-dideoxycytidine 5'-triphosphate (ddCTP) and an primer - template DNA containing a site specific Pt-GG adduct. The first obtained structure shows the ddCTP positioned in a loosely bound conformation in the active site, hydrogen bonded to the templating base. Realizing the importance of the 3’ hydroxy group for positioning the NTP and the DNA correctly inside the polymerase, the complex was crystallized again with a 2’-deoxynucleoside 5’-triphosphate (dNTP). To prevent nucleotidyl transfer, primer strands which terminate at the 3’-end with a 2’,3’ dideoxy ribose were prepared by reverse DNA synthesis and used for cocrystallization. The resulting crystals diffracted typically to 3.1-3.3Å resolution at a synchrotron light source. A Pol eta specific arginine (Arg73 in yeast Pol eta) was identified for its importance to position the dNTP correctly in the active site and was shown to be necessary for lesion bypass. In contrast to the fixed preorientation of the dNTP in the active site, the damaged DNA is bound flexibly in a rather open DNA binding cleft. Nucleotidyl transfer requires a revolving of the DNA, energetically driven by hydrogen bonding of the templating base to the dNTP. For the 3’dG of the Pt-GG, this step is accomplished by bona fide Watson-Crick base pairs to dCTP and is biochemically efficient and accurate. In contrast, bypass of the 5’dG of the Pt-GG is less efficient and promiscuous for dCTP and dATP. Structurally, this can be attributed to misalignment of the templating 5’dG due to the rigid Pt crosslink. In cooperation with the Cramer group the structural reasons for the blockage of RNA Polymerase II (RNAP II) by the cisplatin lesion were elucidated. Using structural as well as biochemical methods it could be shown that stalling results from a translocation barrier that prevents delivery of the lesion to the active site. AMP misincorporation occurs at the barrier and also at an abasic site, suggesting that it arises from nontemplated synthesis according to an 'A-rule' known for DNA polymerases. RNAP II can bypass a cisplatin lesion that is artificially placed beyond the translocation barrier, even in the presence of a G A mismatch. Thus, the barrier prevents transcriptional mutagenesis.

Gene expression encompasses a multitude of different steps, starting with transcription in the nucleus, co-transcriptional processing and packaging of the mRNA into a mature mRNP, export of the mRNP through the nuclear pore and finally the translation of the message in the cytoplasm. The central coordinator for coupling of the nuclear events is the differentially phosphorylated C-terminal domain (CTD) of RNA polymerase II (RNAP II). The phosphorylation pattern of the CTD not only dictates the progression through the transcription cycle but also the recruitment of mRNA processing machineries. Coupling of transcription to mRNA export is achieved by the TREX complex, which consists in the yeast S. cerevisiae of the heterotetrameric THO complex important for transcription elongation, the SR-like proteins Gbp2 and Hrb1, and Tex1 and the mRNA export factors Sub2 and Yra1. By direct interaction with Yra1, the mRNA export receptor Mex67-Mtr2 is then recruited to the mRNP and transports the mRNP through the nuclear pore complex to the cytoplasm. In a genetic screen for factors that are crucial for TREX complex function in the living cell, Ctk1, a cyclin dependent kinase (CDK) that phosphorylates the C-terminal domain (CTD) of RNAP II during transcription elongation, was identified (Hurt et al. 2004). Surprisingly, besides the TREX components Gbp2 and Hrb1, Ctk1 co-purified ribosomal proteins and translation factors. Using sucrose density centrifugation, it could be shown that Ctk1 indeed associates with translating ribosomes in vivo, suggesting a novel function of this protein in translation. This assumption was confirmed by in vitro translation assays: loss of Ctk1 function leads to a reduction in translational activity. More specifically, Ctk1 is important for efficient translation elongation and contributes to the accurate decoding of the message. Cells depleted for Ctk1 are more sensitive towards drugs that impair translational accuracy and show an increase in the frequency of miscoding in vivo. The function of Ctk1 during decoding of the message is most likely direct, as in extracts of cells depleted for Ctk1 the defect in correct decoding of the message can be restored to wild type levels by addition of purified CTDK-I complex. An explanation for the molecular mechanism of Ctk1’s function is provided by the identification of Rps2 as a novel substrate of Ctk1. Rps2 is a protein of the small ribosomal subunit, located at the mRNA entry tunnel and known to be essential for translational accuracy. Importantly, Rps2 is phosphorylated on serine 238 by Ctk1, and cells containing an rps2-S238A mutation show an increased sensitivity towards drugs that affect translational accuracy and an increase in miscoding as determined by in vitro translation extracts. The role of Ctk1 in translation is probably conserved as CDK9, the mammalian homologue of Ctk1, also associates with polysomes. Since Ctk1 interacts with the TREX complex, which functions at the interface of transcription and mRNA export, Ctk1 might bind to the mRNP during transcription and accompany the mRNP to the ribosomes, where Ctk1 enhances efficient and accurate translation of the mRNA. This study could be an example of a novel layer of control in gene expression: the composition of the mRNP determines its translational fate, including efficiency and accuracy of translation.

RNA polymerase II (RNAP II) transiently associates with many different proteins and multiprotein complexes during the mRNA transcription cycle, which includes three phases, initiation, elongation, and termination. This thesis describes structural studies of two factors that facilitate transcription through chromatin. The heterodimeric Saccharomyces cerevisiae elongation factor Spt4-Spt5 (human DSIF) has been identified by biochemical and genetic approaches to help RNAP II transcribe through chromatin. It is assumed that Spt4-Spt5 pauses RNAP II to open a time window for capping enzyme recruitment and addition of a cap to the 5'-end of the nascent RNA. The preparation of milligram quantities of soluble Spt4-Spt5 variants that are suited for structural studies has been achieved. Several strategies to resolve the structure of the RNAP II–Spt4-Spt5 complex were unsuccessful, possibly indicating an intrinsic flexibility of the complex. In addition, there is now evidence for direct links between chromatin modification and transcription elongation. A major player in this process is the histone lysine methyltransferase Set2 which has a modular structure. The catalytic activity of Set2 is mediated by the SET [Su(var)3-9, Enhancer of Zeste, Trithorax] domain. During mRNA elongation, the SRI (Set2 Rpb1-interacting) domain of Set2 binds to the phosphorylated CTD (carboxyl-terminal domain) of RNAP II. The NMR solution structure of yeast Set2 SRI domain has been determined. The structure reveals a novel CTD-binding fold consisting of a left-handed three-helix bundle. Unexpectedly, the SRI domain fold resembles the structure of an RNA polymerase-interacting domain in sigma factors that mediate transcription initiation in bacteria (domain sigma2 in sigma70). NMR titration experiments show that the SRI domain binds a Ser2/Ser5-phosphorylated CTD peptide comprising two heptapeptide repeats and three flanking NH2-terminal residues. Amino acid residues that show strong chemical shift perturbations upon CTD binding cluster in two regions on the SRI surface. The results will enable a detailed analysis of the specific CTD interactions underlying the coupling of transcription and chromatin modification by Set2.