Maria L. Kireeva

Nucleosome Remodeling Induced by RNA Polymerase II: Loss of the H2A/H2B Dimer during Transcription

RNA polymerase II (Pol II) must transcribe genes in a chromatin environment in vivo. We examined transcription by Pol II through nucleosome cores in vitro. At physiological and lower ionic strengths, a mononucleosome imposes a strong block to elongation, which is relieved at increased ionic strength. Passage of Pol II causes a quantitative loss of one H2A/H2B dimer but does not alter the location of the nucleosome. In contrast, bacteriophage SP6 RNA polymerase (RNAP) efficiently transcribes through the same nucleosome under physiological conditions, and the histone octamer is transferred behind SP6 RNAP. Thus, the mechanisms for transcription through the nucleosome by Pol II and SP6 RNAP are clearly different. Moreover, Pol II leaves behind an imprint of disrupted chromatin structure.

Cyr61, a Product of a Growth Factor-Inducible Immediate-Early Gene, Promotes Cell Proliferation, Migration, and Adhesion

cyr61 was first identified as a growth factor-inducible immediate-early gene in mouse fibroblasts. The encoded Cyr61 protein is a secreted, cystein-rich heparin-binding protein that associates with the cell surface and the extracellular matrix, and in these aspects it resembles the Wnt-1 protein and a number of known growth factors. During embryogenesis, cyr61 is expressed most notably in mesenchymal cells that are differentiating into chondrocytes and in the vessel walls of the developing circulatory system. cyr61 is a member of an emerging gene family that encodes growth regulators, including the connective tissue growth factor and an avian proto-oncoprotein, Nov cyr61 also shares sequence similarities with two Drosophila genes, twisted gastrulation and short gastrulation, which interact with decapentaplegic to regulate dorsal-ventral patterning. In this report we describe the purification of the Cyr61 protein in a biologically active form, and we show that purified Cyr61 has the following activities: (i) it promotes the attachment and spreading of endothelial cells in a manner similar to that of fibronectin; (ii) it enhances the effects of basic fibroblast growth factor and platelet-derived growth factor on the rate of DNA synthesis of fibroblasts and vascular endothelial cells, although it has no detectable mitogenic activity by itself; and (iii) it acts as a chemotactic factor for fibroblasts. Taken together, these activities indicate that Cyr61 is likely to function as an extracellular matrix signaling molecule rather than as a classical growth factor and may regulate processes of cell proliferation, migration, adhesion, and differentiation during development.

Activation-dependent Adhesion of Human Platelets to Cyr61 and Fisp12/Mouse Connective Tissue Growth Factor Is Mediated through Integrin αIIbβ3

Cyr61 and connective tissue growth factor (CTGF), members of a newly identified family of extracellular matrix-associated signaling molecules, are found to mediate cell adhesion, promote cell migration and enhance growth factor-induced cell proliferation in vitro, and induce angiogenesis in vivo. We previously showed that vascular endothelial cell adhesion and migration to Cyr61 and Fisp12 (mouse CTGF) are mediated through integrin αvβ3. Both Cyr61 and Fisp12/mCTGF are present in normal blood vessel walls, and it has been demonstrated that CTGF is overexpressed in advanced atherosclerotic lesions. In the present study, we examined whether Cyr61 and Fisp12/mCTGF could serve as substrates for platelet adhesion. Agonist (ADP, thrombin, or U46619)-stimulated but not resting platelets adhered to both Cyr61 and Fisp12/mCTGF, and this process was completely inhibited by prostaglandin I2, which prevents platelet activation. The specificity of Cyr61- and Fisp12/mCTGF-mediated platelet adhesion was demonstrated by specific inhibition of this process with polyclonal anti-Cyr61 and anti-Fisp12/mCTGF antibodies, respectively. The adhesion of ADP-activated platelets to both proteins was divalent cation-dependent and was blocked by RGDS, HHLGGAKQAGDV, or echistatin, but not by RGES. Furthermore, this process was specifically inhibited by the monoclonal antibody AP-2 (anti-αIIbβ3), but not by LM609 (anti-αvβ3), indicating that the interaction is mediated through integrin αIIbβ3. In a solid phase binding assay, activated αIIbβ3, purified by RGD affinity chromatography, bound to immobilized Cyr61 and Fisp12/mCTGF in a dose-dependent and RGD-inhibitable manner. In contrast, unactivated αIIbβ3 failed to bind to either protein. Collectively, these findings identify Cyr61 and Fisp12/mCTGF as two novel activation-dependent adhesive ligands for the integrin αIIbβ3 on human platelets, and implicate a functional role for these proteins in hemostasis and thrombosis.

Transient Reversal of RNA Polymerase II Active Site Closing Controls Fidelity of Transcription Elongation

To study fidelity of RNA polymerase II (Pol II), we analyzed properties of the 6-azauracil-sensitive and TFIIS-dependent E1103G mutant of rbp1 (rpo21), the gene encoding the catalytic subunit of Pol II in Saccharomyces cerevisiae. Using an in vivo retrotransposition-based transcription fidelity assay, we observed that rpb1-E1103G causes a 3-fold increase in transcription errors. This mutant showed a 10-fold decrease in fidelity of transcription elongation in vitro. The mutation does not appear to significantly affect translocation state equilibrium of Pol II in a stalled elongation complex. Primarily, it promotes NTP sequestration in the polymerase active center. Furthermore, pre-steady-state analyses revealed that the E1103G mutation shifted the equilibrium between the closed and the open active center conformations toward the closed form. Thus, open conformation of the active center emerges as an intermediate essential for preincorporation fidelity control. Similar mechanisms may control fidelity of DNA-dependent DNA polymerases and RNA-dependent RNA polymerases.

Shortening of RNA:DNA Hybrid in the Elongation Complex of RNA Polymerase Is a Prerequisite for Transcription Termination

Passage of E. coli RNA polymerase through an intrinsic transcription terminator, which encodes an RNA hairpin followed by a stretch of uridine residues, results in quick dissociation of the elongation complex. We show that folding of the hairpin disrupts the three upstream base pairs of the 8 bp RNA:DNA hybrid, a major stability determinant in the complex. Shortening the weak rU:dA hybrid from 8 nt to 5 nt causes dissociation of the complex. During termination, the hairpin does not directly compete for base pairing with the 8 bp hybrid. Thus, melting of the hybrid seems to result from spatial restrictions in RNA polymerase that couple the hairpin formation with the disruption of the hybrid immediately downstream from the stem. Our results suggest that a similar mechanism disrupts elongation complexes of yeast RNA polymerase II in vitro.

Mechanism of sequence-specific pausing of bacterial RNA polymerase

Sequence-specific pausing of multisubunit RNA polymerases (RNAPs) represents a rate-limiting step during transcription elongation. Pausing occurs on average every 100 bases of DNA. Several models have been proposed to explain pausing, including backtracking of the ternary elongation complex, delay of translocation of the enzyme along DNA, or a conformational change in the active site preventing formation of the phosphodiester bond. Here, we performed biochemical characterization of previously-reported pauses of Escherichia coli RNAP and found that they are not associated with backtracking or a translocation delay. Instead, the paused complex contains the 3′ end of the transcript in the active center and is capable of binding the next cognate NTP. However, bond formation occurs much slower in the paused complex compared with its fully-active counterpart. The pausing is dramatically decreased by a substitution of the base encoding the next incoming NTP and the base encoding the 3′ end of the nascent RNA, suggesting that (mis)-alignment of the 3′ end of the RNA and the incoming NTP in the active site is crucial for pausing. These pause sites are conserved between E. coli and Thermus thermophilus RNAPs, but are not recognized by Saccharomyces cerevisiae RNAP II, indicating that prokaryotic RNAPs might be more sensitive to the changes in the alignment of the nascent transcript and the substrate NTP in the active site.

Synergistic action of RNA polymerases in overcoming the nucleosomal barrier

During gene expression, RNA polymerase (RNAP) encounters a major barrier at a nucleosome and yet must access the nucleosomal DNA. Previous in vivo evidence has suggested that multiple RNAPs might increase transcription efficiency through nucleosomes. Here we have quantitatively investigated this hypothesis using Escherichia coli RNAP as a model system by directly monitoring its location on the DNA via a single-molecule DNA-unzipping technique. When an RNAP encountered a nucleosome, it paused with a distinctive 10–base pair periodicity and backtracked by ∼10–15 base pairs. When two RNAPs elongate in close proximity, the trailing RNAP apparently assists in the leading RNAPs elongation, reducing its backtracking and enhancing its transcription through a nucleosome by a factor of 5. Taken together, our data indicate that histone-DNA interactions dictate RNAP pausing behavior, and alleviation of nucleosome-induced backtracking by multiple polymerases may prove to be a mechanism for overcoming the nucleosomal barrier in vivo.

Mechanism of Translesion Transcription by RNA Polymerase II and Its Role in Cellular Resistance to DNA Damage

UV-induced cyclobutane pyrimidine dimers (CPDs) in the template DNA strand stall transcription elongation by RNA polymerase II (Pol II). If the nucleotide excision repair machinery does not promptly remove the CPDs, stalled Pol II creates a roadblock for DNA replication and subsequent rounds of transcription. Here we present evidence that Pol II has an intrinsic capacity for translesion synthesis (TLS) that enables bypass of the CPD with or without repair. Translesion synthesis depends on the trigger loop and bridge helix, the two flexible regions of the Pol II subunit Rpb1 that participate in substrate binding, catalysis, and translocation. Substitutions in Rpb1 that promote lesion bypass in vitro increase UV resistance in vivo, and substitutions that inhibit lesion bypass decrease cell survival after UV irradiation. Thus, translesion transcription becomes essential for cell survival upon accumulation of the unrepaired CPD lesions in genomic DNA.

Mutations in the Saccharomyces cerevisiae RPB1 Gene Conferring Hypersensitivity to 6-Azauracil

RNA polymerase II (RNAPII) in eukaryotic cells drives transcription of most messenger RNAs. RNAPII core enzyme is composed of 12 polypeptides where Rpb1 is the largest subunit. To further understand the mechanisms of RNAPII transcription, we isolated and characterized novel point mutants of RPB1 that are sensitive to the nucleotide-depleting drug 6-azauracil (6AU). In this work we reisolated the rpo21-24/rpb1-E1230K allele, which reduces the interaction of RNAPII–TFIIS, and identified five new point mutations in RPB1 that cause hypersensitivity to 6AU. The novel mutants affect highly conserved residues of Rpb1 and have differential genetic and biochemical effects. Three of the mutations affect the “lid” and “rudder,” two small loops suggested by structural studies to play a central role in the separation of the RNA–DNA hybrids. Most interestingly, two mutations affecting the catalytic center (rpb1-N488D) and the homology box G (rpb1-E1103G) have strong opposite effects on the intrinsic in vitro polymerization rate of RNAPII. Moreover, the synthetic interactions of these mutants with soh1, spt4, and dst1 suggest differential in vivo effects.

Sensitivity of Mitochondrial Transcription and Resistance of RNA Polymerase II Dependent Nuclear Transcription to Antiviral Ribonucleosides

Ribonucleoside analogues have potential utility as anti-viral, -parasitic, -bacterial and -cancer agents. However, their clinical applications have been limited by off target effects. Development of antiviral ribonucleosides for treatment of hepatitis C virus (HCV) infection has been hampered by appearance of toxicity during clinical trials that evaded detection during preclinical studies. It is well established that the human mitochondrial DNA polymerase is an off target for deoxyribonucleoside reverse transcriptase inhibitors. Here we test the hypothesis that triphosphorylated metabolites of therapeutic ribonucleoside analogues are substrates for cellular RNA polymerases. We have used ribonucleoside analogues with activity against HCV as model compounds for therapeutic ribonucleosides. We have included ribonucleoside analogues containing 2′-C-methyl, 4′-methyl and 4′-azido substituents that are non-obligate chain terminators of the HCV RNA polymerase. We show that all of the anti-HCV ribonucleoside analogues are substrates for human mitochondrial RNA polymerase (POLRMT) and eukaryotic core RNA polymerase II (Pol II) in vitro. Unexpectedly, analogues containing 2′-C-methyl, 4′-methyl and 4′-azido substituents were inhibitors of POLRMT and Pol II. Importantly, the proofreading activity of TFIIS was capable of excising these analogues from Pol II transcripts. Evaluation of transcription in cells confirmed sensitivity of POLRMT to antiviral ribonucleosides, while Pol II remained predominantly refractory. We introduce a parameter termed the mitovir (mitochondrial dysfunction caused by antiviral ribonucleoside) score that can be readily obtained during preclinical studies that quantifies the mitochondrial toxicity potential of compounds. We suggest the possibility that patients exhibiting adverse effects during clinical trials may be more susceptible to damage by nucleoside analogs because of defects in mitochondrial or nuclear transcription. The paradigm reported here should facilitate development of ribonucleosides with a lower potential for toxicity.