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Query: EC:2.7.7.6 (RNA polymerase)
 34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Monoclonal antibodies were raised in mouse against native RNA polymerase A from Saccharomyces cerevisiae. After screening with the spot-immunodetection technique, 14 hybridomas were selected and the antibodies produced in mice. Their specificity, analyzed by blot-immunodetection, was found to be markedly biased towards a few RNA polymerase subunits: A135 , A49 , A43 , and A14.5. A different monoclonal antibody directed against the largest subunit, A190 , was obtained by immunizing a mouse with RNA polymerase A dissociated into its subunits with SDS. Two antibodies, which probably recognized the same antigenic determinant on subunit A135 , inhibited in vitro RNA synthesis. Inhibition was prevented by preincubation of the enzyme with DNA, suggesting a role for the A135 subunit in template binding. The antibody directed against A14.5 interacted with the A14.5 kd subunit present in all three forms of the yeast nuclear RNA polymerases but did not interfere with RNA polymerase activity. These antibody probes will be useful to study subunit function in reconstituted transcription systems.
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PMID:Probing yeast RNA polymerase A subunits with monospecific antibodies. 620 6

There is a natural variation in the molecular structure of RNA polymerase A isolated from several genetically distant yeast species, Saccharomyces cerevisiae, Saccharomyces douglasii, Schizosaccharomyces pombe, and Candida tropicalis. Several biochemical criteria were used to identify their homologous polypeptide components. Based on these correlations, the minimal subunit composition of S. cerevisiae (and Saccharomyces carlsbergensis) RNA polymerase A was tentatively defined as A190, A135, A40, A27, A23, A19, and A14.5. Without the two Saccharomyces species, S. cerevisiae and S. douglasii, 7 of 13 polypeptides of enzyme A(A49, A43, A40, A34.5, A19, A14.5, and A14) differ slightly in molecular weight and can be resolved by electrophoresis on polyacrylamide gel. The RNA polymerase A isolated from the diploid interspecific hybrid contains all the polypeptides characteristic of the two parents. One meiotic segregant had a hybrid RNA polymerase A with five of the polymorphic polypeptides (A49, A43, A19, A14.5, and A14) coming from S. douglasii and two (A40 and A34.5) from S. cerevisiae. In three successive backcrosses with S. cerevisiae, all the genes for S. douglasii polypeptides were shown to recombine although parental ditype tetrads predominated in the four four-spored asci examined. Thus, the genes for the seven polymorphic polypeptides are not clustered: they lie on at least three different chromosomes.
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PMID:Natural variation in yeast RNA polymerase A. Formation of a mosaic RNA polymerase A in a meiotic segregant from an interspecific hybrid. 704 Mar 85

RNA polymerase I of Saccharomyces cerevisiae is composed of 14 subunits. All of the corresponding genes have been cloned with the exception of the RPA14 gene encoding A14, a specific polypeptide of this enzyme. We report the cloning and the characterization of RPA14. The A14 polypeptide was separated from the other RNA polymerase I subunits by reverse-phase high pressure liquid chromatography and digested with proteinase K. Based on the amino acid sequence of one of the resulting peptides, a degenerate oligonucleotide was synthesized and used to isolate the RPA14 gene from a yeast subgenomic DNA library. RPA14 is a single copy gene that maps to chromosome IV and is flanked by CYP1 and HOM2. Disruption of RPA14 is not lethal, but growth of the rpa14::URA3 mutant strain is impaired at 37 and 38 degrees C. RNA polymerase I was purified from the rpa14::URA3 strain. After two purification steps, the enzyme did not contain the subunits A14, ABC23, and A43. This form of the enzyme was not active in a nonspecific in vitro transcription assay. These results demonstrate that A14 is a genuine subunit of RNA polymerase I and suggest that A14 plays a role in the stability of a subgroup of subunits.
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PMID:The association of three subunits with yeast RNA polymerase is stabilized by A14. 776 55

A34.5, a phosphoprotein copurifying with RNA polymerase I (Pol I), lacks homology to any component of the Pol II or Pol III transcription complexes. Cells devoid of A34.5 hardly affect growth and rRNA synthesis and generate a catalytically active but structurally modified enzyme also lacking subunit A49 upon in vitro purification. Other Pol I-specific subunits (A49, A14, and A12.2) are nonessential for growth at 30 degrees C but are essential (A49 and A12.2) or helpful (A14) at 25 or 37 degrees C. Triple mutants without A34.5, A49, and A12.2 are viable, but inactivating any of these subunits together with A14 is lethal. Lethality is rescued by expressing pre-rRNA from a Pol II-specific promoter, demonstrating that these subunits are collectively essential but individually dispensable for rRNA synthesis. A14 and A34.5 single deletions affect the subunit composition of the purified enzyme in pleiotropic but nonoverlapping ways which, if accumulated in the double mutants, provide a structural explanation for their strict synthetic lethality. A34.5 (but not A14) becomes quasi-essential in strains lacking DNA topoisomerase I, suggesting a specific role of this subunit in helping Pol I to overcome the topological constraints imposed on ribosomal DNA by transcription.
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PMID:A34.5, a nonessential component of yeast RNA polymerase I, cooperates with subunit A14 and DNA topoisomerase I to produce a functional rRNA synthesis machine. 912 26

The yeast RNA polymerase I is a multimeric complex composed of 14 distinct subunits, 5 of which are shared by the three forms of nuclear RNA polymerase. The reasons for this structural complexity are still largely unknown. Isolation of an inactive form of RNA Pol I lacking the A43, ABC23, and A14 subunits (RNA Pol I delta) allowed us to investigate the function of the shared subunit ABC23 by in vitro reconstitution experiments. Addition of recombinant ABC23 alone to the RNA Pol I delta reactivated the enzyme to up to 50% of the wild-type enzyme activity. The recombinant subunit was stably and stoichiometrically reassociated within the enzymatic complex. ABC23 was found to be required for the formation of the first phosphodiester bond, but it was not involved in DNA binding by RNA Pol I, as shown by gel retardation and surface plasmon resonance experiments, and did not recycle during transcription. Electron microscopic visualization and electrophoretic analysis of the subunit depleted and reactivated forms of the enzyme indicate that binding of ABC23 caused a major conformational change leading to a transcriptionally competent enzyme. Altogether, our results demonstrate that the ABC23 subunit is required for the structural and functional integrity of RNA Pol I and thus should be considered as part of the core enzyme.
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PMID:A shared subunit belongs to the eukaryotic core RNA polymerase. 913 31

Development of multidrug resistance (MDR) is the major obstacle to successful cancer chemotherapy. We have developed Daudi human lymphoma cells that are 20-fold more resistant than the parent cell line to vincristine (VCR) by infecting cells with pHaMDR1/A retroviral vector (Daudi/MDR20). Three DNA sequences of anti-MDR1 hammerhead ribozymes (Rzs), one cleaving codon 196 of MDR1 mRNA (196MDR1-Rz), the second a stem II base-modified (U9-->Gg, U13-->A13, G14-->A14, A18-->C18) Rz against codon 196 (196MDR1-sRz), and the third a stem II base-modified Rz directed against the -6 approximately -4 GUC sequence of the translation initiation site of the MDR1 mRNA (iMDR1-sRz), were synthesized and cloned into the retroviral vector N2A+tRNAiMet downstream of the RNA polymerase III promoter and adjacent to a tRNA gene sequence, forming the constructs N2A+tRNAiMet-196MDR1-Rz, N2A+tRNAiMet-196MDR1-sRz, and N2A+tRNAiMet-iMDR1-sRz. The three constructs were transfected into GP+envAM 12 cells for packaging the retroviral vectors. The supernatants containing the packaged retrovirus in high titers (1.1-2.5 X 10(5) CFU/ml as determined by infection of NIH 3T3 cells) were used to infect Daudi/MDR20 cells. The iMDR1-sRz- and 196MDR1-sRz-transduced Daudi/MDR20 cells completely restored chemosensitivity to VCR and doxorubicin, and were accompanied by blocked expression of MDR1 mRNA and P-glycoprotein as well as overexpression of anti-MDR1 Rz. In a cell-free system, the chimeric tRNA-sRz molecules were more stable and had more efficient catalytic activities than the corresponding naked Rz molecules. The stem II base-modified Rz were also more stable and efficient in catalytic activities than the unmodified Rz molecules. The base modification in the Rz stem II structure and the development of chimeric tRNA-Rz molecules were identified to enhance the cleavage efficacy. The combination of these two factors, together with the use of a retroviral vector, appear to have contributed to the complete reversal of MDR.
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PMID:Retrovirus-mediated transfer of anti-MDR1 ribozymes fully restores chemosensitivity of P-glycoprotein-expressing human lymphoma cells. 1034 May 50

The spatial distribution of four subunits specifically associated to the yeast DNA-dependent RNA polymerase I (RNA pol I) was studied by electron microscopy. A structural model of the native enzyme was determined by cryo-electron microscopy from isolated molecules and was compared with the atomic structure of RNA pol II Delta 4/7, which lacks the specific polypeptides. The two models were aligned and a difference map revealed four additional protein densities present in RNA pol I, which were characterized by immunolabelling. A protruding protein density named stalk was found to contain the RNA pol I-specific subunits A43 and A14. The docking with the atomic structure showed that the stalk protruded from the structure at the same site as the C-terminal domain (CTD) of the largest subunit of RNA pol II. Subunit A49 was placed on top of the clamp whereas subunit A34.5 bound at the entrance of the DNA binding cleft, where it could contact the downstream DNA. The location of the RNA pol I-specific subunits is correlated with their biological activity.
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PMID:Localization of the yeast RNA polymerase I-specific subunits. 1214 13

A43, an essential subunit of yeast RNA polymerase I (pol I), interacts with Rrn3, a class I general transcription factor required for rDNA transcription. The pol I-Rrn3 complex is the only form of enzyme competent for promoter-dependent transcription initiation. In this paper, using biochemical and genetic approaches, we demonstrate that the A43 polypeptide forms a stable heterodimer with the A14 pol I subunit and interacts with the common ABC23 subunit, the yeast counterpart of the omega subunit of bacterial RNA polymerase. We show by immunoelectronic microscopy that A43, ABC23, and A14 colocalize in the three-dimensional structure of the pol I, and we demonstrate that the presence of A43 is required for the stabilization of both A14 and ABC23 within the pol I. Because the N-terminal half of A43 is clearly related to the pol II Rpb7 subunit, we propose that the A43-A14 pair is likely the pol I counterpart of the Rpb7-Rpb4 heterodimer, although A14 distinguishes from Rpb4 by specific sequence and structure features. This hypothesis, combined with our structural data, suggests a new localization of Rpb7-Rpb4 subunits in the three-dimensional structure of yeast pol II.
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PMID:The A14-A43 heterodimer subunit in yeast RNA pol I and their relationship to Rpb4-Rpb7 pol II subunits. 1240 81

In the archaeal RNA polymerase and the eukaryotic RNA polymerase II, two subunits (E/F and RPB4/RPB7, respectively) form a heterodimer that reversibly associates with the core of the enzyme. Recently it has emerged that this heterodimer also has a counterpart in the other eukaryotic RNA polymerases: in particular two subunits of RNA polymerase I (A14 and A43) display genetic and biochemical characteristics that are similar to those of the RPB4 and RPB7 subunits, despite the fact that only A43 shows some sequence homology to RPB7. We demonstrate that the sequence of A14 strongly suggests the presence of a HRDC domain, a motif that is found at the C-terminus of a number of helicases and RNases. The same motif is also seen in the structure of the F subunit, suggesting a structural link between A14 and the RPB4/C17/subunit F family, even in the absence of direct sequence homology. We show that it is possible to co-express and co-purify large amounts of the recombinant A14/A43 heterodimer, indicating a tight and specific interaction between the two subunits. To shed light on the function of the heterodimer, we performed gel mobility shift assays and showed that the A14/A43 heterodimer binds single-stranded RNA in a similar way to the archaeal E/F complex.
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PMID:Structural and functional homology between the RNAP(I) subunits A14/A43 and the archaeal RNAP subunits E/F. 1288 98

The architecture of eukaryotic rRNA transcription complexes was analyzed, revealing facts significant to the RNA polymerase (pol) I initiation process. Functional initiation and elongation complexes were mapped by site-specific photocross-linking to template DNA. Polymerase I is recruited to the promoter via protein-protein interactions with DNA-bound transcription initiation factor-IB. The latter's TATA-binding protein (TBP) and TAFs photocross-link to the promoter from -78 to +10 relative to the tis (+1). Although TBP does not bind DNA using its TATA-binding saddle, it does photocross-link to a 22-bp sequence that does not resemble a TATA box. Only TAF(I)96 (the mammalian TAF(I) 68, yeast Rrn7p homolog) overlaps significantly with the DNA interaction cleft of pol I based on modeling to the pol II crystal structure. None of the pol I-specific subunits that are localized on the lips of the cleft (A49 and A34.5) or the pol I-specific stalk (A43 and A14) cross-link to DNA. Pol I does not extend significantly upstream of the promoter-proximal border of the factor complex (-11 to -14), and similarly in the promoter proximal elongation complex, the enzyme does not contact DNA upstream of its normal exit from the cleft.
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PMID:Photocross-linking of the RNA polymerase I preinitiation and immediate postinitiation complexes: implications for promoter recruitment. 1516 19


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