RNA polymerase II holoenzymes have been described that consist of RNA polymerase II, a subset of general transcription factors, and four SRB proteins. The SRB proteins, which were identified through a selection for genes involved in transcription initiation by RNA polymerase II in vivo, are a hallmark of the holoenzyme. We report here the isolation and characterization of additional SRB genes. We show that the products of all nine SRB genes identified thus far are components of the RNA polymerase II holoenzyme and are associated with a holoenzyme subcomplex termed the mediator of activation. The holoenzyme is capable of responding to a transcriptional activator, suggesting a model in which activators function, in part, through direct interactions with the holoenzyme. Immunoprecipitation experiments with anti-SRB5 antibodies demonstrate that the acidic activating domain of VPI6 specifically binds to the holoenzyme. Furthermore, the holoenzyme and the mediator subcomplex bind to a VP16 affinity column. These results provide a more complete description of the RNA polymerase II holoenzyme and suggest that this form of the transcription apparatus can be recruited to promoters via direct interactions with activators.[Key Words: RNA polymerase II; holoenzyme; carboxy-terminal domain; genetic suppressors; transcription initiation; SRBs; transcription activation] Received December 13, 1994; revised version accepted March 14, 1995.Large muhisubunit complexes containing RNA polymerase II, a subset of the general transcription factors, and additional factors implicated in regulation of transcription initiation in vivo, can assemble independently of promoter DNA (Kim et al. 1994;Koleske and Young 1994). These complexes, termed RNA polymerase II holoenzymes, have been purified from Saccharomyces cerevisiae. The larger form of holoenzyme contains RNA polymerase II, TFIIB, TFIIF, TFIIH, and SRB (suppressor of RNA polymerase B) proteins (Koleske and Young 1994). Another form of holoenzyme has been described that contains RNA polymerase II, TFIIF, and SRB proteins but lacks TFIIB and TFIIH (Kim et al. 1994). The two holoenzyme forms may exist simultaneously in vivo, or the isolation of the smaller complex may be a consequence of the instability of the RNA polymerase II holoenzyme during purification.Selective transcription initiation in vitro by the 12-subunit core RNA polymerase II was shown previously to require the action of at least five general initiation factors: TATA-binding protein (TBPJ, TFIIB, TFIIE, TFIIF, and TFIIH (for review, see Conaway and Conaway 1993;Zawel and Reinberg 1993). Consistent with these data, selective transcription initiation in vitro with the larger form of RNA polymerase II holoenzyme required TBP and TFIIE (Koleske and Young 1994), and initiation with the smaller form required TBP, TFIIB, TFIIE, and TFIIH (Kim et al. 1994).The holoenzymes were discovered by virtue of their association with SRB proteins. SRB genes were obtained through a genetic selection designed to identify genes involved in RNA ...
Conditional mutations in the Saccharomyces cerevisiae RNA polymerase II large subunit, RPBJ, were obtained by introducing a mutagenized RPBI plasmid into yeast cells, selecting for loss of the wild-type RPBI gene, and screening the cells for heat or cold sensitivity. Sequence analysis of 10 conditional RPBI mutations and 10 conditional RPB2 mutations revealed that the amino acid residues altered by these distinct mutations are nearly always invariant among eucaryotic RPBJ and RPB2 homologs. These results suggest that RNA polymerase mutants might be obtained in other eucaryotic organisms by alteration of these invariant residues.Eucaryotic RNA polymerases I, II, and III are highly conserved enzymes that are responsible for rRNA, premRNA, and small stable RNA synthesis, respectively (16,25). These enzymes are each composed of two very large subunits, which account for much of the molecular mass of the enzyme, and 8 to 11 smaller proteins. The two large subunits of RNA polymerase I are similar in sequence to the two large subunits of RNA polymerases II and III and to the two large subunits of the procaryotic enzyme (2,18,26). The two large subunits of the procaryotic enzyme bind DNA and nucleoside triphosphate substrates, contain the catalytic site for RNA synthesis, and interact with the transcription factor a (8,29). Sequence similarity between the two large procaryotic and eucaryotic subunits and evidence that the eucaryotic large subunits can bind to DNA and nucleoside substrates (7, 10) suggest that the eucaryotic homologs play similar roles.RNA polymerase II is highly conserved in subunit structure and sequence among eucaryotes (14,22,25). Comparison of the sequences of the largest RNA polymerase IL subunits from Saccharomyces cerevisiae (2), Caenorhabditis elegans (5), Drosophila inelanogaster (4, 15), and the mouse (1) reveal that almost 40% of the amino acid residues are invariant. The RNA polymerase II large subunits are also similar to their procaryotic counterparts. Much of the amino acid conservation between the large eucaryotic subunit and the procaryotic RNA polymerase 3' subunit occurs in multiple segments (1,2,4,5). Sequence similarities between the second largest subunits of eucaryotic and procaryotic RNA polymerases also occur in multiple segments (11,26). The presence of multiple segments of sequence similarity may reflect the fact that these large subunits have multiple functions. Invariant amino acid residues in and around these domains probably play essential structural and functional roles.We have begun a detailed genetic investigation of the two large RNA polymerase II subunit genes in S. cerevisiae. A systematic survey of mutations in the two large subunits has permitted the isolation of mutant cells that exhibit conditional and auxotrophic phenotypes. We have found that most of the amino acids altered by these mutations involve residues that are invariant among homologous subunits from * Corresponding author. a broad range of eucaryotes and discuss the implications of this observation her...
cryptic a and Ȋ genes resident at the HML and HMR loci. As a result, sir mutant strains have the properties of a/Ȋ diploids.Non-homologous end-joining (NHEJ) is required in mammals both for V(D)J recombination 2 and for repairing doublestranded DNA breaks. NHEJ also occurs in yeast 3,4 , and it has been reported that Sir proteins are required for this process 5,6 . This observation was interpreted to mean that Sir proteins are involved directly in NHEJ, perhaps by forming a heterochromatin-like structure at double-stranded breaks. But we have found evidence for an alternative interpretation: that the a/Ȋ-state regulates NHEJ and that sir mutations affect NHEJ indirectly.To distinguish between these two possibilities, we performed plasmid-rejoining assays. Plasmids that were linearized by restriction enzymes and contained a doublestranded break in vector sequences lacking homology to the yeast genome were transformed into yeast. The frequency of transformants was used as a measure of NHEJ 5,6 . Results obtained from SIR + and sir ǁ strains were consistent with previous findings 5,6 . NHEJ in sir ǁ strains was 20-fold less efficient than in wild-type strains (Table 1). However, assays performed in SIR + and sir ǁ strains in which all mating-type genes had been inactivated by a promoter deletion (hmla∆p mata∆p hmra∆p, abbreviated here as a ǁ a ǁ a ǁ ) revealed that the absence of mating-type heterozygosity suppressed the defect in NHEJ exhibited by the sir ǁ strains (Table 1).We performed plasmid-rejoining assays on two SIR + diploid strains, an a/Ȋ diploid and a non-a/Ȋ diploid (mata∆p/MATȊ, in which only Ȋ information is expressed). The non-a/Ȋ diploid strain accomplished NHEJ tenfold more efficiently than the a/Ȋ diploid (Table 1). NHEJ was therefore controlled by mating-type heterozygosity, and no cell-type-independent effect of sir mutations was detected.The defect in NHEJ found in a/Ȋ cells indicates that a gene required for NHEJ was regulated by the a1/Ȋ2 repressor. RNA blot analysis of HDF1, HDF2, DNL4, XRS2 and MRE11, the leading candidate genes 7-10 in wild-type, sir3, a ǁ a ǁ a ǁ and a ǁ a ǁ a ǁ sir3 strains, revealed that all five genes were comparably expressed in SIR3 and sir3 strains (data not shown). These genes are therefore not relevant targets for the a1/Ȋ2 repression of NHEJ.Our results provide evidence against a direct role for heterochromatin formation in NHEJ, indicating instead that the efficiency of NHEJ is controlled by cell type. But our data do not exclude the possibility that different strains might yield different results: indeed, the W303 strain we used contains a mild rad5 mutation. However, the a/Ȋ regulation of NHEJ found here can explain problems associated with DNA repair in yeast. Diploid cells that suffer a double-stranded break have a homologous partner that can perform a homologydriven recombinational repair process. In cells that have more than one doublestranded break, NHEJ could lead to exchange-type aberrations 11 , indicating that homology-driven repair should be the pre...
The two large subunits of RNA polymerase H, RPB1 and RPB2, contain regions of extensive homology to the two large subunits of Escherichia coli RNA polymerase. These homologous regions may represent separate protein domains with unique functions. We investigated whether suppressor genetics could provide evidence for interactions between specific segments of RPB1 and RPB2 in Saccharomyces cerevisiae. A plasmid shuffle method was used to screen thoroughly for mutations in RPB2 that suppress a temperature-sensitive mutation, rpbl-1, which is located in region H of RPB1. All six RPB2 mutations that suppress rpbl-1 were clustered in region I of RPB2. The location of these mutations and the observation that they were allele specific for suppression of rpbl-l suggests an interaction between region H of RPB1 and region I of RPB2. A similar experiment was done to isolate and map mutations in RPB1 that suppress a temperature-sensitive mutation, rpb2-2, which occurs in region I of RPB2. These suppressor mutations were not clustered in a particular region. Thus, fine structure suppressor genetics can provide evidence for interactions between specific segments of two proteins, but the results of this type of analysis can depend on the conditional mutation to be suppressed.Yeast RNA polymerase II is composed of 10 polypeptide subunits whose molecular masses range from 190 to 10 kilodaltons (20). The genes that encode RPB1 and RPB2, the two largest subunits, have been cloned and sequenced (2, 21). RPB1 and RPB2 show extensive sequence similarity to the Escherichia coli RNA polymerase subunits P' and P, respectively. The areas of most extensive sequence similarity between the procaryotic and eucaryotic subunits have been termed homology regions (9). RPB1 has eight regions of homology, regions A through H (2), and RPB2 has nine, regions A through I (21). The presence of these regions of conserved amino acids has led to the suggestion that they define separate protein domains, and it is possible that each of these domains has a specific function that is conserved among RNA polymerases (18).Multiple functions have been attributed to the two large subunits of procaryotic and eucaryotic RNA polymerases. The E. coli RNA polymerase has been most extensively studied in this regard (4,23 Suppression of conditional mutations is a genetic means of identifying genes whose protein products interact with the product of a mutated gene (6). Suppression genetics has been used to reveal protein-protein interactions in the large multisubunit complexes of bacterial ribosomes (14) and eucaryotic flagella (10). It has also led to the isolation of new conditional mutations in P22 bacteriophage genes that encode structural components of the phage and permitted an analysis of the path of assembly of these components (8). The a-tubulin gene of Aspergillus nidulans was isolated by suppression of a conditional ,B-tubulin mutation (13). An actin-associating protein was isolated by suppression of a conditional mutation in actin (1). Suppressors of a Salmonel...
RasGRP3 is an exchange factor for Ras-like small GTPases that is activated in response to the second messenger diacylglycerol. As with other diacylglycerol receptors, RasGRP3 is redistributed upon diacylglycerol or phorbol ester binding. Several factors are important in determining the pattern of translocation, including the potency of the diacylglycerol analog, the affinity of the receptor for phospholipids, and in some cases, protein-protein interactions. However, little is known about the mechanisms that play a role in RasGRP3 redistribution aside from the nature of the ligand. To discover potential protein binding partners for RasGRP3, we screened a human brain cDNA library using a yeast two-hybrid approach. We identified dynein light chain 1 as a novel RasGRP3-interacting protein.The interaction was confirmed both in vitro and in vivo and required the C-terminal domain encompassing the last 127 amino acids of RasGRP3. A truncated mutant form of RasGRP3 that lacked this C-terminal domain was unable to interact with dynein light chain 1 and displayed a dramatically altered subcellular localization, with a strong reticular distribution and perinuclear and nuclear localization. These findings suggest that dynein light chain 1 represents a novel anchoring protein for RasGRP3 that may regulate subcellular localization of the exchange factor and, as such, may participate in the signaling mediated by diacylglycerol through RasGRP3.RasGRP3 is one of the members of the RasGRP family, a novel group of exchange factors that catalyzes the formation of the active, GTP-bound form of Ras-like small GTPases (1-4). RasGRP3 has the broadest substrate selectivity of all the Ras-GRP family members and is able to activate H-Ras, R-Ras, and Rap1 (3, 5). An important aspect of RasGRP3 regulation is its binding to the second messenger diacylglycerol. This binding is usually accompanied by subcellular redistribution of RasGRP3, which is believed to contribute to co-localization with its substrates (6). In fact, subcellular redistribution in response to diacylglycerol and its ultrapotent analogs, the phorbol esters, is one of the hallmarks of activation of diacylglycerol receptors, such as protein kinase C, chimaerin, and RasGRP (7). The process of translocation for a given receptor is orchestrated by a combination of factors, among them the nature and lipophilicity of the ligand (8) and the affinity for phospholipids like phosphatidylserine (9, 10). The interaction of the receptor with specific intracellular proteins can also participate in the translocation events. The role of an adaptor protein as a raft or shuttling molecule has been shown in the case of protein kinase C and its adaptor protein RACK (11). It is tempting to speculate that distinct adaptor proteins participate in translocation of the different diacylglycerol receptors, among them RasGRP3. The identification of such adaptors for Ras-GRP3 should provide insights into the mechanisms involved in RasGRP3 regulation and the subsequent activation of downstream signaling c...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.