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Results for “Multiprotein Complexes”

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PubMed1997

A large protein complex containing the yeast Sin3p and Rpd3p transcriptional regulators.

The SIN3 gene is required for the transcriptional repression of diverse genes in Saccharomyces cerevisiae. Sin3p does not bind directly to DNA but is thought to be targeted to promoters by interacting with sequence-specific DNA-binding proteins. We show here that Sin3p is present in a large multiprotein complex with an apparent molecular mass, estimated by gel filtration chromatography, of greater than 2 million Da. Genetic studies have shown that the yeast RPD3 gene has a function similar to that of SIN3 in transcriptional regulation, as SIN3 and RPD3 negatively regulate the same set of genes. The SIN3 and RPD3 genes are conserved from yeasts to mammals, and recent work suggests that RPD3 may encode a histone deacetylase. We show that Rpd3p is present in the Sin3p complex and that an rpd3 mutation eliminates SIN3-dependent repression. Thus, Sin3p may function as a bridge to recruit the Rpd3p histone deacetylase to specific promoters.

Bacterial Proteins↗

PubMed1998

Distinct regulatory mechanisms for interferon-alpha/beta (IFN-alpha/beta)- and IFN-gamma-mediated induction of Ly-6E gene in B cells.

The murine Ly6-E gene is transcriptionally induced by interferon-alpha/beta (IFN-alpha/beta) and IFN-gamma in a variety of distinct cell types. The mechanism of IFN inducibility in B-cell lines was investigated by deletion analysis of the promoter and by identifying DNA binding proteins in mobility shift assays. A region located in the distal part of the promoter at -2.3 kb contributed to inducibility by both types of IFNs. This region contains a novel element in addition to the previously well-characterized IFN-stimulated response element (ISRE). The probes containing ISRE detected IFN-inducible complexes in mobility shift assays and the signal transducer and activator of transcripition-1 was found to be in these complexes from cells treated with either type of IFN. An additional element present in the proximal part of the promoter at position -109 is also required for IFN-alpha/beta-mediated induction. These data suggested a cooperative interaction between these physically disparate regulatory regions. A crucial role for HMGI(Y) protein in this cooperative multiprotein complex is supported by the evidence that inhibition of HMGI(Y) expression via antisense RNA results in the loss of IFN-alpha/beta-mediated induction of the Ly6-E gene. These results show the complexity involved in achieving cell-type specificity in IFN-mediated gene regulation.

Animals↗

PubMed1995

DNA loops induced by cooperative binding of transcriptional activator proteins and preinitiation complexes.

DNA conformational changes are essential for the assembly of multiprotein complexes that contact several DNA sequence elements. An approach based on atomic force microscopy was chosen to visualize specific protein-DNA interactions occurring on eukaryotic class II nuclear gene promoters. Here we report that binding of the transcription regulatory protein Jun to linearized plasmid DNA containing the consensus AP-1 binding site upstream of a class II gene promoter leads to bending of the DNA template. This binding of Jun was found to be essential for the formation of preinitiation complexes (PICs). The cooperative binding of Jun and PIC led to looping of DNA at the protein binding sites. These loops were not seen in the absence of either PICs, Jun, or the AP-1 binding site, suggesting a direct interaction between DNA-bound Jun homodimers and proteins bound to the core promoter. This direct visualization of functional transcriptional complexes confirms the theoretical predictions for the mode of gene regulation by trans-activating proteins.

Binding Sites↗

PubMed1997

A multisubunit 3' end processing factor from yeast containing poly(A) polymerase and homologues of the subunits of mammalian cleavage and polyadenylation specificity factor.

Polyadenylation is the second step in 3' end formation of most eukaryotic mRNAs. In Saccharomyces cerevisiae, this step requires three trans-acting factors: poly(A) polymerase (Pap1p), cleavage factor I (CF I) and polyadenylation factor I (PF I). Here, we describe the purification and subunit composition of a multiprotein complex containing Pap1p and PF I activities. PF I-Pap1p was purified to homogeneity by complementation of extracts mutant in the Fip1p subunit of PF I. In addition to Fip1p and Pap1p, the factor comprises homologues of all four subunits of mammalian cleavage and polyadenylation specificity factor (CPSF), as well as Ptalp, which previously has been implicated in pre-tRNA processing, and several as yet uncharacterized proteins. As expected for a PF I subunit, pta1-1 mutant extracts are deficient for polyadenylation in vitro. PF I also appears to be functionally related to CPSF, as it polyadenylates a substrate RNA more efficiently than Pap1p alone. Possibly, the observed interaction of the complex with RNA tethers Pap1p to its substrate.

Amino Acid Sequence↗

PubMed1992

Three distinct elements within the murine c-myc promoter are required for transcription.

We have undertaken a detailed analysis of the cis-acting elements that are required for optimal transcription initiation from P2, the major promoter of the murine c-myc gene. We find that three elements contribute to promoter strength, termed ME1a2, E2F and ME1a1 at positions -85, -64 and -46 respectively (relative to P2). Individually the elements are weak, but combined they contribute to full promoter activity, all acting in a positive fashion. The E2F element is the site at which the SV40 large T antigen transactivates the c-myc promoter. However, transactivation requires the presence of the ME1a2 or ME1a1 elements in addition to E2F. By a number of criteria it appears that the ME1a2 and ME1a1 elements bind the same or a very closely related protein (monomer Mr 94,000). It was found that Hela cells contain a novel factor that is capable of binding to the ME1a1 and ME1a2 elements but only in the presence of the protein-dissociating agents deoxycholate or formamide. Finally, the E2F factor binds DNA both as a monomer and as a multiprotein complex; the latter is cell type specific. Both types of bound E2F factor form less stable protein-DNA complexes than the ME1a2/ME1a1 factor.

3T3 Cells↗

PubMed1997

Constitutive and strong association of PAF53 with RNA polymerase I.

Mouse RNA polymerase I (Pol I) is composed of 14 polypeptides, 3 of which are thought to be loosely associated with, and may be dislodged from, Pol I. To find out whether these polymerase-associated factors (PAF53, 51, and 49) serve a role in growth-dependent control of rDNA transcription, we generated polyclonal antibodies against three subunits of murine Pol I, RPA116, RPA40 and PAF53, and used different experimental approaches, e.g. immunoblot analysis, immunoprecipitation and immunofluorescence studies, to compare the stoichiometry of individual subunits both in different Pol I preparations and in extracts from cells grown under different conditions. This comparative analysis reveals that the molar ratio of the second largest subunit RPA116 to PAF53 is the same, irrespective of whether crude extracts or highly purified Pol I fractions are analyzed. Significantly, the relative level of PAF53 was comparable in exponentially growing or growth-arrested cells, indicating that growth-dependent fluctuations in Pol I activity are not accompanied by alterations in the amount of PAF53. In addition, we show by high resolution immunofluorescence analysis that, under conditions of repressed rDNA transcription, including serum starvation, actinomycin treatment und during mitosis, PAF53 remains attached to the transcriptional machinery. The finding that the Mr 53,000 protein remains in the multiprotein complex under all experimental conditions tested indicates that PAF53 is not a loosely associated regulatory factor but a bona fide subunit of Pol I.

3T3 Cells↗

PubMed1995

The yeast TATA-binding protein (TBP) core domain assembles with human TBP-associated factors into a functional TFIID complex.

In mammalian and Drosophila cells, the central RNA polymerase II general transcription factor TFIID is a multisubunit complex containing the TATA-binding protein (TBP) and TBP-associated factors (TAFs) bound to the conserved TBP carboxy-terminal core domain. TBP also associates with alternative TAFs in these cells to form general transcription factors required for initiation by RNA polymerases I and III. Although extracts of human HeLa cells contain little TBP that is not associated with TAFs, free TBP is readily isolated from yeast cell extracts. However, recent studies indicate that yeast TBP can also interact with other yeast polypeptides to form multiprotein complexes. We established stable human HeLa cell lines expressing yeast TBP and several yeast-human TBP hybrids to study TBP-TAF interactions. We found that the yeast TBP core domain assembles with a complete set of human TAFs into a stable TFIID complex that can support activated transcription in vitro. The fact that the yeast TBP core, which differs from human TBP core in approximately 20% of its amino acid residues, has the structural features required to form a stable complex with human TAFs implies that Saccharomyces cerevisiae probably contains TAFs that are structurally and functionally analogous to human TAFs. Surprisingly, the non-conserved amino terminus of yeast TBP inhibited association between the yeast core domain and human TAFs.

DNA-Binding Proteins↗

PubMed1993

A developmentally regulated glycoprotein complex from Dictyostelium discoideum.

The monoclonal antibody MUD50 recognizes an epitope on a family of developmentally regulated proteins in Dictyostelium discoideum (Alexander, S., Smith, E., Davis, L., Gooley, A., Por, S. B., Browne, L., and Williams, K. L. (1988) Differentiation 38, 82-90). This epitope is an O-linked oligosaccharide that requires the wild-type modB allele for expression. These glycoproteins are structurally diverse. Some are integral membrane proteins, while others are soluble and do not require detergent for solubilization. One of the soluble glycoproteins, PsB, is also recognized by the monoclonal antibody MUD102, which binds to the polypeptide backbone. The antibody recognizes a single 100-kDa band in Western analysis. In this report we demonstrate that the PsB molecule is part of a specific developmentally regulated multiprotein complex containing six different proteins. The complex is held together by both covalent and noncovalent bonds. Interestingly, only the PsB glycoprotein has the MUD50 O-linked oligosaccharide determinant. None of the proteins in the complex is N-glycosylated as assessed by sensitivity to peptide N-glycosidase F. Only one of the complex proteins is heavily phosphorylated. This work establishes the foundation for additional studies on the biosynthesis, processing, and assembly of the O-glycosylated PsB protein.

Animals↗

PubMed1982

The Leeuwenhoek Lecture, 1981. The biochemical and genetic approach to the study of bioenergetics with the use of Escherichia coli: progress and prospects.

How the 'energy currency' of the cell, adenosine triphosphate (ATP), is produced consequent upon the oxidation of foodstuffs (oxidative phosphorylation) is, despite prolonged research, still a matter of debate and the molecular mechanism of the process is unknown. It appears that the problem of oxidative phosphorylation can be approached with the aid of the biochemical genetics of the bacterium Escherichia coli. The ease of manipulation of bacteria and definitive results obtained by this approach have been invaluable in solving other major biochemical problems. Mutants affected in oxidative phosphorylation have been isolated and characterized by genetic and biochemical techniques. These 'unc' mutants are affected in the adenosine triphosphatase (ATPase) multiprotein complex which is part of the cell membrane and responsible for the terminal stages of ATP synthesis. Seven distinct genes concerned with oxidative phosphorylation have been characterized in E. coli and shown to be part of an operon. The relationships between the different classes of unc genes and the various components of the ATPase have been established. Information about the assembly of the ATP synthesizing complex in the cell membrane has also been obtained and the stage set for further studies on the assembly, control and function of the ATP synthesizing system.

Adenosine Triphosphatases↗

PubMed1991

Purification and characterization of the membrane-associated components of the maltose transport system from Escherichia coli.

Maltose is transported across the cytoplasmic membrane of Escherichia coli by a binding protein-dependent transport system. The three membrane-associated components of the transport system, the MalK, MalF, and MalG proteins, have been solubilized from the membrane and maltose transport activity has been reconstituted in proteoliposome vesicles (Davidson, A. L., and Nikaido, H. (1990) J. Biol. Chem. 265, 4254-4260). A modification of the reconstitution technique is presented which permits reconstitution from the detergent dodecyl maltoside. Utilizing reconstitution of maltose transport as an assay, we have purified these proteins in the presence of n-dodecyl-beta-D-maltoside. The purified proteins catalyze both maltose transport activity and ATP hydrolysis. In all experiments, the MalF, MalG, and MalK proteins behaved as a multiprotein complex; all three proteins were immunoprecipitated using antibody prepared against MalF, and they copurified, eluting from a gel filtration column between markers of Mr 160,000 and 200,000. Each complex contains two MalK, one MalF, and one MalG proteins, providing two putative sites for ATP hydrolysis. Chemical cross-linking detected specific interactions between MalF and MalG and between MalF and MalK.

ATP-Binding Cassette Transporters↗

PubMed1997

Mercuric ion inhibits the activity and fidelity of the human cell DNA synthesome.

Mercuric ion is cytotoxic and mutagenic to cells; however, the mechanisms of mercuric ion-induced cytotoxicity are not well understood. Numerous studies have suggested that these effects may be due in part to the alteration and inhibition of a variety of cellular processes including DNA replication, DNA repair, RNA transcription, and protein synthesis. Studies utilizing whole cells to examine these activities are not able to specifically identify the precise mechanism or site of the effect. Other studies carried out using whole cell extracts and variously purified DNA polymerases are not able to adequately represent the highly ordered environment in which DNA replication occurs in the intact cell. We report here, for the first time, the use of an intact human cell multiprotein complex (which we have termed the DNA synthesome) to carry out full-length DNA replication and DNA synthesis in the presence of Hg2+ ion in vitro. In this study we report that DNA replication and DNA polymerase activity, as well as DNA replication fidelity of the human cell DNA synthesome, are specifically inhibited by physiologically attainable concentrations of mercuric ion.

Antigens, Viral, Tumor↗

PubMed1993

Purification of the cellular C1 factor required for the stable recognition of the Oct-1 homeodomain by the herpes simplex virus alpha-trans-induction factor (VP16).

The assembly of specific multiprotein complexes on the herpes simplex virus alpha/IE (immediate early) enhancer elements requires the interactions of the Oct-1 POU homeodomain, the viral alpha TIF (alpha-trans-induction factor) (VP16), and at least one additional cellular factor, the C1 factor. The C1 factor interacts directly with alpha TIF, likely forming an intermediate protein complex that recognizes the Oct-1 homeodomain-DNA complex. The biochemical purification of the mammalian C1 factor suggests that it is composed of multiple subunits of related, but heterogeneous, polypeptides. The interaction of a subset of these polypeptides with alpha TIF is stimulated by post-translational modifications of the C1 proteins, suggesting that this factor may be a critical target for the regulation of the herpes simplex virus alpha/IE transcription.

Amino Acid Sequence↗

PubMed1991

Isolation of coactivators associated with the TATA-binding protein that mediate transcriptional activation.

A key step in the regulation of transcription involves interactions between promoter-selective factors and various components of the transcriptional apparatus. Here we report the requirements for transcriptional activation directed by NTF-1, a developmentally regulated transcription factor in Drosophila. Reconstituted transcription with fractionated Drosophila basal factors reveals that activation by NTF-1 requires factors present in the endogenous TFIID fraction that are distinct from the purified TATA-binding protein (TBP). Glycerol gradient sedimentation and immunoprecipitation analyses indicate that TFIID is a multiprotein complex containing TBP and at least six tightly bound TBP-associated factors (TAFs). Preparations of TBP lacking TAFs after fractionation with denaturants no longer support activation by NTF-1 but retain basal level activity. Addition of immunopurified and renatured TAFs to free TBP restores the ability of NTF-1 to activate transcription without influencing basal transcription. These results suggest that one or more of the TAF polypeptides confer coactivator function.

Animals↗

PubMed1992

Control of steroid receptor function and cytoplasmic-nuclear transport by heat shock proteins.

As targeted proteins that move within the cell, the steroid receptors have become very useful probes for understanding the linked phenomena of protein folding and transport. From the study of steroid receptor-associated proteins it has become clear over the past two years that these receptors are bound to a multiprotein complex containing at least two heat shock proteins, hsp90 and hsp56. Attachment of receptors to this complex in a cell-free system appears to require the protein unfolding/folding activity of a third heat shock protein, hsp70. Like the oncogenic tyrosine kinase pp60src, steroid receptors bind to this complex of chaperone proteins at the time of their translation. Binding of the receptor to the hsp90 component of the system occurs through the hormone binding domain and is under strict hormonal control. The hormone binding domain of the receptor acts as a transferable regulatory unit that confers both tight hormonal control and hsp90 binding onto chimaeric proteins. The model of folding and transport being developed for steroid receptors leads to some general suggestions regarding the folding and transport of targeted proteins in the cell.

Animals↗

PubMed1997

Histones H3 and H4 are components of upstream activation factor required for the high-level transcription of yeast rDNA by RNA polymerase I.

RNA polymerase I (Pol I) transcription in the yeast Saccharomyces cerevisiae is greatly stimulated in vivo and in vitro by the multiprotein complex, upstream activation factor (UAF). UAF binds tightly to the upstream element of the rDNA promoter, such that once bound (in vitro), UAF does not readily exchange onto a competing template. Of the polypeptides previously identified in purified UAF, three are encoded by genes required for Pol I transcription in vivo: RRN5, RRN9, and RRN10. Two others, p30 and p18, have remained uncharacterized. We report here that the N-terminal amino acid sequence, its mobility in gel electrophoresis, and the immunoreactivity of p18 shows that it is histone H3. In addition, histone H4 was found in UAF, and myc-tagged histone H4 could be used to affinity-purify UAF. Histones H2A and H2B were not detectable in UAF. These results suggest that histones H3 and H4 probably account for the strong binding of UAF to DNA and may offer a means by which general nuclear regulatory signals could be transmitted to Pol I.

Amino Acid Sequence↗

PubMed1981

Conformation and assembly characteristics of tubulin and microtubule protein from bovine brain.

The conformational requirements for the efficient assembly of bovine brain tubulin into microtubules have been investigated by using near-UV circular dichroism. Microtubule protein was prepared by the assembly-disassembly method of Shelanski et al. [Shelanski, M. L., Gaskin, F., & Cantor, C. R. (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 765-768]. Tubulin dimer, isolated from this multiprotein complex by phosphocellulose ion-exchange chromatography in the presence and absence of Mg2+, was compared with tubulin dimer (WT dimer) prepared by the method of Weisenberg & Timasheff [Weisenberg, R. C., & Timasheff, S. N. (1970) Biochemistry 9, 4110-4116]. The tubulin from both dimer preparations showed identical electrophoretic patterns in which high molecular weight protein was undetectable. However, reproducible and significant differences were found in the near-UV CD spectra. Phosphocellulose-treated tubulin resembles the original microtubule protein more closely than does WT dimer, although this latter material has been widely accepted as being representative of the native protein. The phosphocellulose-treated tubulin and WT dimer are not readily interconvertible by simple physical or chemical treatments. The assembly capability of the various tubulin dimer preparations was compared by measuring the enhancement by tubulin dimer of assembly of ring fraction (isolated from microtubule protein by gel filtration of Sepharose 6B). Again phosphocellulose-treated tubulin is found to have more like native microtubule protein than does WT dimer.

Animals↗

PubMed1996

The human breast cell DNA synthesome: its purification from tumor tissue and cell culture.

In this report, we describe for the first time the isolation and purification of a multiprotein complex for DNA replication from MDA MB-468 human breast cancer cells. This complex, which we designate the DNA synthesome, fully supports the in vitro replication of simian virus 40 (SV40) origin-containing DNA in the presence of the viral large T-antigen. Since the SV40 virus utilizes the host's cellular proteins for its own DNA replication, our results indicate that the DNA synthesome may play a role not only in viral DNA synthesis but in human breast cell DNA replication as well. Our studies demonstrate that the following DNA replication proteins constitute the DNA synthesome: DNA polymerase alpha, DNA primase, DNA polymerase delta, proliferating cell nuclear antigen, replication protein A, replication factor C, DNA topoisomerases I, II, and DNA polymerase epsilon. In addition, we successfully isolated the DNA synthesome from human breast tumor tissue as well as from xenografts from nude mice injected with the human breast cancer cell line MCF-7. The DNA synthesome purified from the breast cancer tissues fully supports SV40 DNA replication in vitro. Furthermore, our results obtained from a novel forward mutagenesis assay suggest that the DNA synthesome isolated from a nonmalignant breast cell line mediates SV40 DNA replication by an error-resistant mechanism. In contrast, the DNA synthesome derived from malignant breast cells and tissue exhibited a lower fidelity for DNA synthesis in vitro. Overall, our data support the role of the DNA synthesome as mediating breast cell DNA replication in vitro and in vivo.

Animals↗

PubMed1997

Structure of cDNAs encoding human eukaryotic initiation factor 3 subunits. Possible roles in RNA binding and macromolecular assembly.

The mammalian translation initiation factor 3 (eIF3), is a multiprotein complex of approximately 600 kDa that binds to the 40 S ribosome and promotes the binding of methionyl-tRNAi and mRNA. cDNAs encoding 5 of the 10 subunits, namely eIF3-p170, -p116, -p110, -p48, and -p36, have been isolated previously. Here we report the cloning and characterization of human cDNAs encoding the major RNA binding subunit, eIF3-p66, and two additional subunits, eIF3-p47 and eIF3-p40. Each of these proteins is present in immunoprecipitates formed with affinity-purified anti-eIF3-p170 antibodies. Human eIF3-p66 shares 64% sequence identity with a hypothetical Caenorhabditis elegans protein, presumably the p66 homolog. Deletion analyses of recombinant derivatives of eIF3-p66 show that the RNA-binding domain lies within an N-terminal 71-amino acid region rich in lysine and arginine. The N-terminal regions of human eIF3-p40 and eIF3-p47 are related to each other and to 17 other eukaryotic proteins, including murine Mov-34, a subunit of the 26 S proteasome. Phylogenetic analyses of the 19 related protein sequences, called the Mov-34 family, distinguish five major subgroups, where eIF3-p40, eIF3-p47, and Mov-34 are each found in a different subgroup. The subunit composition of eIF3 appears to be highly conserved in Drosophila melanogaster, C. elegans, and Arabidopsis thaliana, whereas only 5 homologs of the 10 subunits of mammalian eIF3 are encoded in S. cerevisiae.

Amino Acid Sequence↗