50S Complex Research Articles

Nucleolar proteins Bfr2 and Enp2 interact with DEAD-box RNA helicase Dbp4 in two different complexes

Different pre-ribosomal complexes are formed during ribosome biogenesis, and the composition of these complexes is highly dynamic. Dbp4, a conserved DEAD-box RNA helicase implicated in ribosome biogenesis, interacts with nucleolar proteins Bfr2 and Enp2. We show that, like Dbp4, Bfr2 and Enp2 are required for the early processing steps leading to the production of 18S ribosomal RNA. We also found that Bfr2 and Enp2 associate with the U3 small nucleolar RNA (snoRNA), the U3-specific protein Mpp10 and various pre-18S ribosomal RNA species. Thus, we propose that Bfr2, Dbp4 and Enp2 are components of the small subunit (SSU) processome, a large complex of ∼80S. Sucrose gradient sedimentation analyses indicated that Dbp4, Bfr2 and Enp2 sediment in a peak of ∼50S and in a peak of ∼80S. Bfr2, Dbp4 and Enp2 associate together in the 50S complex, which does not include the U3 snoRNA; however, they associate with U3 snoRNA in the 80S complex (SSU processome). Immunoprecipitation experiments revealed that U14 snoRNA associates with Dbp4 in the 50S complex, but not with Bfr2 or Enp2. The assembly factor Tsr1 is not part of the ‘50S’ complex, indicating this complex is not a pre-40S ribosome. A combination of experiments leads us to propose that Bfr2, Enp2 and Dbp4 are recruited at late steps during assembly of the SSU processome.

639 Optimizing pathological diagnosis with new biological tools: examples in breast cancer

We have investigated the effects of intermolecular disulfide crosslinking and temperature-dependent insolubilization of nuclear proteins in vitro on the association of the polyoma large T antigen with the nuclear matrix in polyomavirus-infected mouse 3T6 cells. Nuclear matrices, prepared from polyomavirus-infected 3T6 cells by sequential extraction of isolated nuclei with 1% Triton X-100 (Triton wash), DNase I, and 2 M NaCl (high salt extract) at 4 °C, represented 18% of total nuclear protein. Incubation of nuclei with 1 mM sodium tetrathionate (NaTT) to induce disulfide crosslinks or at 37 °C to induce temperature-dependent insolubilization prior to extraction, transferred an additional 9–18% of the nuclear protein from the high salt extract to the nuclear matrix. This additional protein represented primarily an increased recovery of the same nuclear protein subset present in nuclear matrices prepared from untreated nuclei. Major constituents of chromatin including histones, hnRNP core proteins, and 98% of nuclear DNA were removed in the high salt extract following either incubation. Polyoma large T antigen was quantified in subcellular fractions by immunoblotting with rat anti-T ascites. Approximately 60–70% of the T antigen was retained in nuclei isolated in isotonic sucrose buffer at pH 7.2. Most (>95 %) of the T antigen retained in untreated nuclei was extracted by DNase-high salt treatment. Incubation at 37 °C or with NaTT transferred most (>95%) of the T antigen to the nuclear matrix. T antigen solubilized from NaTT-treated matrices with 1% SDS sedimented on sucrose gradients as a large (50-S) complex. These complexes, isolated by immunoprecipitation with anti-T sera, were dissociated by reduction with 2-mercaptoethanol, and SDS-PAGE analysis revealed that T antigen was crosslinked in stoichiometric amounts to several host proteins: 150, 129, 72, and 70 kDa. These host proteins were not present in anti-T immunoprecipitates of solubilized nuclear matrices prepared from iodoacetamidetreated cells. Our results suggest that the majority of polyomavirus large T antigen in infected cells is localized to a specific subnuclear domain which is distinct from the bulk chromatin and is closely associated with the nuclear matrix.

Antimicrobial and clinical effect of linezolid (ZYVOX), new class of synthetic antibacterial drug

Linezolid (ZYVOX), a novel synthesized antibacterial drug, was first approved in April 2001, as an antibacterial against vancomycin (VCM)--resistant enterococci in Japan. LZD has a wide spectrum of antibacterial activity against gram-positive bacteria with MIC90 of 0.5-4 mcg/mL. These antibacterial activities of LZD are similar to those of vancomycin (VCM). LZD also has similar antibacterial activities against drug-resistant bacteria including VRE and MRSA. Protein-synthesis inhibitors, e.g., macrolides, tetracycline, aminoglycosides, and chloramphenicol, are known to bind the 30S and 50S subunits of ribosomes and inhibit the elongation cycle of protein synthesis. In contrast, LZD was found to inhibit the process of formation of the 50S, 30S-mRNA, and fMet-tRNA complex in the ribosome cycle, but not the elongation cycle. Due to this novel mechanism of action, LZD does not have a cross-resistance to drug-resistant bacteria and development of its resistance is quite slow. The antibacterial activity of LZD against VRE is bacteriostatic. In vivo antibacterial activity of orally administered LZD was demonstrated in a mouse model of systemic infection by VRE. When administered orally prior to the abscess formation in a mouse model of soft tissue infection by VRE, LZD showed similar antibacterial activity against VRE infection to that against VCM-susceptible enterococci. LZD is rapidly absorbed following oral administration and bioavailability when compared with intravenous administration is almost 100%. LZD administered orally twice-daily showed excellent efficacy in clinical trials with VRE-infected patients.

Identification of 50S components neighboring 23S rRNA nucleotides A2448 and U2604 within the peptidyl transferase center of Escherichia coli ribosomes.

The 23S rRNA nucleotides 2604-12 and 2448-58 fall within the central loop of domain V, which forms a major part of the peptidyl transferase center of the ribosome. We report the synthesis of radioactive, photolabile 2'-O-methyloligoRNAs, PHONTs 1 and 2, complementary to these nucleotides and their exploitation in identifying 50S ribosomal subunit components neighboring their target sites. Photolysis of the 50S complex with PHONT 1, complementary to nts 2604-12, leads to target site-specific photoincorporation into protein L2 and 23S rRNA nucleotides A886, Alpha1918, A1919, G1922-C1924, U2563, U2586, and C2601. Photolysis of the 50S complex with PHONT 2, complementary to nts 2448-58, leads to target site-specific probe photoincorporation into proteins L2, L3, one or more of proteins L17, L18, L21, and of proteins L9, L15, L16, and 23S rRNA nucleotides C2456 and psi2457. Chemical footprinting studies show that 2'-O-methyloligoRNA binding causes little distortion of the peptidyl transferase center but do provide suggestive evidence for the location of flexible regions within 23S rRNA. The significance of these results for the structure of the peptidyl transferase center is considered.

Host and phi X 174 mutations affecting the morphogenesis or stabilization of the 50S complex, a single-stranded DNA synthesizing intermediate.

The morphogenetic pathway of bacteriophage phi X 174 was investigated in rep mutant hosts that specifically block stage III single-stranded DNA synthesis. The defects conferred by the mutant rep protein most likely affect the formation or stabilization of the 50S complex, a single-stranded DNA synthesizing intermediate, which consists of a viral prohead and a DNA replicating intermediate (preinitiation complex). phi X 174 mutants, ogr (rep), which restore the ability to propagate in the mutant rep hosts, were isolated. The org (rep) mutations confer amino acid substitutions in the viral coat protein, a constituent of the prohead, and the viral A protein, a constituent of the preinitiation complex. Four of the six coat protein substitutions are localized on or near the twofold axis of symmetry in the atomic structure of the mature virion.

Sedimentation analysis of polyadenylation-specific complexes.

Precursor RNA containing the adenovirus L3 polyadenylation site is assembled into a 50S complex upon incubation with HeLa nuclear extract at 30 degrees C. The cofactor and sequence requirements for 50S complex formation are similar to those of the in vitro polyadenylation reaction. Assembly of this complex requires ATP but is not dependent upon synthesis of a poly(A) tract. In addition, a 50S complex does not form on substrate RNA in which the AAUAAA hexanucleotide upstream of the poly(A) site has been mutated to AAGAAA or on RNA in which sequences between +5 and +48 nucleotides downstream of the site have been removed. These mutations also prevent in vitro processing of substrate RNA. Kinetic studies suggest that the 50S complex is an intermediate in the polyadenylation reaction. It forms at an early stage in the reaction and at later times contains both poly(A)+ RNA as well as unreacted precursor. U-type small nuclear ribonucleoprotein particles are components of the 50S complex, as shown by immunoprecipitation with antiserum specific to the trimethyl cap of these small nuclear RNAs.

Sedimentation analysis of polyadenylation-specific complexes.

Precursor RNA containing the adenovirus L3 polyadenylation site is assembled into a 50S complex upon incubation with HeLa nuclear extract at 30 degrees C. The cofactor and sequence requirements for 50S complex formation are similar to those of the in vitro polyadenylation reaction. Assembly of this complex requires ATP but is not dependent upon synthesis of a poly(A) tract. In addition, a 50S complex does not form on substrate RNA in which the AAUAAA hexanucleotide upstream of the poly(A) site has been mutated to AAGAAA or on RNA in which sequences between +5 and +48 nucleotides downstream of the site have been removed. These mutations also prevent in vitro processing of substrate RNA. Kinetic studies suggest that the 50S complex is an intermediate in the polyadenylation reaction. It forms at an early stage in the reaction and at later times contains both poly(A)+ RNA as well as unreacted precursor. U-type small nuclear ribonucleoprotein particles are components of the 50S complex, as shown by immunoprecipitation with antiserum specific to the trimethyl cap of these small nuclear RNAs.

Protein disulfide crosslinking stabilizes a polyoma large T antigen-host protein complex on the nuclear matrix

We have investigated the effects of intermolecular disulfide crosslinking and temperature-dependent insolubilization of nuclear proteins in vitro on the association of the polyoma large T antigen with the nuclear matrix in polyomavirus-infected mouse 3T6 cells. Nuclear matrices, prepared from polyomavirus-infected 3T6 cells by sequential extraction of isolated nuclei with 1% Triton X-100 (Triton wash), DNase I, and 2 M NaCl (high salt extract) at 4 °C, represented 18% of total nuclear protein. Incubation of nuclei with 1 m M sodium tetrathionate (NaTT) to induce disulfide crosslinks or at 37 °C to induce temperature-dependent insolubilization prior to extraction, transferred an additional 9–18% of the nuclear protein from the high salt extract to the nuclear matrix. This additional protein represented primarily an increased recovery of the same nuclear protein subset present in nuclear matrices prepared from untreated nuclei. Major constituents of chromatin including histones, hnRNP core proteins, and 98% of nuclear DNA were removed in the high salt extract following either incubation. Polyoma large T antigen was quantified in subcellular fractions by immunoblotting with rat anti-T ascites. Approximately 60–70% of the T antigen was retained in nuclei isolated in isotonic sucrose buffer at pH 7.2. Most (>95 %) of the T antigen retained in untreated nuclei was extracted by DNase-high salt treatment. Incubation at 37 °C or with NaTT transferred most (>95%) of the T antigen to the nuclear matrix. T antigen solubilized from NaTT-treated matrices with 1% SDS sedimented on sucrose gradients as a large (50-S) complex. These complexes, isolated by immunoprecipitation with anti-T sera, were dissociated by reduction with 2-mercaptoethanol, and SDS-PAGE analysis revealed that T antigen was crosslinked in stoichiometric amounts to several host proteins: 150, 129, 72, and 70 kDa. These host proteins were not present in anti-T immunoprecipitates of solubilized nuclear matrices prepared from iodoacetamidetreated cells. Our results suggest that the majority of polyomavirus large T antigen in infected cells is localized to a specific subnuclear domain which is distinct from the bulk chromatin and is closely associated with the nuclear matrix.

Stepwise assembly of a pre-mRNA splicing complex requires U-snRNPs and specific intron sequences

We have investigated the early events of pre-mRNA splicing in vitro by sucrose gradient sedimentation analysis. Time course experiments revealed the assembly, in two steps, of a large (50S) pre-mRNA splicing complex, preceded by formation of two other complexes that sediment at approximately 22S and 35S. Pre-mRNA and the intermediates and products of the in vitro splicing reaction cosediment with the 50S complex, while only pre-mRNA is associated with the 22S and 35S complexes. No splicing is observed in the absence of a 50S complex. Formation of the 50S complex requires ATP, whereas formation of the 22S and 35S complexes does not. U-snRNPs are necessary for assembly of the 35S and the 50S complexes but not for assembly of the 22S complex. Analysis with mutant substrate RNAs demonstrated that a polypyrimidine stretch near the 3' splice site and an intact 5' splice site are absolutely required for splicing complex formation.

Studies on the functional relationship between .PHI.X174 and S13 coded proteins using an in vitro DNA synthesis system.

The functional relationship between bacteriophage φX174 and SI3 gene products was investigated using an in vitro DNA synthesis system in two stages: (a), replication of the closed circular duplex form DNA (RF to RF) and (b) synthesis of single-stranded DNA from RF and formation of the intermediate 50S complex in phage morphogenesis (RF to 50S complex). Partially purified gene A products of φX174 and S13 were able to function reciprocally in the stage from RF to RF replication. In the stage of RF to 50S complex, extracts from Escherichia coli infected with φX174 catalyzed the formation of 50S complex from S13 RF DNA. Extracts from E. coli infected with SI3 also had the ability to catalyze the formation of 50S complex from φX174 RF DNA. Thus, all gene products required for DNA replication and phage morphogenesis were found to function reciprocally between φX174 and S13.

Virus-Like 30S RNA in Mouse Cells

Uninfected JLS-V9 mouse cells are known to express high levels of viral sequences that hybridize to complementary DNA made by the BrdU-induced virus of JLS-V9 cells. The genome in the BrdU-induced virus has been found to consist mainly of an RNA species that migrates as 30S RNA material during electrophoresis through agarose gels. This virus-like 30S RNA, designated VL30 RNA, apparently represents a new class of endogenous defective retroviruses that are not generally evident because of their defectiveness and lack of biological function. Fingerprint analysis and hybridization studies show that VL30 RNA does not have homology with the standard nondefective murine leukemia viruses. Upon superinfection with a nondefective murine leukemia virus, or upon induction of endogenous virus with BrdU, VL30 RNA is rescued into virions by phenotypic mixing. When VL30 RNA is rescued by BrdU induction, the VL30 RNA is mainly organized as a 50S complex, but when VL30 is rescued by superinfection, VL30 is also found in 70S RNA. Rescued VL30 RNA sequences can be reverse transcribed by the virion-associated DNA polymerase in an endogenous reaction. Many mouse cells express the sequences, whereas heterologous cells such as rat or rabbit cells do not contain them. By using hybridization of a complementary DNA probe to cellular RNA immobilized on paper, no subgenomic RNA related to the VL30 RNA could be found in cells expressing the VL30 sequences. From 20 to 50 copies of these sequences were found to be contained in the mouse genome. VL30 RNA is probably present in most stocks of leukemia and sarcoma viruses made in mouse cells.

Synthesis of infectious phiX-174 bacteriophage in vitro.

STUDY of virus assembly has indicated the involvement of a diverse spectrum of protein–protein and protein–nucleic acid interactions (for review see ref. 1). In Escherichia coli cells infected with bacteriophage ΦX-174, synthesis of single-stranded circular viral DNA requires the functions of at least seven phage-coded genes which include genes specifying viral structural proteins F, G and H non-virion proteins A, B, C and D. The only known phage gene that is not involved in single-stranded DNA synthesis is gene E (lysis) (for review see ref. 2). It seems that synthesis of single-stranded circular viral DNA of ΦX-174 is very tightly coupled with the phage morphogenetic pathway. The synthesis of ΦX-174 viral DNA involves a replicative form (RF) DNA with an extended tail of single-stranded viral DNA up to one genome in length (‘rolling circle’ or a structure DNA)3–7. Fujisawa and Hayashi8,9 found that σ DNA in infected cells is associated with a number of phage and host proteins as a 50S complex which is a precursor of mature phage particles. They have proposed that synthesis and packaging of ΦX-174 single-stranded DNA occur in the 50S complex. Formation of the 50S complex requires the association of a functional RF DNA template with a 108S capsomeric structure containing the protein products of genes D, F, G and H (ref. 10). The functions of genes B and C are also required for formation of the 50S complex. The gene B protein is necessary to catalyse assembly of gene F and gene G proteins11 to form a precursor of the 108S structure10–12. In the absence of gene C protein, the 108S structure is produced but the 50S complex is not formed10. Apparently, RF DNA cannot act as a functional template for formation of the SOS complex and, consequently, for synthesis of single-stranded DNA if the gene C protein is absent. To elucidate the function of ΦX-174-coded proteins during phage morphogenesis and single-stranded DNA synthesis, we have developed an in vitro system that synthesizes circular, single-stranded viral DNA13. The system contained an extract prepared from cells infected with a lysis-defective mutant of ΦX-174. We show here that synthesis of circular viral DNA in the system does depend on the functions of the proteins encoded by ΦX-174 genes B and C. The circular single-stranded viral DNA synthesised in vitro is contained in infectious particles whose appearance is very much like that of mature ΦX-174 phage particles when examined by electron microscopy.

Synthesis of phiX174 viral DNA in vitro depends on phiX replicative form DNA.

A cell-free system that catalyzes phiX174 replicative form I (supercoiled circular duplex, RFI)-dependent phiX174 DNA synthesis has been isolated from Escherichia coli infected with phiX174 phage. The products formed with such preparations are viral strands as judged by hybridization to poly(U,G) followed by equilibrium centrifugation in CsCl. This phiX174 DNA-synthesizing involves formation of DNA-protein complexes that sediment in neutral sucrose with S values of 50, 60-70, and higher. The 50S complex contained a rolling-circle replicative intermediate DNA with an extended tail of single-stranded viral DNA. The DNA contained in the 60-70S region was a mixture of circular and linear single-stranded DNA, RFI, and RFII with an extended single-stranded tail. Such complexes have been isolated during in vivo progeny phiX174 DNA synthesis [Fujisawa, H. & Hayashi, M. (1976) J. Vriol. 19,409]. In vitro, maximal phiX174 DNA synthesis was shown to require the genetically defined proteins E. coli dna B, dna C, dna G, dna Z, rep. phiX174 gene A product, and other phiX174 coded proteins. The synthesis of phiX174 DNA is ATP-dependent and is inhibited by nalidixic acid and novobiocin but is resistant to rifampicin.

Gene A product of phi X174 is required for site-specific endonucleolytic cleavage during single-stranded DNA synthesis in vivo.

A functional gene A product of phi X174 was found to be required at the stage of single-stranded DNA synthesis. A precursor complex that synthesizes single-stranded DNA (50S complex [Fujisawa and Hayashi, 1976]) was isolated from cells infected with wild-type or with temperature-sensitive gene A mutant phage. Proper cleavage of the single-stranded viral DNA did not occur in cells infected with the temperature-sensitive gene A mutant under restrictive conditions. This resulted in (i) accumulation of linear viral DNA molecules of 2 units in length in the 50S complex and (ii) cessation of elongation of viral-strand DNA after one complete cycle of single-stranded DNA synthesis.

Viral DNA-synthesizing intermediate complex isolated during assembly of bacteriophage phi X174.

A DNA protein complex that is a precursor of mature phi X174 phage was isolated. The complex sedimented with an S value of 50 in a sucrose gradient and contained phage DNA consisting of a replicative form molecule with an extended tail of single-stranded viral DNA. The viral-strand DNA ranged from one to two genomes in length. Proteins coded on the phi X174 genome as well as the host genome were associated with the viral DNA in the 50S precursor complex. Our results indicated that both viral DNA synthesis and cleavage of the growing viral-strand DNA occurred in the 50S complex.