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J Greenblatt

Publications and source records attributed to J Greenblatt.

At least 91 records · Page 5Linked to original sources
PubMed1988

Purification and properties of the NusB protein of Escherichia coli.

Mutations in the nusB gene of Escherichia coli block transcriptional antitermination mediated by the N gene protein of bacteriophage lambda. We describe here two methods of overproducing the NusB protein in E. coli and a method of purifying NusB to apparent homogeneity on a large scale. Purified NusB directly stimulates transcriptional antitermination by the lambda N protein in vitro. It behaves as a monomer (Mr = 15,689) during gel permeation chromatography and gradient sedimentation. The number of NusB molecules in a wild type E. coli K12 cell ranges from about 3,000 to about 6,000 molecules/cell, depending on the growth medium, and is about 50-80% of the number of molecules of the core component of RNA polymerase in the cell. This implies that NusB has a major role in regulating chain elongation during the transcription of E. coli genes. Many E. coli strains with nusB mutations cannot grow at low temperature. However, a sup+ strain with the suppressible amber mutation nusBam115 can grow at 42 degrees C. Since such a strain does not produce NusB protein detectable by immunoprecipitation with anti-NusB, normal amounts of NusB are not essential for the survival of E. coli at 42 degrees C.

Bacterial Proteins↗
PubMed1988

RAP30/74: a general initiation factor that binds to RNA polymerase II.

We have previously shown by affinity chromatography that RAP30 and RAP74 are the mammalian proteins that have the highest affinity for RNA polymerase II. Here we show that RAP30 binds to RAP74 and that the RAP30-RAP74 complex (RAP30/74) is required for accurate initiation by RNA polymerase II. RAP30/74 is required for accurate transcription from the following promoters: the adenovirus major late promoter, the long terminal repeat of human immunodeficiency virus, P2 of the human c-myc gene, the mouse beta maj-globin promoter (all of which have TATA boxes), and the mouse dihydrofolate reductase promoter (which lacks a TATA box). RAP30/74 is not required for initiation by RNA polymerase III at the adenovirus virus-associated RNA promoters. Therefore, RAP30/74 is a general initiation factor that binds to RNA polymerase II.

Carrier Proteins↗
PubMed1987

An elongation control particle containing the N gene transcriptional antitermination protein of bacteriophage lambda.

The N gene transcriptional antitermination protein of bacteriophage lambda is incorporated in vitro into transcriptional elongation complexes containing the E. coli proteins NusA and NusB. The binding of NusA to elongating RNA polymerase is sequence-independent and follows the release of sigma 70. Incorporation of N into the elongation complex requires an N utilization site (nut site) on the DNA template. Incorporation of NusB into the complex requires NusA, ribosomal protein S10, and the boxA component of the nut site. T1 RNAase releases N, but not NusB, from the elongation complex. We therefore propose that an N-modified termination-resistant elongation complex includes an elongation control particle (ECP) containing at least NusA, NusB, S10, N, and an RNA transcript of the nut site.

Bacterial Proteins↗
PubMed1986

Proteins that bind to RNA polymerase II are required for accurate initiation of transcription at the adenovirus 2 major late promoter.

Extracts prepared from HeLa cell nuclei are inactivated for accurate transcription from the adenovirus 2 major late promoter (Ad2 MLP) by repeated passage over a column containing immobilized calf thymus RNA polymerase II. The capacity for accurate transcription is restored to these extracts by a fraction that is eluted from the RNA polymerase II column with 0.5 M KC1. This fraction contains RNA polymerase II-associating proteins (RAPs). RAPs are not required for the formation of a stable, promoter-associated pre-initiation complex. However, at least one RAP is required for the formation of the first phosphodiester bond at the Ad2 MLP, both when initiation depends only on the TATAAA box, and when initiation is stimulated by the upstream sequence of the promoter. Initiation occurs approximately 1 min after the addition of RAPs to initiation complexes formed in the absence of RAPs. Therefore, the RAP(s) that is an RNA polymerase II initiation factor functions late in the initiation pathway to convert a stable preinitiation complex to an initiation-competent complex.

Adenoviruses, Human↗
PubMed1985

Isolation of three proteins that bind to mammalian RNA polymerase II.

We have used affinity chromatography on columns containing immobilized calf thymus RNA polymerase II to isolate three phosphoproteins (RAP72, RAP38, and RAP30) that bind directly to RNA polymerase II. All could be isolated from cell nuclei, and all three could be detected in mouse and human tissue culture cell lines, but only RAP38 and RAP30 have so far been isolated from calf thymus. RAP38 stimulates nonspecific transcription of native DNA templates by RNA polymerase II in the presence of Mn2+; it appears to be similar or identical to SII, a previously identified RNA polymerase II stimulatory factor (Nakanishi, Y., Mitsuhashi, Y., Sekimizu, K., Yokoi, H., Tanaka, Y., Horikoshi, M., and Natori, S. (1981) FEBS Lett. 130, 69-72). Unlike RAP38, RAP72 and RAP30 do not affect nonspecific transcription by RNA polymerase II. However, RAP30 may have a role in regulating some alterations of transcription that accompany cellular differentiation; RAP30 is partially dephosphorylated when murine erythroleukemia cells are induced with dimethyl sulfoxide to undergo terminal erythroid differentiation. We suggest that phosphate groups in RNA polymerase II-binding proteins may regulate transcription by modulating the interaction of RNA polymerase II with other regulatory proteins that possess sequence recognition specificity.

Adrenal Cortex Neoplasms↗
PubMed1985

Efficient modification of E. coli RNA polymerase in vitro by the N gene transcription antitermination protein of bacteriophage lambda.

The N gene protein of bacteriophage lambda prevents termination of transcription by E. coli RNA polymerase. We describe here the conditions of a cell-free reaction system in which pure N stimulates net transcription up to tenfold and therefore nearly stoichiometrically modifies transcribing RNA polymerase molecules. The reaction contains micrococcal nuclease-treated S100 extract derived from E. coli and a plasmid template DNA containing the lambda early promoter PL, the N utilization site nutL, and the Rho-dependent terminator tL1. Stimulation by N in this system is specific and biologically relevant since it is absent with vector pBR322 DNA and with extracts derived from E. coli strains bearing the nusA1 and nusE71 mutations known to block N function in vivo. We use the system to provide further evidence that ribosomes are not necessary for N function and to demonstrate the direct involvement in N function of the NusA protein of E. coli.

Ammonia↗
PubMed1982

Properties of the N gene transcription antitermination protein of bacteriophage lambda.

The product of the N gene of bacteriophage lambda prevents the termination of lambda early transcription. Here we describe the physical properties of pure lambda N protein. N protein is small and very basic. The apparent Mr of N protein during electrophoresis in the presence of sodium dodecyl sulfate is 12,500. It contains 22 mol % (arginine plus lysine) and only one methionine. The methionine residue is at the blocked NH2 terminal since N protein is not detectably shortened by reaction with cyanogen bromide. When use is made of the DNA sequence of the N gene region of lambda DNA (Franklin, N. C., and Bennett, G. N. (1979) Gene 8, 107-119), the lack of an internal methionine residue, the size, and the amino acid composition of N protein can be used to predict that N protein contains 107 amino acids (calculated Mr = 12,241) and that its coding sequence begins at position 223 of the lambda pL operon mRNA. N protein can be assayed by its ability to stimulate endolysin synthesis in vitro in a reaction programmed with lambda N- DNA. N protein activity is heat-stable and trypsin-sensitive. Its sedimentation velocity in a sucrose gradient and its Stokes' radius indicate that N protein is an extremely asymmetric monomer (f/fmin = 1.6). The relationship between this high degree of molecular asymmetry and the sequence which N protein must recognize in lambda nucleic acid is discussed.

Amino Acids↗
PubMed1982

Effects of NusA protein on transcription termination in the tryptophan operon of Escherichia coli.

Termination of transcription at the end of the tryptophan (trp) operon of E. coli at the trp t site is very efficient in vivo, but is only 25% efficient in vitro. To try to resolve this discrepancy, we have altered numerous parameters and report here on the modifications that bring the in vitro results into closer agreement with the in vivo ones. Lowering the concentration of UTP (but not ATP, CTP or GTP) in the transcription mix can greatly improve termination at trp t. With three other terminators structurally similar to trp t, there is no detectable effect of reducing the concentration of any of the four triphosphates. This response at trp t to low UTP is therefore both nucleotide-specific and terminator-specific, suggesting that apparently minor structural differences may still have profound effects upon termination. Increased specificity and sensitivity may also be provided by the NusA protein, which causes RNA polymerase to pause at trp t and at the 1:2 stem of the trp attenuator. NusA protein also enhances termination at trp t, an effect similar to the low UTP response. Further, termination can be slightly improved by including rho factor, resulting in an overall efficiency of almost 100% at trp t.

Bacterial Proteins↗
PubMed1981

Termination of transcription by nusA gene protein of Escherichia coli.

The nusA gene protein of Escherichia coli and N gene protein of bacteriophage lambda interact in vitro and cooperate in vivo to prevent transcription termination. In vitro the nusA gene protein causes RNA polymerase to pause in the tR2 terminator region of lambda DNA. A completed termination event at tR2 requires both the nusA gene protein and the previously described E. coli termination factor rho. The nusA gene protein is therefore both a transcription termination factor and a protein which couples antitermination factors to the elongating transcription complex.

Bacterial Proteins↗
PubMed1981

Interaction of the sigma factor and the nusA gene protein of E. coli with RNA polymerase in the initiation-termination cycle of transcription.

The nusA gene protein of E. coli is involved in regulating termination of transcription in vivo. In vitro it causes termination of transcription in the tR2 region of the PR operon of bacteriophage lambda. We have now used a nusA-Sepharose affinity column and gradient sedimentation experiments to show that the nusA protein binds directly, with great specificity, and with equimolar stoichiometry to the E. coli RNA polymerase core enzyme beta beta' alpha 2. The RNA polymerase sigma subunit is able to displace the nusA protein from a beta beta' alpha 2-nusA complex, regenerating RNA polymerase holoenzyme beta beta alpha 2-sigma able selectively to initiate transcription at promoter sites. It is proposed that beta beta' alpha 2-nusA and beta beta' alpha 2-sigma are complementary forms of RNA polymerase that interchange with one another in the initiation-termination cycle of transcription.

Bacterial Proteins↗
PubMed1980

L factor that is required for beta-galactosidase synthesis is the nusA gene product involved in transcription termination.

The DNA-dependent in vitro synthesis of Escherichia coli beta-galactosidase requires the presence of a soluble protein referred to as L factor [Kung, H., Spears, C. & Weissbach, H. (1975) J. Biol. Chem. 250, 1556-1562]. In the present study, comparison of physical, immunological, and biological properties shows that L factor is the product of the E. coli nusA gene. The nusA gene product is known to interact with bacteriophage lambda N gene protein and to prevent premature termination of transcription from the early lambda promoters. Our results suggest that premature transcription termination in the lac operon of E. coli may also be overcome by the nusA protein.

Bacterial Proteins↗
PubMed1978

Activation of the alternate complement pathway by peptidoglycan from streptococcal cell wall.

Activation of the alternate complement pathway in human serum by several bacterial components was compared. Peptidoglycan from group A streptococcal cell walls was the most active material, on a weight basis, followed by cell walls, protoplast membranes, and whole cells. The group-specific carbohydrate was inactive. Treatment of peptidoglycan with low concentrations of lysozyme or short periods of sonic treatment enhanced complement activation. High concentrations of lysozyme or extended sonic treatment of peptidoglycan destroyed or greatly reduced the capacity to activate complement. Lysozyme treatment of group A streptococcal cell walls or lipopolysaccharide had no measurable effect. Activation of the alternate complement pathway by group D streptococcal cell walls was destroyed by lysozyme. Activity of peptidoglycan was not inhibited by N-acetyl glucosamine, N-acetyl muramic acid, or D-alanine-D-alanine. Conversion of C3 and factored B by peptidoglycan was shown to occur by immunoelectrophoresis and crossed immunoelectrophoresis.

Animals↗
PubMed1978

malB region in Escherichia coli K-12: specialized transducing bacteriophages and first restriction map.

By starting from an Escherichia coli K-12 strain with a lambda phage integrated in the malB region, series of transducing phages carrying part or all of the malB region have been isolated. Genetic mapping of the transduced malB fragments was accomplished by complementation and recombination with known mutations in the region. By using the DNA of these phages, it was found that the malB region is cleaved by the restriction enzymes BglII, EcoRI, HaeII, HincII, SalI, and SstI, but not BamHI, HindIII, KpnI, PstI, XbaI, or XhoI. A physical map was constructed and tentatively correlated with the genetic map.

Chromosome Mapping↗
PubMed1976

Two forms of simian-virus-40-specific T-antigen in abortive and lytic infection.

Simian-virus-40-specific T-antigen was isolated by immunoprecipitation. From other studies we have proof that the T-antigen described in this work is coded by the viral DNA. The molecular weight estimated from electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels of T-antigen isolated from nonpermissive mouse cells in abortive infection is 86,000 and from permissive monkey cells in lytic infection is 82,000. The 86 kilodalton T-antigen is readily converted in vitro into an 82 kilodalton form by incubation with extracts from permissive monkey cells but not with extracts from nonpermissive mouse or hamster cells. This and the results of fingerprinting analysis of tryptic peptides suggest that T-antigen may be processed in permissive cells.

Animals↗