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J Greenblatt

Publications and source records attributed to J Greenblatt.

At least 73 records · Page 4Linked to original sources
PubMed1992

A cDNA encoding RAP74, a general initiation factor for transcription by RNA polymerase II.

RAP30/74 (also known as TFIIF, beta gamma and FC is one of several general factors required for initiation by RNA polymerase II. The small RAP30 subunit of RAP30/74 binds directly to polymerase and appears structurally and functionally homologous to bacterial sigma factors in their RNA polymerase-binding region. RAP30/74 or recombinant RAP30 suppresses nonspecific binding of RNA polymerase II to DNA and is required for RNA polymerase II to assemble stably into a preinitiation complex containing promoter DNA and the general factors TFIID, TFIIA and TFIIB; both RAP30 and RAP74 are physical components of the preinitiation complex. A complementary DNA encoding human RAP30 has been isolated, and here we report the isolation of a cDNA encoding human RAP74. RAP30 and RAP74 produced in Escherichia coli can be used in place of natural human RAP30/74 to direct accurate transcription initiation by RNA polymerase II in vitro.

Amino Acid Sequence↗
PubMed1992

The NusA and NusG proteins of Escherichia coli increase the in vitro readthrough frequency of a transcriptional attenuator preceding the gene for the beta subunit of RNA polymerase.

The genes for the beta (rpoB) and beta' (rpoC) subunits of Escherichia coli RNA polymerase are the distal members of a complex transcriptional unit that contains four upstream ribosomal protein genes. The RNA polymerase subunit genes are transcribed at a lower frequency than the ribosomal protein genes as a result of termination at an attenuator preceding rpoB. A purified in vitro transcription system was developed using linear DNA templates that carry the attenuator. The ability of known termination and antitermination proteins to modulate termination at the attenuator was tested. Both NusA and NusG increase the frequency of transcriptional readthrough at the attenuator whereas NusB, S10, and Rho had no significant effect in this system.

Bacterial Proteins↗
PubMed1992

Direct interaction between two Escherichia coli transcription antitermination factors, NusB and ribosomal protein S10.

The Escherichia coli proteins NusB and ribosomal protein S10 are important for transcription antitermination by the bacteriophage lambda N protein. We have used sucrose gradient co-sedimentation and affinity chromatography with immobilized ribosomal protein S10, a glutathione S-transferase-S10 fusion protein, and NusB to show that NusB binds directly and very selectively to S10. The interaction is non-ionic and has an estimated Kd value of 10(-7) M. We hypothesize that NusB binds to N-modified transcription complexes primarily by interacting with S10.

Bacterial Proteins↗
PubMed1992

Primary care physicians and children's blood cholesterol.

BACKGROUND. In a national survey sponsored by the National Heart, Lung, and Blood Institute, 62% of primary care physicians of children (under age 18 years) believed that high levels of low-density lipoprotein cholesterol in childhood had a great effect on subsequent heart disease risk. RESULTS. About 75% believed high blood pressure, smoking, and diabetes had similar effects. Although routine cholesterol screening in children under age 10 was infrequent, 72% of physicians screened high risk children. The age at which screening was done varied markedly; more pediatricians screened children younger than 5 years. The majority of physicians who saw children with high blood cholesterol instituted nondrug therapy, with pediatricians being most apt to do so. Low saturated fat diets were prescribed by 26% of these physicians and 9% of physicians prescribed increased polyunsaturated diets. Twelve percent of physicians treating hypercholesterolemic children used lipid-lowering drugs. Among those using drugs, 9% based drug use on total blood cholesterol measurements only. Factors that affected physician treatment of childhood hypercholesterolemia included physician specialty type, organization of practice (group or solo), and the age distribution of the pediatric patient population.

Age Factors↗
PubMed1992

Identification of three mammalian proteins that bind to the yeast TATA box protein TFIID.

The TATA box binding transcription factor TFIID of S. cerevisiae was used as a ligand for affinity chromatography. Polypeptides that bind specifically to yeast TFIID (TFIID-associated proteins, DAPs) were purified from human HeLa (heDAPs) and calf thymus (ctDAPs) whole cell extracts. Both heDAP and ctDAP fractions altered the binding of TFIID to the TATA element, and substituted for the TFIIA transcription activity in a reconstituted in vitro system. The heDAP fraction also behaved like TFIIA in its ability to form a promoter-TFIID-TFIIA complex and to recruit TFIIB to such a complex. The interaction of DAPs with TFIID can confer heat-resistance (47 degrees C) on recombinant yeast or human TFIID. SDS-PAGE analysis revealed that three polypeptides from HeLa extracts specifically bound to yTFIID columns (heDAP35, heDAP21, and heDAP12). These data suggest that a multi-subunit transcription factor with the properties of TFIIA can bind to TFIID in the absence of DNA.

Base Sequence↗
PubMed1991

The small subunit of transcription factor IIF recruits RNA polymerase II into the preinitiation complex.

We found that transcription factor IIF mediates the association of RNA polymerase II with promoter sequences containing transcription factors IID, IIB, and IIA (DAB complex). The resulting DNA-protein complex contained RNA polymerase II and the two subunits of transcription factor IIF (RAP 30 and RAP 74). Cloned human RAP 30 was sufficient for the recruitment of RNA polymerase II to the DAB complex. This ability of RAP 30 to recruit RNA polymerase to a promoter is also a characteristic of sigma factors in prokaryotes.

Adenoviridae↗
PubMed1991

RNA polymerase II-associated proteins are required for a DNA conformation change in the transcription initiation complex.

Proteins purified on the basis of their affinity for RNA polymerase II effectively substitute for previously defined transcription initiation factors. In two assays, formation of initiation complexes and transcription in vitro, the RNA polymerase II-associated proteins behaved identically to a fraction containing transcription factors IIE and IIF. Both fractions greatly stabilized the association of polymerase with the promoter and were required for the formation of complete initiation complexes. By using the DNA-cleaving reagent phenanthroline.copper in footprinting reactions, the RNA polymerase II-associated proteins were shown to be required for a DNA conformation change near the initiation site of the promoter. Based on similarity to the prokaryotic transcription complex, this conformation change is likely to represent a transition from a closed to an open complex.

Cellulose↗
PubMed1991

Related RNA polymerase-binding regions in human RAP30/74 and Escherichia coli sigma 70.

RAP30/74 is a heteromeric general transcription initiation factor that binds to mammalian RNA polymerase II. The RAP30 subunit contains a region that is similar in amino acid sequence to the RNA polymerase-binding domain of the Escherichia coli transcription initiation factor sigma 70 (sigma 70). Mammalian RNA polymerase II specifically protected a serine residue in the sigma 70-related region of RAP30 from phosphorylation in vitro. In addition, human RAP30/74 bound to Escherichia coli RNA polymerase and was displaced by sigma 70. These results suggest that RAP30 and sigma 70 have functionally related RNA polymerase-binding regions.

Amino Acid Sequence↗
PubMed1991

Reduced binding of TFIID to transcriptionally compromised mutants of VP16.

Activator proteins that control transcription initiation by RNA polymerase II usually have two domains: one binds to DNA, and the other activates transcription. A particularly potent acidic activation domain at the C terminus of the herpes simplex virus protein VP16 binds directly and selectively to the human and yeast TATA box-binding factor TFIID. We have now investigated the biological significance of this in vitro interaction by using mutant forms of VP16. For changes at the critical phenylalanine residue at position 442 of VP16 there was a good correlation between transactivation activity in vivo and the binding of VP16 to TFIID in vitro. In contrast, mutants with reduced negative charge were more defective for binding than for activation.

DNA Mutational Analysis↗
PubMed1991

RNA polymerase-associated transcription factors.

Proteins that bind to RNA polymerase regulate initiation and termination of transcription in bacteria. Recently, such RNA polymerase-associated proteins were also found to be essential for accurate transcription by eukaryotic RNA polymerase II.

Animals↗
PubMed1991

The nut site of bacteriophage lambda is made of RNA and is bound by transcription antitermination factors on the surface of RNA polymerase.

The boxA and boxB components of the lambda nut site are important for transcriptional antitermination by the phage N protein. We show here that boxA and boxB RNA in N-modified transcription complexes are inaccessible to ribonucleases and have altered sensitivity to dimethylsulfate. N and NusA suffice to weakly protect boxB, independently of boxA and other factors. However, efficient protection of the entire nut site from ribonucleases requires boxA and boxB, N, NusA, NusB, S10, and NusG. Mutations in RNA polymerase, which inhibit antitermination by N in vivo, disallow protection of the nut site during transcription in vitro; therefore, the surface of RNA polymerase must coordinate the formation of complexes containing the antitermination factors and nut site RNA.

Bacteriophage lambda↗
PubMed1991

Assembly of transcription elongation complexes containing the N protein of phage lambda and the Escherichia coli elongation factors NusA, NusB, NusG, and S10.

The transcription antitermination protein, N, of bacteriophage lambda; the Escherichia coli elongation factors NusA, NusB, ribosomal protein S10, and NusG; and a DNA template containing a lambda nut (N-ututilization) site are necessary and sufficient for the highly cooperative formation in vitro of stable transcription complexes containing all five elongation factors. Mutations in the nut site, NusA, or the beta-subunit of RNA polymerase (RNAP) that impair antitermination in vivo also abolish the assembly of a stable complex containing the antitermination factors in vitro. The effects of RNAP mutations on assembly imply that the antitermination factors assemble on the surface of RNAP. We have shown previously that NusA binds directly to transcribing RNAP (Ka approximately 10(7) M-1); Ka = association constant and we show here that S10 also binds directly and specifically to RNAP with an apparent Ka of 10(6) M-1. These observations led to a model for the ordered assembly of the N-modified transcription complex.

Bacterial Proteins↗
PubMed1989

Structure and associated DNA-helicase activity of a general transcription initiation factor that binds to RNA polymerase II.

RAP30/74 is a heteromeric general transcription initiation factor which binds to RNA polymerase II. Here we report that preparations of RAP30/74 contain an ATP-dependent DNA helicase whose probable function is to melt the DNA at transcriptional start sites. The sequence of the RAP30 subunit of RAP30/74 indicates that RAP30 may be distantly related to bacterial sigma factors.

Amino Acid Sequence↗
PubMed1989

Passive smoking on commercial airline flights.

In-flight exposure to nicotine, urinary cotinine levels, and symptom self-reports were assessed in a study of nine subjects (five passengers and four attendants) on four routine commercial flights each of approximately four hours' duration. Urine samples were collected for 72 hours following each flight. Exposures to nicotine measured during the flights using personal exposure monitors were found to be variable, with some nonsmoking areas attaining levels comparable to those in smoking sections. Attendants assigned to work in nonsmoking areas were not protected from smoke exposure. The type of aircraft ventilation was important in determining the levels of in-flight nicotine exposure. The environmental tobacco smoke levels that occurred produced measurable levels of cotinine (a major metabolite of nicotine) in the urine of passengers and attendants. Passengers who experienced the greatest smoke exposure had the highest levels of urinary cotinine. Changes in eye and nose symptoms between the beginning and end of the flights were significantly related both to nicotine exposure during the flight and to the subsequent urinary excretion of cotinine. In addition, subjects' perceptions of annoyance and smokiness in the airplane cabin were also related to in-flight nicotine exposure and urinary excretion measures.

Air Pollutants↗
PubMed1988

Factors involved in specific transcription by mammalian RNA polymerase II. RNA polymerase II-associating protein 30 is an essential component of transcription factor IIF.

Transcription from class II promoters requires five general factors, IIA, IIB, IID, IIE, and IIF, in addition to RNA polymerase II for basal levels of transcription (Reinberg, D., Flores, O., and Buckbinder, L. (1987) in Molecular Biology of RNA: New Perspectives (Inouye, M., and Dudock, B., eds) pp. 423-439, Academic Press, Orlando, FL). A protein fraction containing transcription factors (TF) IIE and IIF was able to reconstitute transcription from the adenovirus major late promoter when added to extracts depleted of the RNA polymerase II-associating proteins RAP 30 and RAP 74 (Sopta, M., Carthew, R.W., and Greenblatt, J. (1985) J. Biol. Chem. 260, 10353-10360). Studies with monoaffinity-purified antibodies directed against RAP 30 demonstrated, by Western blot analysis, that RAP 30 copurifies on five columns with transcription factor IIF. That RAP 30 is a functional component of TFIIF was also demonstrated; preincubation of anti-RAP 30 antibodies with purified TFIIF inhibited transcription. Inhibition of transcription was overcome by the addition of purified TFIIF. RAP 30 is an integral part of a preinitiation complex; the incubation of all the general transcription factors with a promoter-containing DNA, prior to the addition of the anti-RAP 30 antibodies, resulted in the formation of a DNA-protein complex that was not inhibited by the antibodies. Incubation of the transcription factors in the absence of a promoter-containing DNA resulted in a complex that was partially resistant to the antibodies.

Adenoviruses, Human↗