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D Reinberg

Publications and source records attributed to D Reinberg.

122 records · Page 7Linked to original sources
PubMed1988

EivF, a factor required for transcription of the adenovirus EIV promoter, binds to an element involved in EIa-dependent activation and cAMP induction.

The isolation of a cellular factor that was specifically required for transcription from the promoter of the adenovirus early gene iv, an EIa-activated promoter, is described. This factor (EivF) was purified from HeLa cells using a functional transcription assay and identified as a 72,000- to 65,000-dalton protein. DNase I footprinting experiments demonstrated that purified EivF bound to the sequence 5'-GT(G/T)ACGT-3' present two times upstream of the Eiv TATA box. Nuclear extracts prepared from HeLa cells contained more than one factor capable of binding to the EivF recognition site. Previous studies have indicated that a sequence similar to the EivF-binding site was recognized by a 43,000-dalton protein and participated in the cAMP response of the somatostatin promoter. The purified and transcriptionally active EivF also bound to DNA sequence elements present in the somatostatin and alpha-gonadotropin promoters shown previously to be responsive to cAMP.

Adenoviridae↗
PubMed1987

Purification and functional characterization of transcription factor SII from calf thymus. Role in RNA polymerase II elongation.

SII was purified from calf thymus tissue to apparent homogeneity by a rapid procedure. The 38-kDa protein stimulated RNA synthesis by purified calf thymus RNA polymerase II 4-fold. The calf thymus SII had similar chromatographic properties and molecular size and cross-reacted immunologically with antibodies to mouse SII (Sekimizu, K., Nakanishi, Y., Mizuno, D., and Natori, S. (1979) Biochemistry 18, 1582-1588). We have substituted the purified calf thymus SII for the partially purified HeLa transcription factor IIS fraction in a HeLa (human) transcription system reconstituted with purified factors and RNA polymerase II. The purified protein stimulated specific transcription from the adenovirus 2 major late promoter by increasing the efficiency of the elongation reaction.

Animals↗
PubMed1987

Factors involved in specific transcription by mammalian RNA polymerase II. Purification and functional analysis of initiation factors IIB and IIE.

Two general transcription factors (IIE and IIB) (TF) were purified from HeLa cell nuclear extracts and shown to be absolutely required, along with two additional factors (IIA and IID) and RNA polymerase II, for specific transcription initiation at the adenovirus major late promoter. TFIIB and TFIIE were also required, in addition to TFIIA, TFIID, RNA polymerase II, and the adenovirus 2 major late promoter, for the formation of a (preinitiation) complex that could initiate transcription (upon addition of nucleoside triphosphates) in the presence of heparin concentrations which inhibited the action of unbound factors. Glycerol gradient analyses indicated independent interactions of TFIIE with TFIIB and with the purified RNA polymerase II, but not with RNA polymerase III. Transcription factors IIB and IIE were also shown to be required for specific initiation of transcription from several cellular and viral class II promoters.

Adenoviridae↗
PubMed1987

Factors involved in specific transcription in mammalian RNA polymerase II. Functional analysis of initiation factors IIA and IID and identification of a new factor operating at sequences downstream of the initiation site.

Transcription from the major late promoter of adenovirus type 2 DNA (including DNA sequences from 56 nucleotides upstream to 33 nucleotides downstream of the CAP site) was reconstituted with transcription factors purified from HeLa cells. Five components, transcription factors (TF) IIA, -B, -E, -D and RNA polymerase II, were required for accurate initiation of transcription. Kinetic analyses combined with order of addition experiments suggested that TFIIA acted first during the initiation reaction and that this interaction was followed by the action of TFIID. In agreement with these conclusions, both TFIIA and TFIID were required to render a transcription reaction partially resistant to concentrations of Sarkosyl previously shown to inhibit an early step in the formation of a preinitiation complex. Related Sarkosyl studies indicated that the inferred complex was subsequently recognized by RNA polymerase II, which resulted in an increased level of Sarkosyl-resistant transcription (in the presence of TFIIA and TFIID), and that this interaction occurred independently of TFIIB and TFIIE. However, TFIIB and TFIIE were implicated, along with the other factors and RNA polymerase II, in the subsequent formation of a highly stable preinitiation complex, which was inferred from its ability to initiate (with added nucleotides) in the presence of heparin concentrations which blocked unbound factors. The identification of a new transcription factor, which was required only when viral sequences 3' to the major late promoter were part of the transcription unit, is also reported.

Adenoviridae↗
PubMed1987

Factors involved in specific transcription by mammalian RNA polymerase II. Transcription factor IIS stimulates elongation of RNA chains.

A factor that stimulates random transcription of purified DNAs by RNA polymerase II has been partially purified and analyzed with respect to its possible role in specific transcription from class II promoters. Studies of the effect of this factor (transcription factor IIS) on transcription from the adenovirus major late promoter in a system reconstituted with RNA polymerase II and purified factors (IIA, IIB, IIE, and IID) indicated that it acted subsequent to the initiation step and that it stimulated the rate of elongation. Kinetic experiments indicated that the factor affected the efficiency with which the RNA polymerase II passed through pausing sites. The relationship of transcription factor IIS to a protein previously purified from Erlich ascites tumor cells (Sekimizu, K., Nakanishi, Y., Mizuno, D., and Natori, S. (1979) Biochemistry 18, 1582-1588) was also studied.

Adenoviridae↗
PubMed1985

The enzymatic conversion of 3'-phosphate terminated RNA chains to 2',3'-cyclic phosphate derivatives.

The enzyme, RNA cyclase, has been purified from cell-free extracts of HeLa cells approximately 6000-fold. The enzyme catalyzes the conversion of 3'-phosphate ends of RNA chains to the 2',3'-cyclic phosphate derivative in the presence of ATP or adenosine 5'-(gamma-thio)triphosphate (ATP gamma S) and Mg2+. The formation of 1 mol of 2',3'-cyclic phosphate ends is associated with the disappearance of 1 mol of 3'-phosphate termini and the hydrolysis of 1 mol of ATP gamma S to AMP and thiopyrophosphate. No other nucleotides could substitute for ATP or ATP gamma S in the reaction. The reaction catalyzed by RNA cyclase was not reversible and exchange reactions between [32P]pyrophosphate and ATP were not detected. However, an enzyme-AMP intermediate could be identified that was hydrolyzed by the addition of inorganic pyrophosphate or 3'-phosphate terminated RNA chains but not by 3'-OH terminated chains or inorganic phosphate. 3'-[32P](Up)10Gp* could be converted to a form that yielded, (Formula: see text) after degradation with nuclease P1, by the addition of wheat germ RNA ligase, 5'-hydroxylpolynucleotide kinase, RNA cyclase, and ATP. This indicates that the RNA cyclase had catalyzed the formation of the 2',3'-cyclic phosphate derivative, the kinase had phosphorylated the 5'-hydroxyl end of the RNA, and the wheat germ RNA ligase had catalyzed the formation of a 3',5'-phosphodiester linkage concomitant with the conversion of the 2',3'-cyclic end to a 2'-phosphate terminated residue.

Adenosine Triphosphate↗
PubMed1984

Analysis of bacteriophage phi X174 gene A protein-mediated termination and reinitiation of phi X DNA synthesis. I. Characterization of the termination and reinitiation reactions.

The phi X174 (phi X) gene A protein-mediated termination and reinitiation of single-stranded circular (SS(c] phi X viral DNA synthesis in vitro were directly and independently analyzed. Following incubation together with purified DNA replication enzymes from Escherichia coli, ATP, [alpha-32P]dNTPs, and either the phi X A protein and phi X replicative form I (RF I) DNA, or the purified RF II X A complex, the phi X A protein was detected covalently linked to newly synthesized 32P-labeled DNA. Formation of the phi X A protein-[32P]DNA covalent complex required all the factors necessary for phi X (+) SS(c) DNA synthesis in vitro. Thus, it was a product of the reinitiation reaction and an intermediate of the replication cycle. Identification of this complex provided direct evidence that reinitiation of phi X (+) strand DNA synthesis involved regeneration of the RF II X A complex. Substitution of 2',3'-dideoxyguanosine triphosphate (ddGTP) for dGTP in reaction mixtures resulted in the formation of covalent phi X A protein 32P-oligonucleotide complexes; these complexes were trapped analogues of the regenerated RF II X A complex. They could not act catalytically due to the presence of ddGMP residues at the 3'-termini of the oligonucleotide moieties. Reaction mixtures containing ddGTP also yielded nonradioactive (+) SS(c) DNA products derived from circularization of the displaced (+) strand of the input parental template DNA. The formation of the phi X A protein-32P-oligonucleotide complexes and nonradioactive (+) SS(c) DNA were used to assay both reinitiation and termination reactions, respectively. Both reactions required DNA synthesis from the 3'-hydroxyl primer at nucleotide residue 4305 which was formed by cleavage of phi X RF I DNA by the phi X A protein. Elongation of this primer by 18, but not 11 nucleotides was sufficient to support each reaction. Reinitiation reactions proceeded rapidly and were essentially complete after 90 s. In contrast, when ddGTP was replaced with dGTP in reaction mixtures, DNA synthesis proceeded with linear kinetics for up to 10 min. These results suggested that in the presence of all four dNTPs, active templates supported more than 40 rounds of DNA synthesis.

Bacteriophage phi X 174↗
PubMed1983

DNA sequences which support activities of the bacteriophage phi X174 gene A protein.

The DNA sequence of 30 nucleotides which surrounds the origin of viral strand DNA replication is highly conserved amongst the icosahedral single-stranded DNA bacteriophages. The A gene of these phages encodes a protein which is required for initiation and termination of viral strand DNA synthesis and acts as a nicking-closing activity specifically within this 30-nucleotide sequence. A system of purified Escherichia coli host proteins and phi X174 gene A protein has been developed which specifically replicates in vitro the viral strand of phi X174 from RF (replicative form) I template DNA and yields single-stranded circular DNA products (RF leads to SS(c) DNA replication system). Recombinant plasmids carrying inserts derived from phage phi X174 or G4 DNA which range in length from 49 to 1175 base pairs and contain the 30-nucleotide conserved sequence have been shown to support phi X A protein-dependent DNA synthesis in vitro in this replication system. We report here that insertion of the 30-nucleotide sequence alone into pBR322 allows the resulting recombinant plasmids to support phi X A protein-dependent in vitro DNA synthesis as efficiently as phi X174 template DNA in the RF leads to SS(c) replication system. The 30-nucleotide sequence functions as a fully wild type DNA replication origin as determined by the rate of DNA synthesis and the structure of resulting DNA products. Furthermore, the DNA sequence requirements for nicking of RF I DNA by the phi X A protein and for supporting replication origin function have been partially separated. Homology to positions 1, 29, and 30 of the 30-nucleotide conserved sequence are not required for cleavage of RF I DNA by the A protein; homology to position 1 but not 29 or 30 is required for efficient DNA replication.

Bacteriophage phi X 174↗
PubMed1983

Studies on the phi X174 gene A protein-mediated termination of leading strand DNA synthesis.

Recombinant RF (replicate form) I DNAs containing the bacteriophage phi X174 gene A protein-recognition sequence are cleaved by the phi X A protein yielding a phi X RF II X A protein complex (Zipursky, S.L., Reinberg, D., and Hurwitz, J. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 5182-5186). Such complexes support DNA synthesis in both RF I leads to SS(c) and RF I leads to RF I phi X DNA replication reactions in vitro. Two phi X A protein-recognition sequences were inserted into plasmid pBR322. Both sequences were contiguous with the same strand of the vector DNA and separated by 667 and 4275 base pairs. This recombinant plasmid (G27-4) was cleaved by the phi X A protein at either insert and both inserts support the initiation of RF leads to SS(c) DNA synthesis. This was verified by the finding that replication products were circular molecules of 667 and 4275 nucleotides. This finding is in keeping with the multifunctional activities associated with the phi X A protein; these include the site-specific nicking of RF I DNA which initiates DNA synthesis and site-specific termination resulting in the circularization of the displaced DNA strand. The phi X A protein and the Escherichia coli rep and SSb proteins catalyze the unwinding of phi X RF I DNA in vitro (Scott, J.F., Eisenberg, S., Bertsch, L.L., and Kornberg, A. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 193-197). Recombinant plasmid G27-4 RF I DNA was also unwound in vitro by this enzyme system; in this case, both circular and linear single-stranded DNA molecules of 667 and 4275 nucleotides, as well as full length circular single-stranded DNA were formed. Full length linear DNA was not detected. The two single-stranded circular DNA products formed as leading strands in RF leads to SS(c) reaction mixtures containing G27-4 RF I DNA differed in their ability to support lagging strand DNA synthesis. It was shown that the large single-stranded circular product included DNA sequences homologous to a replication factor Y effector sequence required for RF leads to RF and SS(c) leads to RF replication (Zipursky, S.L., and Marians, K.J. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 6521-6525). The 4275-nucleotide, but not the 667-nucleotide, single-stranded circular DNA product was converted to a duplex structure.

Bacteriophage phi X 174↗
PubMed1980

Initiation of DNA replication by the dnaG protein.

Highly purified preparations of dnaG protein from Escherichia coli prime minus strand synthesis of phage alpha 3 DNA in vitro. This protein synthesizes primer oligonucleotides which may be composed of ribonucleotide or deoxyribonucleotide moieties or both. The presence of deoxyribonucleotide moieties in the chain limits primer chain length; this effect occurs even when ribonucleoside triphosphates are included in the priming reaction. The dnaG protein can use ADP in place of ATP. Primer formation by dnaG protein is strictly stoiochiometric in vitro; one molecule of dnaG protein is required to prime one molecule of alpha 3 DNA. All of these primers are equally efficient in the subsequent elongation reaction with DNA elongation factors I and III, dnaZ gene product, and DNA polymerase III to form RFII. The site recognized by dnaG protein on alpha 3 DNA in vitro is within the same region of the alpha 3 chromosome as the origin of replication in vivo. Structural properties of this site are crucial to dnaG action in vitro. No other enzymatic activity for dnaG protein has been detected.

Bacterial Proteins↗
PubMed1980

In vitro DNA replication of recombinant plasmid DNAs containing the origin of progeny replicative form DNA synthesis of phage phi X174.

The origin of phage phi X174 progeny replicative form (RF) DNA synthesis has been inserted into the plasmid vector pBR322 and cloned. In direct contrast to pBR322, the recombinant superhelical plasmids can substitute for phi X174 RFI DNA as template in phi X174-specific reactions in vitro. We have shown that the recombinant plasmids: (i) are cleaved by the phi X174 A protein; (ii) support net synthesis of unit-length single-stranded circular DNA in the presence of the phi X174 A protein and Escherichia coli rep protein, DNA-binding protein, and DNA polymerase III elongation system; (iii) support replication of duplexes catalyzed by the phi X174 A protein and extracts of E. coli.

Bacterial Proteins↗