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W S Dynan

Publications and source records attributed to W S Dynan.

At least 19 recordsLinked to original sources
PubMed1992

Ku autoantigen is the regulatory component of a template-associated protein kinase that phosphorylates RNA polymerase II.

The carboxyl-terminal domain of RNA polymerase II contains a tandemly repeated heptapeptide sequence. Previous work has shown that this sequence is phosphorylated at multiple sites by a template-associated protein kinase, in a reaction that is closely associated with the initiation of RNA synthesis. We have purified this kinase to apparent homogeneity from human (HeLa) cells. The purified kinase phosphorylates native RNA polymerase II only in the presence of DNA and the general transcription factors TFIID (TBP), TFIIB, and TFIIF. Two kinase components are required for full activity: a catalytic component and a DNA-binding regulatory component. The regulatory component has been identified as Ku autoantigen, based on the molecular weights of its component polypeptides, its DNA-binding properties, and its reactivity with anti-Ku monoclonal antibodies. The Ku autoantigen recruits the catalytic component of the kinase to the template. Ku autoantigen has been previously proposed to interact with DNA by a characteristic bind-and-slide mechanism. This mode of interaction may provide a mechanism for targeting the kinase to the transcription complex and other DNA-bound substrates.

Antigens, Nuclear↗
PubMed1992

DNA binding provides a signal for phosphorylation of the RNA polymerase II heptapeptide repeats.

Isolated transcription complexes contain a protein kinase that phosphorylates the heptapeptide repeats of the carboxy-terminal domain (CTD) of the RNA polymerase II (RNAP II) large subunit in an apparently promoter-dependent manner. We now show that the essential features of this reaction can be reproduced in a reconstituted system containing three macromolecular components: a fusion protein consisting of the CTD of RNAP II fused to a heterologous DNA-binding domain, an activating DNA fragment containing the recognition sequence for the fusion protein, and a protein kinase that binds nonspecifically to DNA. This kinase closely resembles a previously known DNA-dependent protein kinase. Evidently, the association of the CTD with DNA provides a key signal for phosphorylation. There appears to be no absolute requirement for specific contacts with other DNA-bound transcription factors.

Animals↗
PubMed1992

In vitro activation of transcription by the human T-cell leukemia virus type I Tax protein.

The human T-cell leukemia virus type I (HTLV-I) regulatory protein Tax activates transcription of the proviral long terminal repeats and a number of cellular promoters. We have developed an in vitro system to characterize the mechanism by which Tax interacts with the host cell transcription machinery. Tax was purified from cells infected with a baculovirus expression vector. Addition of these Tax preparations to nuclear extracts from uninfected human T lymphocytes activated transcription of the HTLV-I long terminal repeat approximately 10-fold. Transcription-stimulatory activity copurified with the immunoreactive 40-kDa Tax polypeptide on gel filtration chromatography, and, as expected, the effect of recombinant Tax was diminished in HTLV-I-infected T-lymphocyte extracts containing endogenous Tax. Tax-mediated transactivation in vivo has been previously shown to require 21-bp-repeat Tax-responsive elements (TxREs) in the promoter DNA. Stimulation of transcription in vitro was also strongly dependent on these sequences. To investigate the mechanism of Tax transactivation, cellular proteins that bind the 21-bp-repeat TxREs were prepared by DNA affinity chromatography. Recombinant Tax markedly increased the formation of a specific host protein-DNA complex detected in an electrophoretic mobility shift assay. These data suggest that Tax activates transcription through a direct interaction with cellular proteins that bind to the 21-bp-repeat TxREs.

Base Sequence↗
PubMed1991

Promoter-dependent phosphorylation of RNA polymerase II by a template-bound kinase. Association with transcriptional initiation.

The largest subunit of eukaryotic RNA polymerase II (RNAP II) has a serine- and threonine-rich C-terminal domain (CTD) that may interact both with DNA and with the activating region of transcription factors. It has been proposed, in one model, that a protein kinase phosphorylates the promoter-associated CTD, facilitating the transition between promoter-binding and RNA-elongating forms of RNAP II. An immobilized template transcription system was used to test the predictions of this model directly. A protein kinase that phosphorylated the CTD at multiple sites was detected. This activity was tightly bound to the template, as evidenced by continued association after multiple rounds of washing. Phosphorylation was promoter sequence-dependent and exhibited the same nucleotide substrate specificity as the previously characterized ATP-requiring step in initiation. It was necessary for [gamma-32P]ATP and initiating rNTPs to be present simultaneously in the reaction in order to efficiently chase-radiolabel into elongating RNAP II-containing complexes, consistent with the idea that initiation and phosphorylation are temporally associated reactions.

Adenosine Triphosphate↗
PubMed1991

Human T-cell leukemia virus (HTLV) type II Rex protein binds specifically to RNA sequences of the HTLV long terminal repeat but poorly to the human immunodeficiency virus type 1 Rev-responsive element.

The human T-cell leukemia viruses (HTLVs) encode a trans-regulatory protein, Rex, which differentially regulates viral gene expression by controlling the cytoplasmic accumulation of viral mRNAs. Because of insufficient amounts of purified protein, biochemical characterization of Rex activity has not previously been performed. Here, utilizing the baculovirus expression system, we purified HTLV type II (HTLV-II) Rex from the cytoplasmic fraction of recombinant baculovirus-infected insect cells by heparin-agarose chromatography. We directly demonstrated that Rex specifically bound HTLV-II 5' long terminal repeat RNA in both gel mobility shift and immunobinding assays. Sequences sufficient for Rex binding were localized to the R-U5 region of the HTLV-II 5' long terminal repeat and correlate with the region required for Rex function. The human immunodeficiency virus type 1 (HIV-1), has an analogous regulatory protein, Rev, which directly binds to and mediates its action through the Rev-responsive element located within the HIV-1 env gene. We demonstrated that HTLV-II Rex rescued an HIV-1JR-CSF Rev-deficient mutant, although inefficiently. This result is consistent with a weak binding activity to the HIV-1 Rev-responsive element under conditions in which it efficiently bound the HTLV-II long terminal repeat RNA.

Animals↗
PubMed1991

BK virus and JC virus shed during pregnancy have predominantly archetypal regulatory regions.

Twenty-three BK virus and JC virus DNA samples obtained from urine of pregnant women had almost exclusively archetypal transcriptional control regions. Rearrangements characteristic of laboratory strains are apparently not required for reactivation in humans. Unexpectedly, alignment shows that many elements identified previously in the BK virus enhancer are conserved in the JC virus archetype.

BK Virus↗
PubMed1990

Interaction of host cell proteins with the human T-cell leukemia virus type I transcriptional control region. II. A comprehensive map of protein-binding sites facilitates construction of a simple chimeric promoter responsive to the viral tax2 gene product.

We present a map describing the binding of cellular proteins to a 300-base pair (bp) region of the human T-cell leukemia virus type I (HTLV-I) long terminal repeat. The map accounts for nearly all of the DNase I protection reported in a previous study using crude nuclear extracts. Notable features include a complex arrangement of overlapping binding sites encompassing the 21-bp repeat elements (see accompanying paper) as well as binding sites for the transcription factors Sp1 and NF-I that significantly deviate from the previously defined consensus recognition sequences. Based on the binding results, we constructed simple chimeric promoters containing 21-bp repeat elements, Sp1-, and nuclear factor I-binding sites upstream of a TATA box. Transient transfection experiments show that these promoters are expressed in T-cells and are regulated by the viral tax2 gene product. Deletion of the Sp1 and nuclear factor I sites abolishes tax-induction, suggesting that one or both of these proteins play a role in mediating the tax-responsiveness conferred by the 21-bp repeat element.

Base Sequence↗
PubMed1990

Interaction of cellular proteins with the human T-cell leukemia virus type I transcriptional control region. Purification of cellular proteins that bind the 21-base pair repeat elements.

Nuclear extracts of cultured human cells contain multiple proteins that recognize specific sequence elements within the transcriptional control region of the human retrovirus, human T-cell leukemia virus type I (HTLV-I). Here we report the purification of host expression factor 1T (HEF-1T), a DNA-binding protein from human T-lymphocytes that binds to each of the three 21-base pair repeat enhancer elements in the proviral long terminal repeats of HTLV-I and the related virus, HTLV-II. HEF-1T is composed of two polypeptides of 41 and 59 kDa. We show that HEF-1T is distinct from a second protein, present in non-lymphoid cells, that binds to overlapping sites in and near the 21-base pair repeats. This second protein is composed of a single 47-kDa polypeptide and appears to be identical to the previously described transcription factor AP-2. A third protein, also distinct from HEF-1T, binds within the first repeat. The present results suggest that there may be multiple modes of HTLV-I promoter recognition involving distinct sets of cellular proteins.

Base Sequence↗
PubMed1990

Promoter evolution in BK virus: functional elements are created at sequence junctions.

The archetypal strain of BK virus directed very little early gene expression compared with rearranged laboratory strains of the virus. One laboratory strain that was analyzed in detail contained newly created AP-1 binding enhancer modules spanning the junction between adjacent sequence repeats. Introduction of these sequences into the archetype activated the previously quiescent early promoter.

BK Virus↗
PubMed1990

A downstream-element-binding factor facilitates assembly of a functional preinitiation complex at the simian virus 40 major late promoter.

Recent work has shown that many promoters recognized by eucaryotic RNA polymerase II contain essential sequences located downstream of the transcriptional initiation site. We show here that the activity of a promoter element centered 28 base pairs downstream of the simian virus 40 major late initiation site appears to be mediated by a DNA-binding protein, which was isolated by affinity chromatography from HeLa cell nuclear extracts. In the absence of the other components of the transcriptional machinery, the protein bound specifically but weakly to its recognition sequence, with a Kd of approximately 10(-8) M. Analysis of kinetic data showed that mutation of the downstream element decreased the number of functional preinitiation complexes assembled at the promoter without significantly altering the time required for half the complexes to assemble. This suggests that in the absence of the downstream activating protein, preinitiation complexes are at least partially assembled but are not transcriptionally competent.

Base Sequence↗
PubMed1989

Measurement of the binding of transcription factor Sp1 to a single GC box recognition sequence.

The equilibrium constant was determined for the binding of the transcription factor Sp1 to a single consensus GC box DNA recognition site, (5'-GGGGCGGGGC-3'). For these experiments, single copies of the recognition site were synthesized and cloned in a standard plasmid background. Binding was measured either by a footprinting assay modified so that the binding reaction was at equilibrium, or by a gel mobility shift assay. The concentration of active Sp1 in the reactions and the dissociation constant were determined by computer-assisted fitting to theoretical curves. Values for the dissociation constant obtained in different experiments ranged from 4.1 X 10(-10) M to 5.3 X 10(-10) M. Several variants of the consensus recognition site were also tested. An A-substituted variant (5'-GGGGAGGGGC-3') and a T-substituted variant (5'-GGGGTGGGGC-3') were bound 3-fold and 6-fold more weakly than the consensus site, respectively. A G-substituted variant (5'-GGGGGGGGGC-3') was bound at least 30-fold more weakly than the consensus site. These findings help distinguish between alternative models for Sp1-DNA recognition. They are consistent with the presence of specific hydrogen-bond contacts between Sp1 and the central C-G base pair, but provide no particular evidence to support a model where local DNA structure is the dominant factor in the interaction.

Base Composition↗
PubMed1989

Promoter-dependent transcription by RNA polymerase II using immobilized enzyme complexes.

DNA fragments containing the adenovirus 2 major late or simian virus 40 early promoters were attached to a solid support via a biotin-streptavidin linkage at one end of the fragment, upstream of the RNA start site. Templates immobilized in this manner were incubated with HeLa cell nuclear extracts to form preinitiation complexes containing RNA polymerase II and accessory proteins required for faithful in vitro transcription. These complexes did not require ATP or dATP for assembly, were sensitive to 0.25% Sarkosyl, and were stable to extensive washing. Their incubation with specific combinations of nucleoside triphosphates resulted in the initiation of RNA chain polymerization in situ, while addition of the remaining nucleoside triphosphates was necessary to produce a full length runoff RNA. Transcriptional activity associated with preinitiation complexes was purified approximately 300-fold, relative to the unfractionated nuclear extract. The use of immobilized template permits considerable flexibility in experimental design, as substrates and inhibitors can be added and washed out of the reaction at each step. We exploited this property of the system to dissect the temporal substrate requirements for initiation of RNA synthesis. It is known from prior work that at least one step in the promoter-dependent RNA synthesis reaction requires an adenosine nucleotide that is hydrolyzable at the beta, gamma-position. This requirement is independent of the initiating nucleotide and can be satisfied by dATP, which is not ordinarily incorporated into the RNA product. We show here that the beta, gamma-hydrolyzable adenosine nucleotide must be present simultaneously with the initiating nucleoside triphosphates. No reaction occurred when complexes were incubated with dATP, washed to remove dATP, and incubated subsequently with the two initiating nucleotides.

Adenine Nucleotides↗
PubMed1989

Characterization of a minimal simian virus 40 late promoter: enhancer elements in the 72-base-pair repeat not required.

A 272-base-pair (bp) portion of the simian virus 40 regulatory region containing the replication origin, Sp1-binding region, and part of the 72-bp direct repeats makes up a minimal late promoter that is able to direct late-direction RNA synthesis in vivo and in vitro. Fourteen linker-scan mutants within this region were characterized. Mutations in the Sp1-binding region decreased late expression both in vivo and in vitro. By contrast, mutations that eliminate genetically defined elements of the early transcriptional enhancer or that prevent binding of the transcription factors AP-1, AP-2, and AP-3 in the 72-bp repeat region had little or no effect on late-direction expression. These results argue that, at least under certain circumstances, the early transcriptional enhancer sequences are not required for simian virus 40 late gene expression.

Biological Evolution↗
PubMed1988

Binding of host-cell factors to DNA sequences in the long terminal repeat of human T-cell leukemia virus type I: implications for viral gene expression.

Efficient expression of human T-cell leukemia virus type I (HTLV-I) genes requires both host and viral proteins and is dependent on DNA sequences in the proviral long terminal repeats (LTRs). We have used DNase I-protection assays (footprinting) to construct a map of protein-DNA interactions over a 250-nucleotide region of the LTR upstream of the start site for viral RNA synthesis. We find that a host factor (host expression factor 1, or HEF-1) binds to the imperfect 21-nucleotide repeats that have previously been implicated in HTLV-I gene expression. HEF-1 binding activity is present in preparations from both lymphoid and nonlymphoid cell lines. However, the boundaries of the protected regions and the presence of a flanking DNase-hypersensitive site vary with cell type. Several regions of binding are detected in addition to the HEF-1 sites, including a complex group of sites 40-90 nucleotides upstream of the RNA start site. A comparison of HTLV-I-transformed T lymphocytes that do and do not express the viral trans-activating protein p40xI shows that none of the observed features of the DNase I footprint pattern correlate directly with the presence of this protein in the extract. These results suggest (i) that the primary recognition of promoter elements in the HTLV-I LTR involves specific interactions with host-cell proteins and (ii) that p40xI influences the activity of one or more of these proteins, rather than interacting directly with the DNA.

Binding Sites↗
PubMed1988

Binding of cellular proteins to the regulatory region of BK virus DNA.

The human papovavirus BK has a noncoding regulatory region located between the divergently transcribed early and late coding regions. Many strains of BK virus (BKV) have direct DNA sequence repeats in the regulatory region, although the number and extent of these repeats varies widely between independent isolates. Until recently, little was known about the individual functional elements within the BKV regulatory region, and the biological significance of the variable repeat structure has been unclear. To characterize the interaction between sequences in the BKV regulatory region and host cell transcription factors, we have carried out DNase I footprinting and competitive binding experiments on three strains of BKV, including one strain that does not contain direct sequence repeats. We have used relatively crude fractions from HeLa cell nuclear extracts, as well as DNA affinity-purified preparations of proteins. Our results demonstrate that BK(Dunlop), BK(WW), and BK(MM) each contain multiple binding sites for a factor, NF-BK, that is a member of the nuclear factor 1 family of transcription factors. We predict the presence of three to eight binding sites for NF-BK in the other strains of BKV for which a DNA sequence is available. This suggests that the binding of this protein is likely to be required for biological activity of the virus. In addition to NF-BK sites, BK(WW) and BK(MM) each contain a single binding site for transcription factor Sp1, and BK(Dunlop) contains two binding sites for transcription factor AP-1. The AP-1 sites in BK(Dunlop) span the junction of adjacent direct repeats, suggesting that repeat formation may be an important mechanism for de novo formation of binding sites not present in a parental strain.

BK Virus↗
PubMed1988

Simian virus 40 major late promoter: a novel tripartite structure that includes intragenic sequences.

Unlike most genes transcribed by RNA polymerase II, the simian virus 40 late transcription unit does not have a TATA box. To determine what sequences are required for initiation at the major late mRNA cap site of simian virus 40, clustered point mutations were constructed and tested for transcriptional activity in vitro and in vivo. Three promoter elements were defined. The first is centered 31 base pairs upstream of the cap site in a position normally reserved for a TATA box. The second is at the cap site. The third occupies a novel position centered 28 base pairs downstream of the cap site within a protein-coding sequence. The ability of RNA polymerase II to recognize this promoter suggests that there is greater variation in promoter architecture than had been believed previously.

Base Sequence↗