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D Reinberg

Publications and source records attributed to D Reinberg.

At least 91 records · Page 5Linked to original sources
PubMed1993

DNA topoisomerase I is involved in both repression and activation of transcription.

Reconstituted transcription reactions containing the seven general transcription factors, in addition to RNA polymerase II, respond poorly to transcriptional activators. Two factors, Dr2 and ACF, necessary for high levels of transcription in response to an activator have been identified. ACF can enhance basal and activated transcription. Dr2 represses basal transcription, but this can be overcome by transcriptional activators or TFIIA. Dr2 is human DNA topoisomerase I. The DNA relaxation activity of topoisomerase I is dispensable for transcriptional repression. The effect of Dr2 is specific for TATA-box-containing promoters and is mediated by the TATA-binding protein.

Amino Acid Sequence↗
PubMed1993

Interaction between an acidic activator and transcription factor TFIIB is required for transcriptional activation.

How eukaryotic promoter-specific activator proteins (activators) stimulate transcription is a central question. We have previously shown that an acidic activator can directly interact with the general transcription factor TFIIB and increase its stable assembly into a preinitiation complex. We have proposed that this increase in TFIIB assembly is at least part of the mechanism by which an acidic activator functions. A prediction of this hypothesis is that a TFIIB mutant unable to interact with an acidic activator could not support activated transcription, and here we present experiments that verify this prediction. In conjunction with previous studies, our results argue that interaction between an acidic activator and TFIIB is necessary for transcriptional activation.

Binding Sites↗
PubMed1993

Oct-2 facilitates functional preinitiation complex assembly and is continuously required at the promoter for multiple rounds of transcription.

Octamer factor 2 (Oct-2, OTF-2, NF-A2) is an 'upstream' promoter factor that binds to the octamer motif (ATGCAAAT) implicated in control of immunoglobulin gene transcription in B-lymphocytes. We have studied the role of Oct-2 in the process of transcription initiation in vitro using both nuclear extracts and purified basal transcription factors. Oct-2 specifically stimulates transcription from octamer-containing promoters in both systems. Thus, Oct-2 is a 'true activator', rather than merely an 'anti-repressor' counteracting the effect of histones. In order-of-addition experiments, Oct-2 is required early, together with TFIID, to allow formation of a preinitiation complex. Oct-2 cannot functionally interact with cloned TATA binding protein (TBP) but rather requires 'coactivators' found in the TFIID fraction. In single-round transcription experiments, early competition for Oct-2 by an octamer oligonucleotide is deleterious, but no effect is seen after assembly of a complete preinitiation complex. However, for multiple rounds of transcription, Oct-2 is continuously required at the promoter; this result argues against a 'hit-and-run' mechanism whereby the activator becomes dispensible after organizing a TFIID-promoter complex. In agreement with our previous studies in vivo, the N-terminal glutamine-rich activation domain of Oct-2 is required for full activity in vitro, indicating that this domain directly interacts with basal transcription factors.

Base Sequence↗
PubMed1993

Regulation of RNA polymerase II transcription.

Transcription initiation plays a central role in the regulation of gene expression. Exciting developments in the last year have furthered our understanding of the interactions between general transcription factors and how these factors respond to modulators of transcription.

Animals↗
PubMed1993

Isolation of a cDNA encoding the largest subunit of TFIIA reveals functions important for activated transcription.

Transcription factor IIA has been shown to interact with the TATA-binding protein and to act early during preinitiation complex formation. The human factor is composed of three subunits (alpha, beta, gamma). A human cDNA clone encoding the largest subunit of TFIIA (alpha) was isolated. The recombinant alpha polypeptide, together with the beta and gamma subunits, was capable of reconstituting TFIIA activity. Studies using antibodies raised against recombinant alpha polypeptide demonstrate that TFIIA can be an integral component of the preinitiation complex. We demonstrate that TFIIA not only interacts with TBP but also can associate with the TFIID complex. Functional assays establish that TFIIA has no apparent role in basal transcription but plays an important role in activation of transcription. Interestingly, amino acid sequence analyses of the beta-subunit demonstrate these residues to be entirely contained within the carboxyl terminus of the cDNA clone encoding the alpha-subunit.

Amino Acid Sequence↗
PubMed1993

Multiple functional domains of human transcription factor IIB: distinct interactions with two general transcription factors and RNA polymerase II.

Transcription factor IIB (TFIIB) plays a pivotal role in the formation of transcription-competent initiation complexes. TFIIB was found to interact with the TATA-binding protein, the small subunit of TFIIF, and RNA polymerase II. These interactions require distinct domains in TFIIB. Using the gel mobility-shift assay, it was found that the amino terminus of TFIIB was necessary for the formation of complexes containing RNA polymerase II and TFIIF, whereas the carboxy-terminal domain, which is composed of two imperfect direct repeats and includes a putative amphipathic alpha-helix, was sufficient for the formation of complexes containing the TATA-binding protein and TFIIB (DB complex). Protein-protein interaction analyses demonstrate that the amphipathic alpha-helix in TFIIB is important for the interaction with the TATA-binding protein. Specific residues mapping to the carboxyl terminus of the second direct repeat were found to be crucial for the interaction of TFIIB and RNA polymerase II. The interaction with the small subunit of TFIIF was mapped to the amino terminus of TFIIB, which includes a zinc finger.

Base Sequence↗
PubMed1992

Human general transcription factor IIH phosphorylates the C-terminal domain of RNA polymerase II.

Phosphorylation of the carboxy-terminal domain of the largest subunit of RNA polymerase II is believed to control the transition from transcription initiation to elongation. The general transcription factor IIH (TFIIH) contains a kinase activity capable of phosphorylating this domain. Factors that promote the association of RNA polymerase II with the preinitiation complex stimulate this activity. The transcription factor IIE, which is required for the stable association of TFIIH with the preinitiation complex, affects the processivity of TFIIH kinase.

Adenosine Triphosphate↗
PubMed1992

Dr1, a TATA-binding protein-associated phosphoprotein and inhibitor of class II gene transcription.

We have discovered a protein termed Dr1 that interacts with the TATA-binding protein, TBP. The association of Dr1 with TBP results in repression of both basal and activated levels of transcription. The interaction of Dr1 with TBP precludes the formation of a transcription-competent complex by inhibiting the association of TFIIA and/or TFIIB with TBP. Dr1 activity is associated with a 19 kd protein. A cDNA clone encoding Dr1 was isolated. Dr1 is phosphorylated in vivo and phosphorylation of Dr1 affected its interaction with TBP. Our results suggest a regulatory role for Dr1 in repression of transcription mediated via phosphorylation.

Amino Acid Sequence↗
PubMed1992

Specific interaction between the nonphosphorylated form of RNA polymerase II and the TATA-binding protein.

Fractionation of a transcription-competent HeLa cell extract on a column containing one copy of the heptamer repeat (YSPTSPS) present in the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II resulted in the loss of transcriptional activity. Fractionation of the extracts on columns containing mutations of the heptamer repeat was without effect. Such transcriptionally inactive extracts regained their ability to specifically transcribe different class II promoters upon the addition of human TFIID, recombinant yeast TATA-binding protein (TBP), or proteins bound to the column. Fractionation of RNA polymerase II on columns containing human or yeast TBP resulted in the specific retention of the nonphosphorylated form of RNA polymerase II. The phosphorylated form of the enzyme was unable to interact with TBP. The specific interaction of RNA polymerase II with TBP was mediated by the CTD of RNA polymerase II.

Amino Acid Sequence↗
PubMed1992

Protein kinases from Aspergillus nidulans that phosphorylate the carboxyl-terminal domain of the largest subunit of RNA polymerase II.

Three serine kinases which phosphorylate the CTD of RNA polymerase II have been identified in Aspergillus nidulans. The kinases (KI, KII, KIII) were identified using a synthetic peptide containing four copies of the CTD consensus heptamer repeat, and differ in chromatographic behavior, and apparent molecular mass (KI approximately 60kDa; KII approximately 82kDa; KIII approximately 43 kDa). KIII utilized, in addition to peptide, histone H1 as substrate, whereas casein was not phosphorylated by any of the three kinases. The kinases appear to be unrelated to the p34cdc2 kinase, as judged by Western blot analysis and the position of serine phosphorylation of the synthetic CTD peptide. KI was highly purified and renaturation experiments have shown that it consists of a single polypeptide of 57 kDa. KI also phosphorylated RNA polymerase II associated in a preinitiation complex.

Amino Acid Sequence↗
PubMed1992

Factors involved in specific transcription by mammalian RNA polymerase II. Identification and characterization of factor IIH.

Two new factors required for transcription of class II genes have been identified. These factors, TFIIH and TFIIJ, were required together with the previously described general factors (TFIIA, TFIIB, TFIID, TFIIE, and TFIIF) and RNA polymerase II for transcription of different class II genes. TFIIH was extensively purified, and the activity appeared to coelute with polypeptides of 33 and 95 kDa. The role of TFIIH and TFIIJ in preinitiation complex assembly was analyzed using mobility shift assays. It was found that TFIIH and TFIIJ association with the preinitiation complex was ordered and required the previous assembly of a preinitiation complex intermediate containing factors IID, IIB, IIF, IIE, and RNA polymerase II. A model for the ordered assembly of the general factors and RNA polymerase II is presented.

Blotting, Western↗
PubMed1992

Advances in RNA polymerase II transcription.

Multiple protein factors are necessary to mediate transcription by RNA polymerase II. Recently, a number of advances have been made in our understanding of how general transcription factors collectively modulate basal transcription in the context of different promoter environments and how this process is activated and repressed by accessory components.

Animals↗
PubMed1992

Transcription by RNA polymerase II: initiator-directed formation of transcription-competent complexes.

Studies of transcription by RNA polymerase II have revealed two promoter elements, the TATA motif and the initiator (Inr), capable of directing specific transcription initiation. Although binding to the TATA motif by one of the components of the transcription machinery has been shown to be the initial recognition step in transcription complex formation, many promoters that lack a traditional TATA motif have recently been described. In such TATA-less promoters, the Inr element is critical in positioning RNA polymerase II. Various Inr elements have been described and classified according to sequence homology. These Inr elements are recognized specifically by Inr-binding proteins. Interaction between these Inr-binding proteins and components of the basal transcription machinery provides a means through which a transcription competent complex can be formed.

Adenoviridae↗
PubMed1992

Factors involved in specific transcription by mammalian RNA polymerase II: purification and analysis of transcription factor IIA and identification of transcription factor IIJ.

The previously described transcription factor IIA (TFIIA) protein fraction was separated into two factors that affect transcription, TFIIA and TFIIJ. TFIIA was found to have a stimulatory effect, and TFIIJ was found to be required for transcription. The requirement of TFIIJ was observed when bacterially produced purified human or yeast (Saccharomyces cerevisiae) TATA-binding protein (TBP) was used in lieu of the endogenous HeLa cell TFIID complex, suggesting that TFIIJ may be part of the TFIID complex. The stimulatory activity of TFIIA was found also to be dependent on the source of the TBP. Transcription reactions reconstituted with TFIID were stimulated by TFIIA; however, when human or yeast TBP was used instead of TFIID, TFIIA had no effect. TFIIA was found to interact with the TBP and was extensively purified by the use of affinity chromatography on columns containing immobilized recombinant yeast TBP. TFIIA is a heterotrimer composed of polypeptides of 34, 19, and 14 kDa. These three polypeptides were required to isolate, by using the gel mobility shift assay, a stable complex between TBP and the TATA box sequence.

Chromatography↗
PubMed1991

Structure and functional properties of human general transcription factor IIE.

The general transcription factor IIE (TFIIE) is an essential component of the eukaryotic RNA polymerase II initiation complex. We have isolated human complementary DNA clones for both the subunits of TFIIE. Using purified recombinant proteins we find that both subunits are essential to form a stable preinitiation complex and to reconstitute basal-level and Sp1-activated transcription in vitro. Analysis of their predicted amino-acid sequences reveals several intriguing structural motifs that could provide insight into the role of TFIIE in transcription initiation.

Adenosine Triphosphate↗