Edio Maldonado

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.

Factors involved in specific transcription by mammalian RNA polymerase II: role of transcription factors IIA, IID, and IIB during formation of a transcription-competent complex.

Human transcription factor TFIID, the TATA-binding protein, was partially purified to a form capable of associating stably with the TATA motif of the adenovirus major late promoter. Binding of the human and yeast TFIID to the TATA motif was stimulated by TFIIA. TFIIA is an integral part of a complex capable of binding other transcription factors. A complex formed with human TFIID and TFIIA (DA complex) was specifically recognized by TFIIB. We found that TFIIB activity was contained in a single polypeptide of 32 kDa and that this polypeptide participated in transcription and was capable of binding to the DA complex to form the DAB complex. Formation of the DAB complex required TFIIA, TFIID, and sequences downstream of the transcriptional start site; however, the DA complex could be formed on an oligonucleotide containing only the adenovirus major late promoter TATA motif. Using anti-TFIIB antibodies and reagents that affect the stability of a transcription-competent complex, we found that yeast and human TFIID yielded DAB complexes with different stabilities.

Solution structure of the c-terminal core domain of human TFIIB: Similarity to cyclin A and interaction with TATA-binding protein

TFIIB is an essential component of the machinery that transcribes protein-coding genes. The three-dimensional structure of the human TFIIB core domain (TFIIBc) has been determined using multidimensional heteronuclear magnetic resonance spectroscopy. The molecule consists of two direct repeats that adopt similar alpha-helical folds, conferring pseudo-twofold symmetry. An extensive, central basic surface including an amphipathic alpha helix is critical to the function of TFIIB as a bridge between the TBP-promoter complex and RNA polymerase II and associated general and regulatory transcription factors. Similarities between the TFIIBc and cyclin A folds indicate that elements of the eukaryotic cell cycle control apparatus evolved from more fundamental transcriptional control components, demonstrating a link between the transcription and cell cycle molecular machineries.

PURIFICATION OF HUMAN RNA POLYMERASE II AND GENERAL TRANSCRIPTION FACTORS

Publisher Summary The identification and isolation of the general transcription factors (GTFs) that govern the activity of the RNA polymerase II (RNAP II) enzyme have paved the way toward understanding gene expression at the molecular level. Conventional biochemical purification schemes have identified five GTFs specifically required for RNAP II-driven basal transcription. These include transcription factor IID (TFIID), which is composed of an array of proteins, called TATA-binding protein-associated factors (TAFs), and tightly bound to the TATA-binding protein (TBP): TFIIB, TFIIE, TFIIF, and TFIIH. The purification of these GTFs has resulted in the isolation of the complementary DNAs (cDNA) for almost all the factors required to assemble the basal transcription apparatus. Recombinant DNA technology has provided convenient methods by which to express and purify the assortment of GTF cDNAs, and reconstitute functionally active factors. The ability to reconstitute a highly purified transcription system in vitro enables to probe the role of specific repressors and activators/coactivators in regulating RNAP II transcription. The chapter highlights purification protocols for the various recombinant and native GTFs and RNAP II from humans.

News on initiation and elongation of transcription by RNA polymerase II.

Transcription by RNA polymerase II is a complex process that requires additional factors to initiate transcription at the promoters. New developments in the past year have furthered our understanding of the functions of the transcription factors and provided more insights into the mechanisms involved in the regulation of initiation and elongation of transcription. One of the most significant advances of the past year was the discovery of the involvement of the general transcription factor TFIIH in DNA excision repair. Surprisingly, studies aimed at identifying the kinase activity within TFIIH responsible for phosphorylating the carboxy-terminal domain of RNA polymerase II revealed it to be the MO15/Cdk7 kinase and its partner, cyclin H. These exciting observations suggest a paradigm for linking transcription, DNA excision repair and cell cycle progression through one pivotal factor.

Functional interaction of the v-Rel and c-Rel oncoproteins with the TATA-binding protein and association with transcription factor IIB.

Rel family proteins regulate the expression of genes linked to kappa B-binding motifs. Little is known, however, of the mechanism by which they enhance transcription. We have investigated the ability of the v-Rel and c-Rel oncoproteins to interact with components of the basal transcription machinery. Here we report that both the acidic transcription activation domain mapping to the unique C terminus of chicken c-Rel and the F9 cell-specific activation region common to both v-Rel and c-Rel interact with the TATA-binding protein (TBP) and transcription factor IIB (TFIIB) in vitro and in vivo. We also demonstrate that TPB interaction with Rel activation regions leads to synergistic activation of transcription of a kappa B-linked reporter gene. Combined with the observation that the mouse c-Rel and human RelA proteins also interact with TBP and TFIIB in vitro, these results suggest that association with basal transcription factors is important for the transcriptional activities of Rel family proteins.

Membrane Electrical Activity Elicits Inositol 1,4,5-Trisphosphate-dependent Slow Ca2+ Signals through a Gβγ/Phosphatidylinositol 3-Kinase γ Pathway in Skeletal Myotubes

Tetanic electrical stimulation of myotubes evokes a ryanodine receptor-related fast calcium signal, during the stimulation, followed by a phospholipase C/inositol 1,4,5-trisphosphate-dependent slow calcium signal few seconds after stimulus end. L-type calcium channels (Cav 1.1, dihydropyridine receptors) acting as voltage sensors activate an unknown signaling pathway involved in phospholipase C activation. We demonstrated that both G protein and phosphatidylinositol 3-kinase were activated by electrical stimulation, and both the inositol 1,4,5-trisphosphate rise and slow calcium signal induced by electrical stimulation were blocked by pertussis toxin, by a Gβγ scavenger peptide, and by phosphatidylinositol 3-kinase inhibitors. Immunofluorescence using anti-phosphatidylinositol 3-kinase γ antibodies showed a clear location in striations within the cytoplasm, consistent with a position near the I band region of the sarcomere. The time course of phosphatidylinositol 3-kinase activation, monitored in single living cells using a pleckstrin homology domain fused to green fluorescent protein, was compatible with sequential phospholipase Cγ1 activation as confirmed by phosphorylation assays for the enzyme. Co-transfection of a dominant negative form of phosphatidylinositol 3-kinase γ inhibited the phosphatidylinositol 3-kinase activity as well as the slow calcium signal. We conclude that Gβγ/phosphatidylinositol 3-kinase γ signaling pathway is involved in phospholipase C activation and the generation of the slow calcium signal induced by tetanic stimulation. We postulate that membrane potential fluctuations in skeletal muscle cells can activate a pertussis toxin-sensitive G protein, phosphatidylinositol 3-kinase, phospholipase C pathway toward modulation of long term, activity-dependent plastic changes.

Affinity Purification of a Human RNA Polymerase II Complex Using Monoclonal Antibodies against Transcription Factor IIF

Five different monoclonal antibodies that immunoreact with RAP74, the large subunit of general transcription factor (TF) IIF, were produced and characterized. Using one of these antibodies, an affinity purification procedure was devised to isolate a human RNA polymerase II complex. This procedure is fast, simple, and reproducible and does not require extensive purification. The RNA polymerase II complex isolated using this procedure contains SRB (suppressor of RNA polymerase B) polypeptides, transcription factors IIE and IIF, limiting amounts of TFIIH, and the TATA-binding protein, but was devoid of TFIIB.

Rrn7 Protein, an RNA Polymerase I Transcription Factor, Is Required for RNA Polymerase II-dependent Transcription Directed by Core Promoters with a HomolD Box Sequence

The region in promoters that specifies the transcription machinery is called the core promoter, displaying core promoter elements (CPE) necessary for establishment of a preinitiation complex and the initiation of transcription. A classical CPE is the TATA box. In fission yeast, Schizosaccharomyces pombe, a new CPE, called HomolD box, was discovered. Collectively, 141 ribosomal protein genes encoding the full set of 79 different ribosomal proteins and more than 60 other housekeeping genes display a HomolD box in the core promoter. Here, we show that transcription directed by the HomolD box requires the RNA polymerase II machinery, including the general transcription factors. Most intriguingly, however, we identify, by DNA affinity purification, Rrn7 as the protein binding to the HomolD box. Rrn7 is an evolutionary conserved member of the RNA polymerase I machinery involved in transcription initiation of core ribosomal DNA promoters. ChIP shows that Rrn7 cross-links to a ribosomal protein gene promoter containing the HomolD box but not to a promoter containing a TATA box. Taken together, our results suggest that Rrn7 is an excellent candidate to be involved in the coordination of ribosomal DNA and ribosomal gene transcription during ribosome synthesis and, therefore, offer a new perspective to study conservation and evolvability of regulatory networks in eukaryotes.