We report here the isolation of a human RNA polymerase II complex containing a subset of the basal transcription factors and the human homologues of the yeast SRB (for suppressors of RNA polymerase B) proteins. The complex contains transcriptional coactivators and increases the activation of transcription. In addition, some components of the RNA polymerase II complex participate in DNA repair.
ABSTRACT5-Capping is an early mRNA modification that has important consequences for downstream events in gene expression. We have isolated mammalian cDNAs encoding capping enzyme. They contain the sequence motifs characteristic of the nucleotidyl transferase superfamily. The predicted mouse and human enzymes consist of 597 amino acids and are 95% identical. Mouse cDNA directed synthesis of a guanylylated 68-kDa polypeptide that also contained RNA 5-triphosphatase activity and catalyzed formation of RNA 5-terminal GpppG. A haploid strain of Saccharomyces cerevisiae lacking mRNA guanylyltransferase was complemented for growth by the mouse cDNA. Conversion of Lys-294 in the KXDG-conserved motif eliminated both guanylylation and complementation, identifying it as the active site. The K294A mutant retained RNA 5-triphosphatase activity, which was eliminated by N-terminal truncation. Full-length capping enzyme and an active C-terminal fragment bound to the elongating form and not to the initiating form of polymerase. The results document functional conservation of eukaryotic mRNA guanylyltransferases from yeast to mammals and indicate that the phosphorylated C-terminal domain of RNA polymerase II couples capping to transcription elongation. These results also explain the selective capping of RNA polymerase II transcripts.Addition of a 5Ј-terminal cap is an important, early event in mRNA formation (1). This structural hallmark of most eukaryotic mRNAs enhances splicing (2-4), transport (5), translation (6), and stability (7,8) and is essential for viability (9).Caps are formed on nascent nuclear pre-mRNAs by conversion of 5Ј-tri-diphosphate to 5Ј-diphosphate ends, followed by addition of GMP and methylation (1, 10). The guanylyltransfer reaction characterized in various systems involves formation of an active enzyme intermediate containing GMP covalently attached to lysine (11). In yeast, mRNA capping enzyme consists of separate subunits for RNA 5Ј-triphosphatase and guanylyltransferase activities (9, 12). cDNA clones coding for mRNA guanylyltransferase in Saccharomyces cerevisiae (9), Schizosaccharomyces pombe (13), and Candida albicans (14) have been sequenced. Each contains the active site lysine in KXDG (13, 15), one of several highly conserved motifs characteristic of a superfamily of nucleotidyl transferases (16). A number of viral capping enzymes also contain these diagnostic sequence motifs, and the recently solved structure of capping enzyme from Chlorella virus PBCV-1 suggests that specific residues in these motifs are important for binding GTP (17). Despite this detail of sequence and structure information, no metazoan capping enzyme previously has been cloned and characterized.To explore the molecular interactions that result in selective capping of RNA polymerase II (pol II) transcripts in mammalian cells, we have isolated and characterized cDNA clones that code for the human and mouse capping enzymes. Functional studies demonstrated that the mammalian enzyme complements the lethality of a S. cerevisiae mu...
Transcription is regulated by the state of phosphorylation of a heptapeptide repeat known as the carboxy-terminal domain (CTD) present in the largest subunit of RNA polymerase II (RNAPII). RNAPII that associates with transcription initiation complexes contains an unphosphorylated CTD, whereas the elongating polymerase has a phosphorylated CTD. Transcription factor IIH has a kinase activity specific for the CTD that is stimulated by the formation of a transcription initiation complex. Here, we report the isolation of a cDNA clone encoding a 150-kD polypeptide, which, together with RNAPII, reconstitutes a highly specific CTD phosphatase activity. Functional analysis demonstrates that the CTD phosphatase allows recycling of RNAPII. The phosphatase dephosphorylates the CTD allowing efficient incorporation of RNAPII into transcription initiation complexes, which results in increased transcription. The CTD phosphatase was found to be active in ternary elongation complexes. Moreover, the phosphatase stimulates elongation by RNAPII; however, this function is independent of its catalytic activity.
We have isolated a human RNA polymerase II complex that contains chromatin structure remodeling activity and histone acetyltransferase activity. This complex contains the Srb proteins, the Swi-Snf complex, and the histone acetyltransferases CBP and PCAF in addition to RNA polymerase II. Notably, the general transcription factors are absent from this complex. The complex was purified by two different methods: conventional chromatography and affinity chromatography using antibodies directed against CDK8, the human homolog of the yeast Srb10 protein. Protein interaction studies demonstrate a direct interaction between RNA polymerase II and the histone acetyltransferases p300 and PCAF. Importantly, p300 interacts specifically with the nonphosphorylated, initiation-competent form of RNA polymerase II. In contrast, PCAF interacts with the elongation-competent, phosphorylated form of RNA polymerase II.
A complex that represses activated transcription and contains the human homologs of the yeast Srb7, Srb10, Srb11, Rgr1, and Med6 proteins was isolated. The complex is devoid of the Srb polypeptides previously shown to be components of the yeast Mediator complex that functions in transcriptional activation. The complex phosphorylates the CTD of RNA polymerase II (RNAPII) at residues other than those phosphorylated by the kinase of TFIIH. Moreover, the complex specifically interacts with RNAPII. The interaction is not mediated by the CTD of RNAPII, but is precluded by phosphorylation of the CTD. Our results indicate that the complex is a subcomplex of the human RNAPII holoenzyme. We suggest that the RNAPII holoenzyme is a transcriptional control panel, integrating and responding to specific signals to activate or repress transcription.
FCP1, a phosphatase specific for the carboxy-terminal domain of RNA polymerase II (RNAP II), was found to stimulate transcript elongation by RNAP II in vitro and in vivo. This activity is independent of and distinct from the elongation-stimulatory activity associated with transcription factor IIF (TFIIF), and the elongation effects of TFIIF and FCP1 were found to be additive. Genetic experiments resulted in the isolation of several distinct fcp1 alleles. One of these alleles was found to suppress the slow-growth phenotype associated with either the reduction of intracellular nucleotide concentrations or the inhibition of other transcription elongation factors. Importantly, this allele of fcp1 was found to be lethal when combined individually with two mutations in the second-largest subunit of RNAP II, which had been shown previously to affect transcription elongation.The engine at the heart of the transcriptional apparatus is RNA polymerase II (RNAP II), a protein complex comprising 12 subunits that are remarkably conserved throughout eukaryotes. Crystallographic studies with Saccharomyces cerevisiae RNAP II have given us the first detailed insights, at the atomic level, of the molecular mechanism by which eukaryotic RNA polymerases transcribe DNA (14, 16). RNA polymerases cannot recognize the promoters of their target genes but rather rely on accessory proteins, known in eukaryotes as general transcription factors (GTFs) (37, 46). These factors recognize conserved core promoter elements present in most proteincoding genes and also interact directly with RNAP II, resulting in the recruitment of RNAP II to the start site of transcription.At least two forms of RNAP II have been detected in cells. These isoforms result from the presence of a unique domain in the C-terminal region of the largest subunit of RNAP II, known as the carboxyl-terminal domain (CTD). In mammals, this domain consists of 52 repeats of the consensus heptapeptide Tyr-Ser-Pro-Thr-Ser-Pro-Ser and appears from crystallographic studies to be unstructured (14). The precise function of the CTD has been elusive, although it was speculated almost a decade ago that it constitutes a binding site for protein factors involved in transcriptional regulation and RNA processing (17,18).The initiation of transcription by RNAP II is a multistep process that involves separation of the DNA strands at the initiation site (promoter melting), formation of the first phosphodiester bond of the transcript, and disruption of the interactions between RNAP II and the promoter (promoter clearance). The isoform of RNAP II that associates with transcription initiation complexes contains an unphosphorylated CTD, whereas the isoform involved in transcription elongation contains a phosphorylated CTD (4,27,36,62). Thus, the transition from initiation of transcription to elongation of the transcript is accompanied by extensive phosphorylation of the CTD of RNAP II.A number of protein kinases have been implicated in the phosphorylation of the CTD during the transition from transcri...
Under conditions of environmental stress, prokaryotes and lower eukaryotes such as the yeast Saccharomyces cerevisiae selectively utilize particular subunits of RNA polymerase II (pol II) to alter transcription to patterns favoring survival. In S. cerevisiae, a complex of two such subunits, RPB4 and RPB7, preferentially associates with pol II during stationary phase; of these two subunits, RPB4 is specifically required for survival under nonoptimal growth conditions. Previously, we have shown that RPB7 possesses an evolutionarily conserved human homolog, hsRPB7, which was capable of partially interacting with RPB4 and the yeast transcriptional apparatus. Using this as a probe in a two-hybrid screen, we have now established that hsRPB4 is also conserved in higher eukaryotes. In contrast to hsRPB7, hsRPB4 has diverged so that it no longer interacts with yeast RPB7, although it partially complements rpb4 ؊ phenotypes in yeast. However, hsRPB4 associates strongly and specifically with hsRPB7 when expressed in yeast or in mammalian cells and copurifies with intact pol II. hsRPB4 expression in humans parallels that of hsRPB7, supporting the idea that the two proteins may possess associated functions. Structure-function studies of hsRPB4-hsRPB7 are used to establish the interaction interface between the two proteins. This identification completes the set of human homologs for RNA pol II subunits defined in yeast and should provide the basis for subsequent structural and functional characterization of the pol II holoenzyme.Selective control of mRNA transcription in response to intracellular and extracellular signals occurs at multiple levels, with targets for regulation including gene-specific transcription factors, general transcription factors, and the RNA polymerase II holoenzyme (15,18,38,55,63). This last mechanism of regulation, involving modification of core RNA polymerase II (pol II) structural composition by altering incorporation of subunits or regulated phosphorylation, has been well documented in prokaryotes and in yeast (17,31,59,62,70). In higher eukaryotes, the majority of transcriptional control studies have focused on characterizing the expression and modification of gene-specific and general transcription factors. However, a growing body of work on mammalian transcriptional control has demonstrated that mammalian pol II is also subject to modification by phosphorylation of the largest subunit, presumably as a means of regulation (10,24,41,42,49). In contrast, the issue of subunit variation has not been actively investigated.Studies of eukaryotic pol II function have depended heavily on paradigms developed through detailed characterization of the yeast Saccharomyces cerevisiae pol II (reviewed in reference 70). Yeast pol II contains 12 subunits (RPB1-12), all of which have been cloned and sequenced and many of which have been subjected to genetic and biochemical functional analysis. Five of these subunits, the common subunits (RPB5, RPB6, RPB8, RPB10, and RPB12), are also incorporated into RNA polymeras...
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.
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