Opposing effects of Ctk1 kinase and Fcp1 phosphatase at Ser 2 of the RNA polymerase II C-terminal domain.

The C-terminal domain (CTD) of the RNA polymerase II (Pol II) largest subunit is hyperphosphorylated during transcription. Using an in vivo cross-linking/chromatin immunoprecipitation assay, we found previously that different phosphorylated forms of RNA Pol II predominate at different stages of transcription. At promoters, the Pol II CTD is phosphorylated at Ser 5 by the basal transcription factor TFIIH. However, in coding regions, the CTD is predominantly phosphorylated at Ser 2. Here we show that the elongation-associated phosphorylation of Ser 2 is dependent upon the Ctk1 kinase, a putative yeast homolog of Cdk9/P-TEFb. Furthermore, mutations in the Fcp1 CTD phosphatase lead to increased levels of Ser 2 phosphorylation. Both Ctk1 and Fcp1 cross-link to promoter and coding regions, suggesting that they associate with the elongating polymerase. Both Ctk1 and Fcp1 have been implicated in regulation of transcription elongation. Our results suggest that this regulation may occur by modulating levels of Ser 2 phosphorylation, which in turn, may regulate the association of elongation factors with the polymerase.

COMPASS: a complex of proteins associated with a trithorax-related SET domain protein.

The trithorax genes encode an evolutionarily conserved family of proteins that function to maintain specific patterns of gene expression throughout cellular development. Members of this protein family contain a highly conserved 130- to 140-amino acid motif termed the SET domain. We report the purification and molecular identification of the subunits of a protein complex in the yeast Saccharomyces cerevisiae that includes the trithorax-related protein Set1. This protein complex, which we have named COMPASS (Complex Proteins Associated with Set1), consists of seven polypeptides ranging from 130 to 25 kDa. The same seven proteins were identified in COMPASS purified either by conventional biochemical chromatography or tandem-affinity tagging of the individual subunits of the complex. Null mutants missing any one of the six nonessential subunits of COMPASS grow more slowly than wild-type cells under normal conditions and demonstrate growth sensitivity to hydroxyurea. Furthermore, gene expression profiles of strains missing either of two nonessential subunits of COMPASS are altered in similar ways, suggesting these proteins have similar roles in gene expression in vivo. Molecular characterization of trithorax complexes will facilitate defining the role of this class of proteins in the regulation of gene expression and how their misregulation results in the development of human cancer.

Characterization of a six-subunit holo-elongator complex required for the regulated expression of a group of genes in Saccharomyces cerevisiae.

The Elongator complex associated with elongating RNA polymerase II in Saccharomyces cerevisiae was originally reported to have three subunits, Elp1, Elp2, and Elp3. Using the tandem affinity purification (TAP) procedure, we have purified a six-subunit yeast Holo-Elongator complex containing three additional polypeptides, which we have named Elp4, Elp5, and Elp6. TAP tapping and subsequent purification of any one of the six subunits result in the isolation of all six components. Purification of Elongator in higher salt concentrations served to demonstrate that the complex could be separated into two subcomplexes: one consisted of Elp1, -2, and -3, and the other consisted of Elp4, -5, and -6. Deletions of the individual genes encoding the new Elongator subunits showed that only the ELP5 gene is essential for growth. Disruption of the two nonessential new Elongator-encoding genes, ELP4 and ELP6, caused the same phenotypes observed with knockouts of the original Elongator-encoding genes. Results of microarray analyses demonstrated that the gene expression profiles of strains containing deletions of genes encoding subunits of either Elongator subcomplex, in which we detected significantly altered mRNA expression levels for 96 genes, are very similar, implying that all the Elongator subunits likely function together to regulate a group of S. cerevisiae genes in vivo.

Interactions of an Arg-rich region of transcription elongation protein NusA with NUT RNA: implications for the order of assembly of the lambda N antitermination complex in vivo.

The E. coli NusA transcription elongation protein (NusA(Ec)), identified because of its requirement for transcription antitermination by the N protein, has an Arg-rich S1 RNA-binding domain. A complex of N and NusA with other host factors binding at NUT sites in the RNA renders RNA polymerase termination-resistant. An E. coli haploid for nusA944, having nine different codons replacing four normally found in the Arg-rich region, is defective in support of N action. Another variant, haploid for the nusAR199A allele, with a change in a highly conserved Arg codon in the S1 domain, effectively supports N-mediated antitermination. However, nusAR199A is recessive to nusA944, while nusA(Ec) is dominant to nusA944 for support of N-mediated antitermination, suggesting a competition between NusA944 and NusAR199A during complex formation. Complex formation with the variant NusA proteins was assessed by mobility gel shifts. NusAR199A, unlike NusA(Ec) and NusA944, fails to form a complex with N and NUT RNA. However, while NusAR199A, like wild-type NusA, forms an enlarged complex with NUT RNA, N, RNA polymerase, and other host proteins required for efficient N-mediated antitermination, NusA944 does not form this enlarged complex. Consistent with the in vivo results, NusA944 prevents NusAR199A but not NusA(Ec) from forming the enlarged complex. The simplest conclusion from these dominance studies is that in the formation of the complete active antitermination complex in vivo, NusA and N binding to the newly synthesized NUT RNA precedes addition of the other factors. Alternative less effective routes to the active complex that allows bypass of this preferred pathway may also exist.

Protein production: feeding the crystallographers and NMR spectroscopists.

Protein purification efforts for structural genomics will focus on automation for the readily-expressed proteins, and process development for the more difficult ones, such as membrane proteins. Thousands of proteins are expected to be produced in the next few years. The purified proteins will be valuable reagents for the entire research community.

The alpha subunit of E. coli RNA polymerase activates RNA binding by NusA.

The Escherichia coli NusA protein modulates pausing, termination, and antitermination by associating with the transcribing RNA polymerase core enzyme. NusA can be covalently cross-linked to nascent RNA within a transcription complex, but does not bind RNA on its own. We have found that deletion of the 79 carboxy-terminal amino acids of the 495-amino-acid NusA protein allows NusA to bind RNA in gel mobility shift assays. The carboxy-terminal domain (CTD) of the alpha subunit of RNA polymerase, as well as the bacteriophage lambda N gene antiterminator protein, bind to carboxy-terminal regions of NusA and enable full-length NusA to bind RNA. Binding of NusA to RNA in the presence of alpha or N involves an amino-terminal S1 homology region that is otherwise inactive in full-length NusA. The interaction of the alpha-CTD with full-length NusA stimulates termination. N may prevent termination by inducing NusA to interact with N utilization (nut) site RNA rather than RNA near the 3' end of the nascent transcript. Sequence analysis showed that the alpha-CTD contains a modified helix-hairpin-helix motif (HhH), which is also conserved in the carboxy-terminal regions of some eubacterial NusA proteins. These HhH motifs may mediate protein-protein interactions in NusA and the alpha-CTD.

A motif shared by TFIIF and TFIIB mediates their interaction with the RNA polymerase II carboxy-terminal domain phosphatase Fcp1p in Saccharomyces cerevisiae.

Transcription by RNA polymerase II is accompanied by cyclic phosphorylation and dephosphorylation of the carboxy-terminal heptapeptide repeat domain (CTD) of its largest subunit. We have used deletion and point mutations in Fcp1p, a TFIIF-interacting CTD phosphatase, to show that the integrity of its BRCT domain, like that of its catalytic domain, is important for cell viability, mRNA synthesis, and CTD dephosphorylation in vivo. Although regions of Fcp1p carboxy terminal to its BRCT domain and at its amino terminus were not essential for viability, deletion of either of these regions affected the phosphorylation state of the CTD. Two portions of this carboxy-terminal region of Fcp1p bound directly to the first cyclin-like repeat in the core domain of the general transcription factor TFIIB, as well as to the RAP74 subunit of TFIIF. These regulatory interactions with Fcp1p involved closely related amino acid sequence motifs in TFIIB and RAP74. Mutating the Fcp1p-binding motif KEFGK in the RAP74 (Tfg1p) subunit of TFIIF to EEFGE led to both synthetic phenotypes in certain fcp1 tfg1 double mutants and a reduced ability of Fcp1p to activate transcription when it is artificially tethered to a promoter. These results suggest strongly that this KEFGK motif in RAP74 mediates its interaction with Fcp1p in vivo.

The acute myeloid leukemia-associated protein, DEK, forms a splicing-dependent interaction with exon-product complexes.

DEK is an approximately 45-kD phosphoprotein that is fused to the nucleoporin CAN as a result of a (6;9) chromosomal translocation in a subset of acute myeloid leukemias (AMLs). It has also been identified as an autoimmune antigen in juvenile rheumatoid arthritis and other rheumatic diseases. Despite the association of DEK with several human diseases, its function is not known. In this study, we demonstrate that DEK, together with SR proteins, associates with the SRm160 splicing coactivator in vitro. DEK is recruited to splicing factor-containing nuclear speckles upon concentration of SRm160 in these structures, indicating that DEK and SRm160 associate in vivo. We further demonstrate that DEK associates with splicing complexes through interactions mediated by SR proteins. Significantly, DEK remains bound to the exon-product RNA after splicing, and this association requires the prior formation of a spliceosome. Thus, DEK is a candidate factor for controlling postsplicing steps in gene expression that are influenced by the prior removal of an intron from pre-mRNA.

Functional importance of regions in Escherichia coli elongation factor NusA that interact with RNA polymerase, the bacteriophage lambda N protein and RNA.

The association of the essential Escherichia coli protein NusA with RNA polymerase increases pausing and the efficiency of termination at intrinsic terminators. NusA is also part of the phage lambda N protein-modified antitermination complex that functions to prevent transcriptional termination. We have investigated the structure of NusA using various deletion fragments of NusA in a variety of in vitro assays. Sequence and structural alignments have suggested that NusA has both S1 and KH homology regions that are thought to bind RNA. We show here that the portion of NusA containing the S1 and KH homology regions is important for NusA to enhance both termination and antitermination. There are two RNA polymerase-binding regions in NusA, one in the amino-terminal 137 amino acids and the other in the carboxy-terminal 264 amino acids; only the amino-terminal RNA polymerase-binding region provides a functional contact that enhances termination at an intrinsic terminator or antitermination by N. The carboxy-terminal region of NusA is also required for interaction with N and is important for the formation of an N-NusA-nut site or N-NusA-RNA polymerase-nut site complex; the instability of complexes lacking this carboxy-terminal region of NusA that binds N and RNA polymerase can be compensated for by the presence of the additional E. coli elongation factors, NusB, NusG and ribosomal protein S10.

An unusual eukaryotic protein phosphatase required for transcription by RNA polymerase II and CTD dephosphorylation in S. cerevisiae.

The carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II is phosphorylated soon after transcriptional initiation. We show here that the essential FCP1 gene of S. cerevisiae is linked genetically to RNA polymerase II and encodes a CTD phosphatase essential for dephosphorylation of RNA polymerase II in vivo. Fcp1p contains a phosphatase motif, psi psi psi DXDX(T/V)psi psi, which is novel for eukaryotic protein phosphatases and essential for Fcp1p to function in vivo. This motif is also required for recombinant Fcp1p to dephosphorylate the RNA polymerase II CTD or the artificial substrate p-nitrophenylphosphate in vitro. The effects of fcp1 mutations in global run-on and genome-wide expression studies show that transcription by RNA polymerase II in S. cerevisiae generally requires CTD phosphatase.

Topological localization of the carboxyl-terminal domain of RNA polymerase II in the initiation complex.

The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAP II) functions at multiple stages of transcription and is involved in the coupling of transcription to pre-mRNA processing. We have used site-specific protein-DNA photocross-linking to determine the position of the CTD along promoter DNA in the transcriptional pre-initiation complex. Comparison of the promoter contacts made by RNAP II with or without the CTD indicate that the CTD approaches promoter DNA downstream of the transcriptional initiation site between positions +16 and +26. Incubation of pre-assembled initiation complexes with antibodies to the CTD prior to UV irradiation led to specific photocross-linking of the IgG heavy chain to nucleotide +17, indicating that the CTD is accessible for protein-protein interactions in a complex containing RNAP II and the general initiation factors. In conjunction with previously published observations, our structural data are fully compatible with the notion that DNA wrapping around RNAP II places the CTD and the RNA exit channel into juxtaposition and provide a rationale for contacts between the SRB-mediator complex and core RNAP II observed in the RNAP II holoenzyme.

GAL4 is regulated by the RNA polymerase II holoenzyme-associated cyclin-dependent protein kinase SRB10/CDK8.

Phosphorylation of the yeast transcription factor GAL4 at S699 is required for efficient galactose-inducible transcription. We demonstrate that this site is a substrate for the RNA polymerase holoenzyme-associated CDK SRB10. S699 phosphorylation requires SRB10 in vivo, and this site is phosphorylated by purified SRB10/ SRB11 CDK/cyclin in vitro. RNA Pol II holoenzymes purified from WT yeast phosphorylate GAL4 at sites observed in vivo whereas holoenzymes from srb10 yeast are incapable of phosphorylating GAL4 at S699. Mutations at GAL4 S699 and srb10 are epistatic for GAL induction, demonstrating that SRB10 regulates GAL4 activity through this phosphorylation in vivo. These results demonstrate a function for the SRB10/ CDK8 holoenzyme-associated CDK that involves regulation of transactivators by phosphorylation during transcriptional activation.

Multivessel spontaneous coronary artery dissection in a patient with severe systolic hypertension: a possible association. A case report.

Spontaneous coronary artery dissection (SCAD) is an uncommon cause of myocardial ischemia and infarction. Hypertension has not been associated with SCAD. The authors report multivessel SCAD in an elderly woman with severe systolic hypertension. They postulate that hypertension of this degree may play a pathophysiologic role in the causation of SCAD.

Activation of the murine dihydrofolate reductase promoter by E2F1. A requirement for CBP recruitment.

The E2F family of heterodimeric transcription factors plays an important role in the regulation of gene expression at the G1/S phase transition of the mammalian cell cycle. Previously, we have demonstrated that cell cycle regulation of murine dihydrofolate reductase (dhfr) expression requires E2F-mediated activation of the dhfr promoter in S phase. To investigate the mechanism by which E2F activates an authentic E2F-regulated promoter, we precisely replaced the E2F binding site in the dhfr promoter with a Gal4 binding site. Using Gal4-E2F1 derivatives, we found that E2F1 amino acids 409-437 contain a potent core transactivation domain. Functional analysis of the E2F1 core domain demonstrated that replacement of phenylalanine residues 413, 425, and 429 with alanine reduces both transcriptional activation of the dhfr promoter and protein-protein interactions with CBP, transcription factor (TF) IIH, and TATA-binding protein (TBP). However, additional amino acid substitutions for phenylalanine 429 demonstrated a strong correlation between activation of the dhfr promoter and binding of CBP, but not TFIIH or TBP. Finally, transactivator bypass experiments indicated that direct recruitment of CBP is sufficient for activation of the dhfr promoter. Therefore, we suggest that recruitment of CBP is one mechanism by which E2F activates the dhfr promoter.

FCP1, the RAP74-interacting subunit of a human protein phosphatase that dephosphorylates the carboxyl-terminal domain of RNA polymerase IIO.

TFIIF (RAP30/74) is a general initiation factor that also increases the rate of elongation by RNA polymerase II. A two-hybrid screen for RAP74-interacting proteins produced cDNAs encoding FCP1a, a novel, ubiquitously expressed human protein that interacts with the carboxyl-terminal evolutionarily conserved domain of RAP74. Related cDNAs encoding FCP1b lack a carboxyl-terminal RAP74-binding domain of FCP1a. FCP1 is an essential subunit of a RAP74-stimulated phosphatase that processively dephosphorylates the carboxyl-terminal domain of the largest RNA polymerase II subunit. FCP1 is also a stoichiometric component of a human RNA polymerase II holoenzyme complex.

Wrapping of promoter DNA around the RNA polymerase II initiation complex induced by TFIIF.

The formation of the RNA polymerase II (Pol II) initiation complex was analyzed using site-specific protein-DNA photo-cross-linking. We show that the RAP74 subunit of transcription factor (TF) IIF, through its RAP30-binding domain and an adjacent region necessary for the formation of homomeric interactions in vitro, dramatically alters the distribution of RAP30, TFIIE, and Pol II along promoter DNA between positions -40 and +26. This isomerization of the complex, which requires both TFIIF and TFIIE, is accompanied by tight wrapping of the promoter DNA for almost a full turn around Pol II. Addition of TFIIH enhances photo-cross-linking of Pol II to a number of promoter positions, suggesting that TFIIH tightens the DNA wrap around the enzyme. We present a general model to describe transcription initiation.

Independent ligand-induced folding of the RNA-binding domain and two functionally distinct antitermination regions in the phage lambda N protein.

The transcriptional antitermination protein N of bacteriophage lambda binds the boxB component of the RNA enhancer nut (boxA + boxB) and the E. coli elongation factor NusA. Efficient antitermination by N requires an RNA-binding domain (amino acids 1-22) and two activating regions for antitermination: a newly identified NusA-binding region (amino acids 34-47) that suppresses NusA's enhancement of termination, and a carboxy-terminal region (amino acids 73-107) that interacts directly with RNA polymerase. Heteronuclear magnetic resonance experiments demonstrate that N is a disordered protein. Interaction with boxB RNA induces only the RNA-binding domain of N to adopt a folded conformation, while the activating regions of the protein remain disordered in the absence of their target proteins.