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S Buratowski

Publications and source records attributed to S Buratowski.

At least 19 recordsLinked to original sources
PubMed2001

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.

Cyclins↗
PubMed2001

A histone fold TAF octamer within the yeast TFIID transcriptional coactivator.

Gene activity in a eukaryotic cell is regulated by accessory factors to RNA polymerase II, which include the general transcription factor complex TFIID, composed of TBP and TBP-associated factors (TAFs). Three TAFs that contain histone fold motifs (yTAF17, yTAF60 and yTAF61) are critical for transcriptional regulation in the yeast Saccharomyces cerevisiae and are found in both TFIID and SAGA, a multicomponent histone acetyltransferase transcriptional coactivator. Although these three TAFs were proposed to assemble into a pseudooctamer complex, we find instead that yTAF17, yTAF60 and yTAF61 form a specific TAF octamer complex with a fourth TAF found in TFIID, yTAF48. We have reconstituted this complex in vitro and established that it is an octamer containing two copies each of the four components. Point mutations within the histone folds disrupt the octamer in vitro, and temperature-sensitive mutations in the histone folds can be specifically suppressed by overexpressing the other TAF octamer components in vivo. Our results indicate that the TAF octamer is similar both in stoichiometry and histone fold interactions to the histone octamer component of chromatin.

Alleles↗
PubMed2001

Specific manipulative therapy treatment for chronic lateral epicondylalgia produces uniquely characteristic hypoalgesia.

The treatment of lateral epicondylalgia, a widely-used model of musculoskeletal pain in the evaluation of many physical therapy treatments, remains somewhat of an enigma. The protagonists of a new treatment technique for lateral epicondylalgia report that it produces substantial and rapid pain relief, despite a lack of experimental evidence. A randomized, double blind, placebo-controlled repeated-measures study evaluated the initial effect of this new treatment in 24 patients with unilateral, chronic lateral epicondylalgia. Pain-free grip strength was assessed as an outcome measure before, during and after the application of the treatment, placebo and control conditions. Pressure-pain thresholds were also measured before and after the application of treatment, placebo and control conditions. The results demonstrated a significant and substantial increase in pain-free grip strength of 58% (of the order of 60 N) during treatment but not during placebo and control. In contrast, the 10% change in pressure-pain threshold after treatment, although significantly greater than placebo and control, was substantially smaller than the change demonstrated for pain-free grip strength. This effect was only present in the affected limb. The selective and specific effect of this treatment technique provides a valuable insight into the physical modulation of musculoskeletal pain and requires further investigation.

Adult↗
PubMed2000

Different phosphorylated forms of RNA polymerase II and associated mRNA processing factors during transcription.

The activities of several mRNA processing factors are coupled to transcription through binding to RNA polymerase II (Pol II). The largest subunit of Pol II contains a repetitive carboxy-terminal domain (CTD) that becomes highly phosphorylated during transcription. mRNA-capping enzyme binds only to phosphorylated CTD, whereas other processing factors may bind to both phosphorylated and unphosphorylated forms. Capping occurs soon after transcription initiation and before other processing events, raising the question of whether capping components remain associated with the transcription complex after they have modified the 5' end of the mRNA. Chromatin immunoprecipitation in Saccharomyces cerevisiae shows that capping enzyme cross-links to promoters but not coding regions. In contrast, the mRNA cap methyltransferase and the Hrp1/CFIB polyadenylation factor cross-link to both promoter and coding regions. Remarkably, the phosphorylation pattern of the CTD changes during transcription. Ser 5 phosphorylation is detected primarily at promoter regions dependent on TFIIH. In contrast, Ser 2 phosphorylation is seen only in coding regions. These results suggest a dynamic association of mRNA processing factors with differently modified forms of the polymerase throughout the transcription cycle.

Models, Genetic↗
PubMed2000

Bromodomain factor 1 corresponds to a missing piece of yeast TFIID.

The basal transcription factor TFIID consists of the TATA-binding protein (TBP) and TBP-associated factors (TAFs). Yeast Taf67 is homologous to mammalian TAF(II)55. Using a yeast two-hybrid screen to identify proteins that interact with Taf67, we isolated Bromodomain factor 1 (Bdf1) and its homolog (Bdf2). The Bdf proteins are genetically redundant, as cells are inviable without at least one of the two BDF genes. Both proteins contain two bromodomains, a motif found in several proteins involved in transcription and chromatin modification. The BDF genes interact genetically with TAF67. Furthermore, Bdf1 associates with TFIID and is recruited to a TATA-containing promoter. Deletion of Bdf1 or the Taf67 Bdf-interacting domain leads to defects in gene expression. Interestingly, the higher eukaryotic TAF(II)250 has an acetyltransferase activity, two bromodomains, and an associated kinase activity. Its yeast homolog, Taf145, has acetyltransferase activity but lacks the bromodomains and kinase. Bdf1, like TAF(II)250, has a kinase activity that maps carboxy-terminal to the bromodomains. The structural and functional similarities suggest that Bdf1 corresponds to the carboxy-terminal region of higher eukaryotic TAF(II)250 and that the interaction between TFIID and Bdf1 is important for proper gene expression.

Fungal Proteins↗
PubMed2000

Snapshots of RNA polymerase II transcription initiation.

Several papers published within the last year utilize innovative techniques for characterizing intermediates in RNA polymerase II transcription. Structural studies of polymerase and its associated factors provide a detailed picture of the transcription machinery, and studies of transcription complex assembly both in vitro and in vivo provide insights into the mechanism of gene expression. A high resolution picture of the transcription complex is likely to be available within the foreseeable future. The challenge is to determine the roles of individual proteins within this surprisingly large molecular machine.

Humans↗
PubMed2000

Kin28, the TFIIH-associated carboxy-terminal domain kinase, facilitates the recruitment of mRNA processing machinery to RNA polymerase II.

The cotranscriptional placement of the 7-methylguanosine cap on pre-mRNA is mediated by recruitment of capping enzyme to the phosphorylated carboxy-terminal domain (CTD) of RNA polymerase II. Immunoblotting suggests that the capping enzyme guanylyltransferase (Ceg1) is stabilized in vivo by its interaction with the CTD and that serine 5, the major site of phosphorylation within the CTD heptamer consensus YSPTSPS, is particularly important. We sought to identify the CTD kinase responsible for capping enzyme targeting. The candidate kinases Kin28-Ccl1, CTDK1, and Srb10-Srb11 can each phosphorylate a glutathione S-transferase-CTD fusion protein such that capping enzyme can bind in vitro. However, kin28 mutant alleles cause reduced Ceg1 levels in vivo and exhibit genetic interactions with a mutant ceg1 allele, while srb10 or ctk1 deletions do not. Therefore, only the TFIIH-associated CTD kinase Kin28 appears necessary for proper capping enzyme targeting in vivo. Interestingly, levels of the polyadenylation factor Pta1 are also reduced in kin28 mutants, while several other polyadenylation factors remain stable. Pta1 in yeast extracts binds specifically to the phosphorylated CTD, suggesting that this interaction may mediate coupling of polyadenylation and transcription.

Cyclin-Dependent Kinases↗
PubMed2000

The essential interaction between yeast mRNA capping enzyme subunits is not required for triphosphatase function in vivo.

The Saccharomyces cerevisiae mRNA capping enzyme consists of two subunits: an RNA 5'-triphosphatase (Cet1) and an mRNA guanylyltransferase (Ceg1). In yeast, the capping enzyme is recruited to the RNA polymerase II (Pol II) transcription complex via an interaction between Ceg1 and the phosphorylated carboxy-terminal domain of the Pol II largest subunit. Previous in vitro experiments showed that the Cet1 carboxy-terminal region (amino acids 265 to 549) carries RNA triphosphatase activity, while the region containing amino acids 205 to 265 of Cet1 has two functions: it mediates dimerization with Ceg1, but it also allosterically activates Ceg1 guanylyltransferase activity in the context of Pol II binding. Here we characterize several Cet1 mutants in vivo. Mutations or deletions of Cet1 that disrupt interaction with Ceg1 are lethal, showing that this interaction is essential for proper capping enzyme function in vivo. Remarkably, the interaction region of Ceg1 becomes completely dispensable when Ceg1 is substituted by the mouse guanylyltransferase, which does not require allosteric activation by Cet1. Although no interaction between Cet1 and mouse guanylyltransferase is detectable, both proteins are present at yeast promoters in vivo. These results strongly suggest that the primary physiological role of the Ceg1-Cet1 interaction is to allosterically activate Ceg1, rather than to recruit Cet1 to the Pol II complex.

Acid Anhydride Hydrolases↗
PubMed1999

The importin/karyopherin Kap114 mediates the nuclear import of TATA-binding protein.

Two high copy suppressors of temperature-sensitive TATA-binding protein (TBP) mutants were isolated. One suppressor was TIF51A, which encodes eukaryotic translation initiation factor 5A. The other high copy suppressor, YGL241W, also known as KAP114, is one of 14 importin/karyopherin proteins in yeast. These proteins mediate the transport of specific macromolecules into and out of the nucleus. Cells lacking Kap114 partially mislocalize TBP to the cytoplasm. Kap114 binds TBP in vitro, and binding is disrupted in the presence of GTPgammaS. Therefore, Kap114 is an importer of TBP into the nucleus, but alternative import pathways must also exist.

Amino Acid Sequence↗
PubMed1999

TFIID-specific yeast TAF40 is essential for the majority of RNA polymerase II-mediated transcription in vivo.

Many questions remain concerning the role of TFIID TBP-associated factors (TAFs) in transcription, including whether TAFs are required at most or only a small subset of promoters. It was shown previously that three histone-like TAFs are broadly required for transcription, but interpretation of this observation is complicated because these proteins are components of both TFIID and the SAGA histone acetyltransferase complex. Here we show that mutations in the yeast TFIID-specific protein Taf40 lead to a general cessation of transcription, even in the presence of excess TBP, suggesting that the TFIID complex is required at most promoters in vivo.

DNA-Binding Proteins↗
PubMed1999

Evidence that transcription factor IIB is required for a post-assembly step in transcription initiation.

Mutation of glutamate 62 to lysine in yeast transcription factor (TF) IIB (Sua7) causes a cold-sensitive phenotype. This mutant also leads to preferential transcription of downstream start sites on some promoters in vivo. To explore the molecular nature of these phenotypes, the TFIIB E62K mutant was characterized in vitro. The mutant interacts with TATA-binding protein normally. In three different assays, the mutant can also interact with RNA polymerase II and recruit it and the other basal transcription factors to a promoter. Despite the ability to assemble a transcription complex, the TFIIB E62K protein is severely defective in transcription in vitro. Therefore, the role of TFIIB must be more than simply bridging TATA-binding protein and polymerase at the promoter. We propose that the region around Glu-62 in yeast TFIIB plays a role in start site selection, perhaps mediating a conformational change in the polymerase or the DNA during the search for initiation sites. This step may be related to the yeast-specific spacing between TATA elements and start sites since mutations of the corresponding glutamate in mammalian TFIIB do not produce a similar effect.

Mutation↗
PubMed1999

Human PIR1 of the protein-tyrosine phosphatase superfamily has RNA 5'-triphosphatase and diphosphatase activities.

A human cDNA was isolated encoding a protein with significant sequence similarity (41% identity) to the BVP RNA 5'-phosphatase from the Autographa californica nuclear polyhedrosis virus. This protein is a member of the protein-tyrosine phosphatase (PTP) superfamily and is identical to PIR1, shown by Yuan et al. (Yuan, Y., Da-Ming, L., and Sun, H. (1998) J. Biol. Chem. 272, 20347-20353) to be a nuclear protein that can associate with RNA or ribonucleoprotein complexes. We demonstrate that PIR1 removes two phosphates from the 5'-triphosphate end of RNA, but not from mononucleotide triphosphates. The specific activity of PIR1 with RNA is several orders of magnitude greater than that with the best protein substrates examined, suggesting that RNA is its physiological substrate. A 120-amino acid segment C-terminal to the PTP domain is not required for RNA phosphatase activity. We propose that PIR1 and its closest homologs, which include the metazoan mRNA capping enzymes, constitute a subgroup of the PTP family that use RNA as a substrate.

Acid Anhydride Hydrolases↗
PubMed1999

A Saccharomyces cerevisiae RNA 5'-triphosphatase related to mRNA capping enzyme.

The Saccharomyces cerevisiae mRNA capping enzyme consists of two subunits: the RNA 5'-triphosphatase (Cet1) and the mRNA guanylyltransferase (Ceg1). Using computer homology searching, a S. cerevisiae gene was identified that encodes a protein resembling the C-terminal region of Cet1. Accordingly, we designated this gene CTL1 (capping enzyme RNAtriphosphatase-like 1). CTL1 is not essential for cell viability and no genetic or physical interactions with the capping enzyme genes were observed. The protein is found in both the nucleus and cytoplasm. Recombinant Ctl1 protein releases gamma-phosphate from the 5'-end of RNA to produce a diphosphate terminus. The enzyme is specific for polynucleotide RNA in the presence of magnesium, but becomes specific for nucleotide triphosphates in the presence of manganese. Ctl1 is the second member of the yeast RNA triphosphatase family, but is probably involved in an RNA processing event other than mRNA capping.

Acid Anhydride Hydrolases↗
PubMed1998

Allosteric interactions between capping enzyme subunits and the RNA polymerase II carboxy-terminal domain.

mRNA capping is a cotranscriptional event mediated by the association of capping enzyme with the phosphorylated carboxy-terminal domain (CTD) of RNA polymerase II. In the yeast Saccharomyces cerevisiae, capping enzyme is composed of two subunits, the mRNA 5'-triphosphatase (Cet1) and the mRNA guanylyltransferase (Ceg1). Here we map interactions between Ceg1, Cet1, and the CTD. Although the guanylyltransferase subunit can bind alone to the CTD, it cannot be guanylylated unless the triphosphatase subunit is also present. Therefore, the yeast mRNA guanylyltransferase is regulated by allosteric interactions with both the triphosphatase and CTD.

Acid Anhydride Hydrolases↗
PubMed1998

A protein tyrosine phosphatase-like protein from baculovirus has RNA 5'-triphosphatase and diphosphatase activities.

The superfamily of protein tyrosine phosphatases (PTPs) includes at least one enzyme with an RNA substrate. We recently showed that the RNA triphosphatase domain of the Caenorhabditis elegans mRNA capping enzyme is related to the PTP enzyme family by sequence similarity and mechanism. The PTP most similar in sequence to the capping enzyme triphosphatase is BVP, a dual-specificity PTP encoded by the Autographa californica nuclear polyhedrosis virus. Although BVP previously has been shown to have modest tyrosine and serine/threonine phosphatase activity, we find that it is much more potent as an RNA 5'-phosphatase. BVP sequentially removes gamma and beta phosphates from the 5' end of triphosphate-terminated RNA, leaving a 5'-monophosphate end. The activity was specific for polynucleotides; nucleotide triphosphates were not hydrolyzed. A mutant protein in which the active site cysteine was replaced with serine was inactive. Three other dual-specificity PTPs (VH1, VHR, and Cdc14) did not exhibit detectable RNA phosphatase activity. Therefore, capping enzyme and BVP are members of a distinct PTP-like subfamily that can remove phosphates from RNA.

Acid Anhydride Hydrolases↗
PubMed1998

DSIF, a novel transcription elongation factor that regulates RNA polymerase II processivity, is composed of human Spt4 and Spt5 homologs.

We report the identification of a transcription elongation factor from HeLa cell nuclear extracts that causes pausing of RNA polymerase II (Pol II) in conjunction with the transcription inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). This factor, termed DRB sensitivity-inducing factor (DSIF), is also required for transcription inhibition by H8. DSIF has been purified and is composed of 160-kD (p160) and 14-kD (p14) subunits. Isolation of a cDNA encoding DSIF p160 shows it to be a homolog of the Saccharomyces cerevisiae transcription factor Spt5. Recombinant Supt4h protein, the human homolog of yeast Spt4, is functionally equivalent to DSIF p14, indicating that DSIF is composed of the human homologs of Spt4 and Spt5. In addition to its negative role in elongation, DSIF is able to stimulate the rate of elongation by RNA Pol II in a reaction containing limiting concentrations of ribonucleoside triphosphates. A role for DSIF in transcription elongation is further supported by the fact that p160 has a region homologous to the bacterial elongation factor NusG. The combination of biochemical studies on DSIF and genetic analysis of Spt4 and Spt5 in yeast, also in this issue, indicates that DSIF associates with RNA Pol II and regulates its processivity in vitro and in vivo.

Amino Acid Sequence↗
PubMed1998

Histone-like TAFs are essential for transcription in vivo.

In yeast, the TBP-associated factors (TAFs) Taf17, Taf60, and Taf61(68) resemble histones H3, H4, and H2B, respectively. To analyze their roles in vivo, conditional alleles were isolated by mutagenizing their histone homology domains. Conditional alleles of TAF17 or TAF60 can be specifically suppressed by overexpression of any of the other histone-like TAFs. This and other genetic evidence supports the model of a histone octamer-like structure within TFIID. Shifting strains carrying the conditional TAF alleles to non-permissive conditions results in degradation of TFIID components and the rapid loss of mRNA production. Therefore, in contrast to previous studies in yeast that found only limited roles for TAFs in transcription, we find that the histone-like TAFs are generally required for in vivo transcription.

Adaptor Proteins, Signal Transducing↗