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S Buratowski

Publications and source records attributed to S Buratowski.

At least 37 records · Page 2Linked to original sources
PubMed1997

mRNA capping enzyme is recruited to the transcription complex by phosphorylation of the RNA polymerase II carboxy-terminal domain.

Capping of mRNA occurs shortly after transcription initiation, preceding other mRNA processing events such as mRNA splicing and polyadenylation. To determine the mechanism of coupling between transcription and capping, we tested for a physical interaction between capping enzyme and the transcription machinery. Capping enzyme is not stably associated with basal transcription factors or the RNA polymerase II (Pol II) holoenzyme. However, capping enzyme can directly and specifically interact with the phosphorylated form of the RNA polymerase carboxy-terminal domain (CTD). This association occurs in the context of the transcription initiation complex and is blocked by the CTD-kinase inhibitor H8. Furthermore, conditional truncation mutants of the Pol II CTD are lethal when combined with a capping enzyme mutant. Our results provide in vitro and in vivo evidence that capping enzyme is recruited to the transcription complex via phosphorylation of the RNA polymerase CTD.

Binding Sites↗
PubMed1997

An RNA 5'-triphosphatase related to the protein tyrosine phosphatases.

mRNA capping requires the sequential action of three enzymatic activities: RNA triphosphatase, guanylyl-transferase, and methyltransferase. Here we characterize a gene (CEL-1) believed to encode the C. elegans capping enzyme. CEL-1 has a C-terminal domain containing motifs found in yeast and vaccinia virus capping enzyme guanylyltransferases. The N-terminal domain of CEL-1 has RNA triphosphatase activity. Surprisingly, this domain does not resemble the vaccinia virus capping enzyme but does have significant sequence similarity to the protein tyrosine phosphatase (PTP) enzyme family. However, CEL-1 has no detectable PTP activity. The mechanism of the RNA triphosphatase is similar to that of PTPs: the active site contains a conserved nucleophilic cysteine required for activity. These results broaden the superfamily of PTP-like phosphatases to include enzymes with RNA substrates.

Acid Anhydride Hydrolases↗
PubMed1997

Common themes in translational and transcriptional regulation.

How transcription and translation initiation complexes are assembled and regulated has been the subject of research for many years. New information on the translation initiation complex has revealed similarities between its organization and regulation with that of the transcription initiation complex. Here, we will summarize these similarities in order to set up a framework for future integration of results from each of these areas.

Binding Sites↗
PubMed1997

Genetic analysis of the large subunit of yeast transcription factor IIE reveals two regions with distinct functions.

Biochemical analysis of proteins necessary for transcription initiation by eukaryotic RNA polymerase II (pol II) has identified transcription factor IIE (TFIIE) as an essential component of the reaction. To better understand the role of TFIIE in transcription, we isolated conditional alleles of TFA1, the gene encoding the large subunit of TFIIE in the yeast Saccharomyces cerevisiae. The mutant Tfa1 proteins fall into two classes. The first class causes thermosensitive growth due to single amino acid substitutions of the cysteines comprising the Zn-binding motif. The second mutant class is made up of proteins that are C-terminally truncated and that cause a cold-sensitive growth phenotype. The behavior of these mutants suggests that Tfa1p possesses at least two domains with genetically distinct functions. The mutations in the Zn-binding motif do not affect the mutant protein's stability at the nonpermissive temperature or its ability to associate with the small subunit of TFIIE. Our studies further determined that wild-type TFIIE can bind to single-stranded DNA in vitro. However, this property is unaffected in the mutant TFIIE complexes. Finally, we have demonstrated the biological importance of TFIIE in pol II-mediated transcription by depleting the Tfa1 protein from the cells and observing a concomitant decrease in total poly(A)+ mRNA.

Binding Sites↗
PubMed1996

Yeast homologues of higher eukaryotic TFIID subunits.

In eukaryotic cells the TATA-binding protein (TBP) associates with other proteins known as TBP-associated factors (TAFs) to form multisubunit transcription factors important for gene expression by all three nuclear RNA polymerases. Computer searching of the complete Saccharomyces cerevisiae genome revealed five previously unidentified yeast genes with significant sequence similarity to known human and Drosophila RNA polymerase II TAFs. Each of these genes is essential for viability. A sixth essential gene (FUN81) has previously been noted to be similar to human TAFII18. Coimmunoprecipitation experiments show that all six proteins are associated with TBP, demonstrating that they are true TAFs. Furthermore, these proteins are present in complexes containing the TAFII130 subunit, indicating that they are components of TFIID. Based on their predicted molecular weights, these genes have been designated TAF67, TAF61(68), TAF40, TAF23(25), TAF19(FUN81), and TAF17. Yeast TAF61 is significantly larger than its higher eukaryotic homologues, and deletion analysis demonstrates that the evolutionarily conserved, histone-like domain is sufficient and necessary to support viability.

Amino Acid Sequence↗
PubMed1996

Conditional mutants of the yeast mRNA capping enzyme show that the cap enhances, but is not required for, mRNA splicing.

The 5' end of eukaryotic mRNAs are modified by the addition of a 7-methyl guanosine (m7G) cap. The role of the cap in translation has been well established. Additionally, studies conducted in vitro or in microinjected Xenopus oocytes have implicated the cap in RNA processing and transport. To determine the fate of uncapped mRNA in intact yeast cells, conditional alleles of the gene encoding the capping enzyme guanylyltransferase subunit (CEG1) were generated. RNA analysis of temperature-sensitive ceg1 strains revealed an accumulation of unspliced pre-mRNAs and a corresponding decrease in spliced mRNAs at the restrictive temperature. A substantial proportion of spliced mRNA was also uncapped. Therefore, the cap appears to stimulate, but is not absolutely required for, splicing in vivo. In addition, steady-state levels of several mRNAs were decreased, perhaps due to increased degradation of uncapped mRNAs. In contrast to splicing, mRNA polyadenylation and transport to the cytoplasm were unaffected.

Alleles↗
PubMed1995

An interaction between the Tfb1 and Ssl1 subunits of yeast TFIIH correlates with DNA repair activity.

Yeast transcription factor TFIIH (also known as factor b) is a component of the RNA polymerase II initiation complex. Several TFIIH subunits (RAD3, SSL2 and SSL1) have also been implicated in DNA repair. Ssl1 interacts directly with another TFIIH subunit, Tfb1, which has not previously been shown to have a role in DNA repair. We isolated mutations in TFB1 that lead to a temperature sensitive phenotype. These mutations result in C-terminal truncations of the Tfb1 protein and disrupt its interaction with Ssl1. The C-terminal 114 amino acids of Tfb1 are necessary and sufficient for this interaction. Interestingly, cells carrying these truncations in Tfb1 cause sensitivity to ultraviolet (UV) light induced DNA damage, as previously observed for mutations in RAD3, SSL1 and SSL2. Many other mutations in RNA polymerase II basal factors were tested and found not to cause an increase in UV sensitivity, indicating that this phenotype is not due to a general defect in transcription.

DNA Repair↗
PubMed1995

The yeast TFB1 and SSL1 genes, which encode subunits of transcription factor IIH, are required for nucleotide excision repair and RNA polymerase II transcription.

The essential TFB1 and SSL1 genes of the yeast Saccharomyces cerevisiae encode two subunits of the RNA polymerase II transcription factor TFIIH (factor b). Here we show that extracts of temperature-sensitive mutants carrying mutations in both genes (tfb1-101 and ssl1-1) are defective in nucleotide excision repair (NER) and RNA polymerase II transcription but are proficient for base excision repair. RNA polymerase II-dependent transcription at the CYC1 promoter was normal at permissive temperatures but defective in extracts preincubated at a restrictive temperature. In contrast, defective NER was observed at temperatures that are permissive for growth. Additionally, both mutants manifested increased sensitivity to UV radiation at permissive temperatures. The extent of this sensitivity was not increased in a tfb1-101 strain and was only slightly increased in a ssl1-1 strain at temperatures that are semipermissive for growth. Purified factor TFIIH complemented defective NER in both tfb1-101 and ssl1-1 mutant extracts. These results define TFB1 and SSL1 as bona fide NER genes and indicate that, as is the case with the yeast Rad3 and Ss12 (Rad25) proteins, Tfb1 and Ssl1 are required for both RNA polymerase II basal transcription and NER. Our results also suggest that the repair and transcription functions of Tfb1 and Ssl1 are separable.

Base Sequence↗
PubMed1994

Active site of the mRNA-capping enzyme guanylyltransferase from Saccharomyces cerevisiae: similarity to the nucleotidyl attachment motif of DNA and RNA ligases.

Nascent mRNA chains are capped at the 5' end by the addition of a guanylyl residue to form a G(5')ppp(5')N ... structure. During the capping reaction, the guanylyltransferase (GTP:mRNA guanylyltransferase, EC 2.7.7.50) is reversibly and covalently guanylylated. In this enzyme-GMP (E-GMP) intermediate, GMP is linked to the epsilon-amino group of a lysine residue via a phosphoamide bond. Lys-70 was identified as the GMP attachment site of the Saccharomyces cerevisiae guanylyltransferase (encoded by the CEG1 gene) by guanylylpeptide sequencing. CEG1 genes with substitutions at Lys-70 were unable to support viability in yeast and produced proteins that were not guanylylated in vitro. The CEG1 active site exhibits sequence similarity to the active sites of viral guanylyltransferases and polynucleotide ligases, suggesting similarity in the mechanisms of nucleotidyl transfer catalyzed by these enzymes.

Amino Acid Sequence↗
PubMed1994

RNA polymerase III transcription in the yeast Saccharomyces cerevisiae.

The yeast RNA polymerase III system is probably the best-characterized eukaryotic transcription system. Nearly all of the components have been identified and the genes for them cloned. Many of the interactions within initiation complexes are coming to light. Considering the many parallels between Pol III transcription and the other polymerase systems, findings in the Pol III system can act as predictions for Pol II and Pol I transcription. Despite the many advances made in the study of transcription by RNA polymerase III, many important questions remain to be answered. It is unclear what are the functions of individual TFIIIC, TFIIIB and polymerase subunits. Why are so many proteins required? Another extremely important mystery is the mechanism by which the factors assemble. What is the molecular mechanism for TFIIIC recruiting TFIIIB, and how does TFIIIB recruit polymerase? These and many other problems will eventually be solved as researchers apply the biochemical and genetic techniques available in the yeast system.

RNA Polymerase III↗
PubMed1993

Dual roles of a multiprotein complex from S. cerevisiae in transcription and DNA repair.

Yeast RNA polymerase II initiation factor b, homolog of human TFIIH, is a protein kinase capable of phosphorylating the C-terminal repeat domain of the polymerase; it possesses a DNA-dependent ATPase activity as well. The 85 kd and 50 kd subunits of factor b are now identified as RAD3 and SSL1 proteins, respectively; both are known to be involved in DNA repair. Factor b interacts specifically with another DNA repair protein, SSL2. The ATPase activity of factor b may be due entirely to that associated with a helicase function of RAD3. Factor b transcriptional activity was unaffected, however, by amino acid substitution at a conserved residue in the RAD3 nucleotide-binding domain, suggesting that the ATPase/helicase function is not required for transcription. These results identify factor b as a core repairosome, which may be responsible for the preferential repair of actively transcribed genes in eukaryotes.

Adenosine Triphosphatases↗
PubMed1993

Functional domains of transcription factor TFIIB.

Transcription factor TFIIB is an essential component of the RNA polymerase II initiation complex. TFIIB carries out at least two functions: it interacts directly with the TATA-binding protein (TBP) and helps to recruit RNA polymerase II into the initiation complex. The sequence of TFIIB reveals a potential zinc-binding domain and an imperfect duplication of approximately 70 amino acids. Mutagenesis of cysteine codons within the putative zinc finger results in mutant proteins that bind normally to TBP but are unable to recruit RNA polymerase II-TFIIF into the initiation complex. Changing the two most highly conserved amino acids in the TFIIB repeats reduces the ability of TFIIB to interact with TBP. Therefore, the two functions of TFIIB can be assigned to two separable functional domains of the protein.

Amino Acid Sequence↗
PubMed1992

A suppressor of TBP mutations encodes an RNA polymerase III transcription factor with homology to TFIIB.

The TDS4 gene of S. cerevisiae was isolated as an allele-specific high copy suppressor of mutations within the basic region of the TATA-binding protein (TBP). The gene is essential for viability and encodes a 596 aa protein. The first 300 aa of the TDS4 protein exhibit significant sequence similarity to the RNA polymerase II transcription factor TFIIB. However, TDS4 is required for RNA polymerase III transcription in vivo and in vitro. Antibodies specific for TDS4 or TBP react with the TFIIIB complex, indicating that both proteins are components of the RNA polymerase III initiation complex. These findings suggest that the RNA polymerase II and III initiation mechanisms are extremely similar, and they explain how the TATA-binding protein can function in both systems.

Amino Acid Sequence↗
PubMed1992

A novel transcription factor reveals a functional link between the RNA polymerase II CTD and TFIID.

The RNA polymerase II large subunit carboxy-terminal domain (CTD) plays a role in transcription initiation, but its mechanism of action is not well understood. We have investigated the function of the SRB2 gene, which was isolated as a dominant suppressor of CTD truncation mutations. The allele specificity of this suppressor indicates that SRB2 and the CTD are involved in the same function. Indeed, cells lacking SRB2 and cells lacking a large portion of the CTD exhibit the same set of conditional growth phenotypes and exhibit very similar defects in gene expression in vivo. The SRB2 protein is a novel transcription factor that has an important role in basal and activated transcription in vitro and is essential for efficient establishment of the transcription initiation apparatus. Template commitment experiments suggest that SRB2 becomes physically associated with the transcription initiation complex. We find that SRB2 binds specifically to TFIID. As SRB2 and the RNA polymerase II CTD are involved in the same function, these results reveal a functional link between the CTD and the TATA-binding factor. This study implicates the CTD in recruitment of RNA polymerase II to the transcription initiation complex.

Amino Acid Sequence↗