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Rubenstein, E.M., R.R. McCartney, C. Zhang, K.M. Shokat, M.K. Shirra, K.M. Arndt, and M.C. Schmidt (2008) Access denied: Snf1 activation loop phosphorylation is controlled by availability of the phosphorylated threonine 210 to the PP1 phosphatase. J. Biol. Chem. 283:222-230 Phosphorylation of the Saccharomyces cerevisiae Snf1 kinase activation loop is determined by the integration of two reaction rates: the rate of phosphorylation by upstream kinases and the rate of dephosphorylation by Glc7. The activities of the Snf1-activating kinases do not appear to be glucose regulated since immune-complex kinases assays with each of the three Snf1-activating kinases show similar levels of activity when prepared from cells grown in either high or low glucose. In contrast, the dephosphorylation of the Snf1 activation loop was strongly regulated by glucose. When de novo phosphorylation of Snf1 was inhibited, phosphorylation of the Snf1 activation loop was found to be stable in low glucose but rapidly lost upon addition of glucose. A greater than 10-fold difference in the rates of Snf1 activation loop dephosphorylation was detected. However, the activity of the Glc7/Reg1 phosphatase may not itself be directly regulated by glucose since the Glc7/Reg1 enzyme was active in low glucose toward another substrate, the transcription factor Mig1. Glucose-mediated regulation of Snf1 activation loop dephosphorylation is controlled by changes in the ability of the Snf1 activation loop to act as a substrate for Glc7. Arndt, K.M. (2007) Molecular biology: genome under surveillance. Nature 450:959-960 Chu, Y., R. Simic, M.H. Warner, K.M. Arndt, and G. Prelich (2007) Regulation of histone modification and cryptic transcription by the Bur1 and Paf1 complexes. EMBO J. 26:4646-4656 The Bur1-Bur2 and Paf1 complexes function during transcription elongation and affect histone modifications. Here we describe new roles for Bur1-Bur2 and the Paf1 complex. We find that histone H3 K36 tri-methylation requires specific components of the Paf1 complex and that K36 tri-methylation is more strongly affected at the 5' ends of genes in paf1delta and bur2delta strains in parallel with increased acetylation of histones H3 and H4. Interestingly, the 5' increase in histone acetylation is independent of K36 methylation, and therefore is mechanistically distinct from the methylation-driven deacetylation that occurs at the 3' ends of genes. Finally, Bur1-Bur2 and the Paf1 complex have a second methylation-independent function, since bur2delta set2delta and paf1delta set2delta double mutants display enhanced histone acetylation at the 3' ends of genes and increased cryptic transcription initiation. These findings identify new functions for the Paf1 and Bur1-Bur2 complexes, provide evidence that histone modifications at the 5' and 3' ends of coding regions are regulated by distinct mechanisms, and reveal that the Bur1-Bur2 and Paf1 complexes repress cryptic transcription through a Set2-independent pathway. Warner, M.H., K.L. Roinick, and K.M. Arndt (2007) Rtf1 is a multifunctional component of the Paf1 complex that regulates gene expression by directing cotranscriptional histone modification. Mol. Cell Biol. 27:6103-6115 Numerous transcription accessory proteins cause alterations in chromatin structure that promote the progression of RNA polymerase II (Pol II) along open reading frames (ORFs). The Saccharomyces cerevisiae Paf1 complex colocalizes with actively transcribing Pol II and orchestrates modifications to the chromatin template during transcription elongation. To better understand the function of the Rtf1 subunit of the Paf1 complex, we created a series of sequential deletions along the length of the protein. Genetic and biochemical assays were performed on these mutants to identify residues required for the various activities of Rtf1. Our results establish that discrete nonoverlapping segments of Rtf1 are necessary for interaction with the ATP-dependent chromatin-remodeling protein Chd1, promoting covalent modification of histones H2B and H3, recruitment to active ORFs, and association with other Paf1 complex subunits. We observed transcription-related defects when regions of Rtf1 that mediate histone modification or association with active genes were deleted, but disruption of the physical association between Rtf1 and other Paf1 complex subunits caused only subtle mutant phenotypes. Together, our results indicate that Rtf1 influences transcription and chromatin structure through several independent functional domains and that Rtf1 may function independently of its association with other members of the Paf1 complex.
Braun, M.A., P.J. Costa, E.M. Crisucci, and K.M. Arndt (2007) Identification of Rkr1, a nuclear RING domain protein with functional connections to chromatin modification in Saccharomyces cerevisiae. Mol. Cell. Biol. 27:2800-2811 Proper transcription by RNA polymerase II is dependent on the modification state of the chromatin template. The Paf1 complex is associated with RNA polymerase II during transcription elongation and is required for several histone modifications that mark active genes. To uncover additional factors that regulate chromatin or transcription, we performed a genetic screen for mutations that cause lethality in the absence of the Paf1 complex component Rtf1. Our results have led to the discovery of a previously unstudied gene, RKR1. Strains lacking RKR1 exhibit phenotypes associated with defects in transcription and chromatin function. These phenotypes include inositol auxotrophy, impaired telomeric silencing, and synthetic lethality with mutations in SPT10, a gene that encodes a putative histone acetyltransferase. In addition, deletion of RKR1 causes severe genetic interactions with mutations that prevent histone H2B lysine 123 ubiquitylation or histone H3 lysine 4 methylation. RKR1 encodes a conserved, nuclear protein with a functionally important RING domain at its carboxy-terminus. In vitro experiments indicate that Rkr1 possesses ubiquitin-protein ligase activity. Taken together, our results identify a new participant in a protein ubiquitylation pathway within the nucleus that acts to modulate chromatin function and transcription.
Sheldon, K.E., D.M. Mauger, and K.M. Arndt (2005) A requirement for the Saccharomyces cerevisiae Paf1 Complex in snoRNA 3' end formation. Mol. Cell 20:225-236 RNA synthesis and processing are coordinated by proteins that associate with RNA polymerase II (pol II) during transcription elongation. The yeast Paf1 complex interacts with RNA pol II and mediates histone modifications during elongation. To elucidate the functions of this complex, we isolated missense mutations in the gene encoding the Rtf1 subunit and used them to identify functionally interacting proteins. We identified NAB3 as a dosage suppressor of rtf1. Nab3, together with Nrd1, directs 3' end formation of nonpolyadenylated RNA pol II transcripts, such as snoRNAs. Deletion of Paf1, but not the Set1, Set2, or Dot1 histone methyltransferases, causes accumulation of snoRNA transcripts that are extended at their 3' ends. The Paf1 complex associates with and facilitates Nrd1 recruitment to the SNR47 gene, suggesting a direct involvement in 3' end formation. Our results reveal a posttranscriptional function for the Paf1 complex, which appears unrelated to its role in histone methylation.
Arndt, K., and F. Winston (2005) An unexpected role for ubiquitylation of a transcriptional activator. Cell 120:733-734 The yeast transcriptional activator Gal4 has served as a paradigm for understanding how eukaryotic cells mount rapid transcriptional responses to environmental changes. In this issue of Cell, Muratani et al. (2005) provide evidence that Gal4 ubiquitylation and destruction are required for activation by Gal4. Surprisingly, this modification is required at a postinitiation step in transcription for the production of mRNAs that are correctly processed and fully functional for translation.
Shirra, M.K., S.E. Rogers, D.E. Alexander, and K.M. Arndt (2005) The Snf1 protein kinase and Sit4 protein phosphatase have opposing functions in regulating TBP association with the Saccharomyces cerevisiae INO1 promoter. Genetics 169:1957-1972 To identify the mechanisms by which multiple signaling pathways coordinately affect gene expression, we are investigating regulation of the S. cerevisiae INO1 gene. Full activation of INO1 transcription occurs in the absence of inositol and requires the Snf1 protein kinase in addition to other signaling molecules and transcription factors. Here, we present evidence that the Sit4 protein phosphatase negatively regulates INO1 transcription. A mutation in SIT4 was uncovered as a suppressor of the inositol auxotrophy of snf1 strains. We found that sit4 mutant strains exhibit an Spt- phenotype, suggesting a more general role for Sit4 in transcription. In fact, like the gene-specific regulators of INO1 transcription, Opi1, Ino2, and Ino4, both Snf1 and Sit4 regulate binding of TBP to the INO1 promoter, as determined by chromatin immunoprecipitation analysis. Experiments involving double mutant strains indicate that the negative effect of Sit4 on INO1 transcription is unlikely to occur through dephosphorylation of histone H3 or Opi1. Sit4 is a known component of the target of rapamycin (TOR) signaling pathway, and treatment of cells with rapamycin reduces INO1 activation. However, analysis of rapamycin-treated cells suggests that Sit4 represses INO1 transcription through multiple mechanisms, only one of which may involve inhibition of TOR signaling.
Arndt, K.M., and C.M. Kane (2003) Running with RNA polymerase: eukaryotic transcript elongation. Trends Genet. 19:543-550 Long recognized as a target of regulation in prokaryotes, transcript elongation has recently become the focus of many investigators interested in eukaryotic gene expression. The growth of this area has been fueled by the availability of new methods and molecular structures, expanding sequence databases and an appreciation for the exquisite coordination required among different processes in the nucleus. Our article collates new information on regulatory accessory factors, as well as their ultimate target, RNA polymerase, in the nucleus of eukaryotic cells. How this regulation influences the biology of the organism is quite profound, and from single cell to multicellular eukaryotes significant similarities exist in the molecular responses to extracellular signals during transcript elongation. The most advanced genetic knowledge in this area comes from Saccharomyces cerevisiae, but the biochemistry and cell biology results from other organisms are also highlighted.
Simic, R., D.L. Lindstron, H.G. Tran, K.L. Roinick, P.J. Costa, A.D. Johnson, G.A. Hartzog, and K.M. Arndt (2003) Chromatin remodeling protein Chd1 interacts with transcription elongation factors and localizes to transcribed genes. EMBO J. 22:1846-4856 Transcription in eukaryotes is influenced by the chromatin state of the template, and chromatin remodeling factors have well-documented roles in regulating transcription initiation by RNA polymerase (pol) II. Chromatin also influences transcription elongation; however, little is known about the role of chromatin remodeling factors in this process. Here, we present evidence that the Saccharomyces cerevisiae chromatin remodeling factor Chd1 functions during transcription elongation. First, we identified Chd1 in a two-hybrid screen for proteins that interact with Rtf1, a member of the Paf1 complex that associates with RNA pol II and regulates transcription elongation. Secondly, we show through co-immunoprecipitation studies that Chd1 also interacts with components of two essential elongation factors, Spt4-Spt5 and Spt16-Pob3. Thirdly, we demonstrate that deletion of CHD1 suppresses a cold-sensitive spt5 mutation that is also suppressed by defects in the Paf1 complex and RNA pol II. Finally, we demonstrate that Chd1, Rtf1 and Spt5 associate with actively transcribed regions of chromatin. Collectively, these findings suggest an important role for Chd1 and chromatin remodeling in the control of transcription elongation.
Spencer, J.V., and K.M. Arndt (2002) A TATA binding protein mutant with increased affinity for DNA directs transcription from a reversed TATA sequence in vivo. Mol. Cell. Biol. 22:8744-8755 The TATA-binding protein (TBP) nucleates the assembly and determines the position of the preinitiation complex at RNA polymerase II-transcribed genes. We investigated the importance of two conserved residues on the DNA binding surface of Saccharomyces cerevisiae TBP to DNA binding and sequence discrimination. Because they define a significant break in the twofold symmetry of the TBP-TATA interface, Ala100 and Pro191 have been proposed to be key determinants of TBP binding orientation and transcription directionality. In contrast to previous predictions, we found that substitution of an alanine for Pro191 did not allow recognition of a reversed TATA box in vivo; however, the reciprocal change, Ala100 to proline, resulted in efficient utilization of this and other variant TATA sequences. In vitro assays demonstrated that TBP mutants with the A100P and P191A substitutions have increased and decreased affinity for DNA, respectively. The TATA binding defect of TBP with the P191A mutation could be intragenically suppressed by the A100P substitution. Our results suggest that Ala100 and Pro191 are important for DNA binding and sequence recognition by TBP, that the naturally occurring asymmetry of Ala100 and Pro191 is not essential for function, and that a single amino acid change in TBP can lead to elevated DNA binding affinity and recognition of a reversed TATA sequence.
Squazzo, S.L., P.J. Costa, D. Lindstrom, K.E. Kumer, R. Simic, J.L. Jennings, A.K. Link, K.M. Arndt, and G. Hartzog (2002) The Paf1 complex physically and functionally associates with transcription elongation factors in vivo. EMBO J. 21:1764-1774 We are using biochemical and genetic approaches to study Rtf1 and the Spt4-Spt5 complex, which independently have been implicated in transcription elongation by RNA polymerase II. Here, we report a remarkable convergence of these studies. First, we purified Rtf1 and its associated yeast proteins. Combining this approach with genetic analysis, we show that Rtf1 and Leo1, a protein of unknown function, are members of the RNA polymerase II-associated Paf1 complex. Further analysis revealed allele-specific genetic interactions between Paf1 complex members, Spt4-Spt5, and Spt16-Pob3, the yeast counterpart of the human elongation factor FACT. In addition, we independently isolated paf1 and leo1 mutations in an unbiased genetic screen for suppressors of a cold-sensitive spt5 mutation. These genetic interactions are supported by physical interactions between the Paf1 complex, Spt4-Spt5 and Spt16-Pob3. Finally, we found that defects in the Paf1 complex cause sensitivity to 6-azauracil and diminished PUR5 induction, properties frequently associated with impaired transcription elongation. Taken together, these data suggest that the Paf1 complex functions during the elongation phase of transcription in conjunction with Spt4-Spt5 and Spt16-Pob3.
Shirra, M.K., J. Patton-Vogt, A. Ulrich, O. Liuta-Tehlivets, S.D. Kohlwein, S.A. Henry, and K.M. Arndt (2001) Inhibition of acetyl coenzyme A carboxylase activity restores expression of the Ino1 gene in a snf1 mutant strain of Saccharomyces cerevisiae. Mol Cell Biol 21:5710-5722 Mutations in the Saccharomyces cerevisiae SNF1 gene affect a number of cellular processes, including the expression of genes involved in carbon source utilization and phospholipid biosynthesis. To identify targets of the Snf1 kinase that modulate expression of INO1, a gene required for an early, rate-limiting step in phospholipid biosynthesis, we performed a genetic selection for suppressors of the inositol auxotrophy of snf1-D strains. We identified mutations in ACC1 and FAS1, two genes important for fatty acid biosynthesis in yeast; ACC1 encodes acetyl coenzyme A carboxylase (Acc1), and FAS1 encodes the beta subunit of fatty acid synthase. Acc1 was shown previously to be phosphorylated and inactivated by Snf1. Here we show that snf1-D strains with increased Acc1 activity exhibit decreased INO1 transcription. Strains carrying the ACC1 suppressor mutation have reduced Acc1 activity in vitro and in vivo, as revealed by enzymatic assays and increased sensitivity to the Acc1-specific inhibitor soraphen A. Moreover, a reduction in Acc1 activity, caused by addition of soraphen A, provision of exogenous fatty acid, or conditional expression of ACC1, suppresses the inositol auxotrophy of snf1Delta strains. Together, these findings indicate that the inositol auxotrophy of snf1-D strains arises in part from elevated Acc1 activity and that a reduction in this activity restores INO1 expression in these strains. These results reveal a Snf1-dependent connection between fatty acid production and phospholipid biosynthesis, identify Acc1 as a Snf1 target important for INO1 transcription, and suggest models in which metabolites that are generated or utilized during fatty acid biosynthesis can significantly influence gene expression in yeast.
Costa, P.J., and K.M. Arndt (2000) Synthetic lethal interactions suggest a role for the Saccharomyces cerevisiae Rtf1 Protein in transcription elongation. Genetics 156:535-547 Strong evidence indicates that transcription elongation by RNA polymerase II (pol II) is a highly regulated process. Here we present genetic results that indicate a role for the Saccharomyces cerevisiae Rtf1 protein in transcription elongation. A screen for synthetic lethal mutations was carried out with an rtf1 deletion mutation to identify factors that interact with Rtf1 or regulate the same process as Rtf1. The screen uncovered mutations in SRB5, CTK1, FCP1, and POB3. These genes encode an Srb/mediator component, a CTD kinase, a CTD phosphatase, and a protein involved in the regulation of transcription by chromatin structure, respectively. All of these gene products have been directly or indirectly implicated in transcription elongation, indicating that Rtf1 may also regulate this process. In support of this view, we show that RTF1 functionally interacts with genes that encode known elongation factors, including SPT4, SPT5, SPT16, and PPR2. We also show that a deletion of RTF1 causes sensitivity to 6-azauracil and mycophenolic acid, phenotypes correlated with a transcription elongation defect. Collectively, our results suggest that Rtf1 may function as a novel transcription elongation factor in yeast.
Liu, Q., S.E. Gabriel, K.L. Roinick, R.D. Ward, and K.M. Arndt (1999) Analysis of TFIIA function in vivo: evidence for a role in TBP recruitment and gene-specific activation. Mol. Cell. Biol. 19:8673-8685 Activation of transcription can occur by the facilitated recruitment of TFIID to promoters by gene-specific activators. To investigate the role of TFIIA in TFIID recruitment in vivo, we exploited a class of yeast TATA-binding protein (TBP) mutants that is activation and DNA binding defective. We found that co-overexpression of TOA1 and TOA2, the genes that encode yeast TFIIA, overcomes the activation defects caused by the TBP mutants. Using a genetic screen, we isolated a new class of TFIIA mutants and identified three regions on TFIIA that are likely to be involved in TBP recruitment or stabilization of the TBP-TATA complex in vivo. Amino acid replacements in only one of these regions enhance TFIIA-TBP-DNA complex formation in vitro, suggesting that the other regions are involved in regulatory interactions. To determine the relative importance of TFIIA in the regulation of different genes, we constructed yeast strains to conditionally deplete TFIIA levels prior to gene activation. While the activation of certain genes, such as INO1, was dramatically impaired by TFIIA depletion, activation of other genes, such as CUP1, was unaffected. These data suggest that TFIIA facilitates DNA binding by TBP in vivo, that TFIIA may be regulated by factors that target distinct regions of the protein, and that promoters vary significantly in the degree to which they require TFIIA for activation.
Shirra, M.K., and K.M. Arndt (1999) Evidence for the involvement of the Glc7-Reg1 phosphatase and the Snf1-Snf4 kinase in the regulation of INO1 transcription in Saccharomyces cerevisiae. Genetics 152:73-87 Binding of the TATA-binding protein (TBP) to the promoter is a pivotal step in RNA polymerase II transcription. To identify factors that regulate TBP, we selected for suppressors of a TBP mutant that exhibits promoter-specific defects in activated transcription in vivo and severely reduced affinity for TATA boxes in vitro. Dominant mutations in SNF4 and recessive mutations in REG1, OPI1, and RTF2 were isolated that specifically suppress the inositol auxotrophy of the TBP mutant strains. OPI1 encodes a repressor of INO1 transcription. REG1 and SNF4 encode regulators of the Glc7 phosphatase and Snf1 kinase, respectively, and have well-studied roles in glucose repression. In two-hybrid assays, one SNF4 mutation enhances the interaction between Snf4 and Snf1. Suppression of the TBP mutant by our reg1 and SNF4 mutations appears unrelated to glucose repression, since these mutations do not alleviate repression of SUC2, and glucose levels have little effect on INO1 transcription. Moreover, mutations in TUP1, SSN6, and GLC7, but not HXK2 and MIG1, can cause suppression. Our data suggest that association of TBP with the TATA box may be regulated, directly or indirectly, by a substrate of Snf1. Analysis of INO1 transcription in various mutant strains suggests that this substrate is distinct from Opi1.
Stolinski, L.A., D.M. Eisenmann, and K.M. Arndt (1997) Identification of RTF1, a novel gene important for TATA site selection by TATA-box binding protein in Saccharomyces cerevisie. Mol. Cell. Biol. 17:4490-4500 Interaction of the TATA box-binding protein (TBP) with promoters of RNA polymerase II-transcribed genes is an early and essential step in mRNA synthesis. Previous studies have demonstrated that the rate-limiting binding of TBP to a TATA element can be influenced by transcriptional regulatory proteins. To identify additional factors that may regulate DNA binding by TBP in vivo, we performed a genetic selection for extragenic suppressors of a yeast TBP mutant that exhibits altered and relaxed DNA binding specificity. This analysis has led to the discovery of a previously unidentified gene, RTF1. The original rtf1 suppressor mutation, which encodes a single amino acid change in Rtf1, and an rtf1 null allele suppress the effects of the TBP specificity mutant by altering transcription initiation. Differences in the patterns of transcription initiation in these strains strongly suggest that the rtf1 missense mutation is distinct from a simple loss-of-function allele. The results of genetic crosses indicate that suppression of TBP mutants by mutations in RTF1 occurs in an allele-specific fashion. In a strain containing wild-type TBP, the rtf1 null mutation suppresses the transcriptional effects of a Ty delta insertion mutation in the promoter of the HIS4 gene, a phenotype also conferred by the TBP altered-specificity mutant. Finally, as shown by indirect immunofluorescence experiments, Rtf1 is a nuclear protein. Taken together, our findings suggest that Rtf1 either directly or indirectly regulates the DNA binding properties of TBP and, consequently, the relative activities of different TATA elements in vivo.
Arndt, K.M., S. Ricupero-Hovasse, and F. Winston (1995) TBP mutants defective in activated transcription in vivo. EMBO J. 14:1490-1497 The TATA box binding protein (TBP) plays a central and essential role in transcription initiation. At TATA box-containing genes transcribed by RNA polymerase II, TBP binds to the promoter and initiates the assembly of a multiprotein preinitiation complex. Several studies have suggested that binding of TBP to the TATA box is an important regulatory step in transcription initiation in vitro. To determine whether TBP is a target of regulatory factors in vivo, we performed a genetic screen in yeast for TBP mutants defective in activated transcription. One class of TBP mutants identified in this screen comprises inositol auxotrophs that are also defective in using galactose as a carbon source. These phenotypes are due to promoter-specific defects in transcription initiation that are governed by the upstream activating sequence (UAS) and apparently not by the sequence of the TATA element. The finding that these TBP mutants are severely impaired in DNA binding in vitro suggests that transcription initiation at certain genes is regulated at the level of TATA box binding by TBP in vivo.
Arndt, K.M., C.R. Wobbe, S. Ricupero-Hovasse, K. Struhl, and F. Winston (1994) Equivalent mutations in the two repeats of yeast TATA-binding protein confer distinct TATA recognition specificities. Mol. Cell. Biol. 14:3719-3728 To investigate the process of TATA box recognition by the TATA box-binding protein (TBP), we have performed a detailed genetic and biochemical analysis of two Saccharomyces cerevisiae TBP mutants with altered DNA-binding specificity. The mutant proteins have amino acid substitutions (Leu-205 to Phe and Leu-114 to Phe) at equivalent positions within the two repeats of TBP that are involved in TATA element binding. In an in vivo assay that employs a nearly complete set of single point mutations of the consensus TATAAA sequence, one of the TBP mutants (TBP-L114F) recognizes the sequence TATAAG, while the other TBP mutant (TBP-L205F) recognizes one substitution at the first position of the TATA element, CATAAA, and three substitutions at the 3' end of the TATA box. Specificity patterns determined from in vitro transcription experiments with purified recombinant wild-type TBP and TBP-L205F agree closely with those observed in vivo, indicating that altered TATA utilization in the mutant strains is a direct consequence of altered TATA recognition by the mutant TBPs. The distinct TATA recognition patterns exhibited by TBP-L114F and TBP-L205F strongly suggest that in vivo, TBP binds to the TATA element in a specific orientation. The orientation predicted from these studies is further supported by the identification of intragenic suppressors that correct the defect of TBP-L205F. This orientation is consistent with that observed in vitro by crystallographic analyses of TBP-TATA box complexes. Finally, the importance of altered DNA-binding specificity in transcriptional regulation at the S. cerevisiae his4-912 delta promoter was addressed for TBP-L205F. A mutational analysis of this promoter region demonstrates that the nonconsensus TATA element CATAAA is required for a transcriptional effect of TBP-L205F in vivo. This finding suggests that the interaction of TBP with nonconsensus TATA elements may play an important regulatory role in transcription initiation.
Arndt, K.M., S.L. Ricupero, D.M. Eisenmann, and F. Winston (1992) Biochemical and genetic characterization of a yeast TFIID mutant that alters transcription in vivo and DNA binding in vitro. Mol. Cell Biol. 12:2372-2382 A mutation in the gene that encodes Saccharomyces cerevisiae TFIID (SPT15), which was isolated in a selection for mutations that alter transcription in vivo, changes a single amino acid in a highly conserved region of the second direct repeat in TFIID. Among eight independent spt15 mutations, seven cause this same amino acid change, Leu-205 to Phe. The mutant TFIID protein (L205F) binds with greater affinity than that of wild-type TFIID to at least two nonconsensus TATA sites in vitro, showing that the mutant protein has altered DNA binding specificity. Site-directed mutations that change Leu-205 to five different amino acids cause five different phenotypes, demonstrating the importance of this amino acid in vivo. Virtually identical phenotypes were observed when the same amino acid changes were made at the analogous position, Leu-114, in the first repeat of TFIID. Analysis of these mutations and additional mutations in the most conserved regions of the repeats, in conjunction with our DNA binding results, suggests that these regions of the repeats play equivalent roles in TFIID function, possibly in TATA box recognition.
Eisenmann, D.M., K.M. Arndt, S.L. Ricupero, J.W. Rooney, and F. Winston (1992) SPT3 interacts with TFIID to allow normal transcription in Saccharomyces cerevisiae. Genes Dev. 6:1319-1331 Mutations in the Saccharomyces cerevisiae gene SPT15, which encodes the TATA-binding protein TFIID, have been shown to cause pleiotropic phenotypes and to lead to changes in transcription in vivo. Here, we report the cloning and analysis of one such mutation, spt15-21, which causes a single-amino-acid substitution in a conserved residue of TFIID. Surprisingly, the spt15-21 mutation does not affect the stability of TFIID, its ability to bind to DNA or to support basal transcription in vitro, or the ability of an upstream activator to function in vivo. To study further the spt15-21 defect, extragenic suppressors of this mutation were isolated and analyzed. All of the extragenic suppressors of spt15-21 are mutations in the previously identified SPT3 gene. Suppression of spt15-21 by these spt3 mutations is allele-specific, suggesting that TFIID and SPT3 interact and that spt15-21 impairs this interaction in some way. Consistent with these genetic data, coimmunoprecipitation experiments demonstrate that the TFIID and SPT3 proteins are physically associated in yeast extracts. Taken together, these results suggest that SPT3 is a TFIID-associated protein, required for TFIID to function at particular promoters in vivo. Arndt, K.M., and M.J. Chamberlain (1990) RNA chain elongation by Escherichia coli RNA polymerase. Factors affecting the stability of elongating ternary complexes. J. Mol. Biol. 213:79-108 We have devised a method to follow the stability of individual ternary transcription complexes containing Escherichia coli RNA polymerase halted at many different sites along a DNA template during the transcription process. Studies of complexes formed with phage T7 DNA templates reveal at least three general classes of ternary complexes that differ dramatically in their properties. Complexes of one sort (normal complexes) are highly stable to dissociation and denaturation under a variety of solution conditions. They remain intact and active for up to 24 hours even in salt concentrations up to 1 M-K+. This suggests that they are stabilized to a significant extent by non-ionic interactions between RNA polymerase and the nucleic acids. We consider these to be the normal complexes formed during RNA chain elongation. Complexes of a second sort (release complexes) dissociate rapidly, releasing free RNA transcripts and active RNA polymerase. The rate of dissociation is substantially enhanced by elevated concentrations of K+, hence the interaction between RNA polymerase and nucleic acids in these complexes is stabilized predominantly by ionic interactions. However, release complexes are stabilized by millimolar concentrations of Mg2+, which as been implicated in stabilization of the binding of RNA to free RNA polymerase. These complexes are formed at DNA sequences that we refer to as release sites. Analysis of DNA sequences at release sites reveals that all share a common feature, the potential to form an RNA hairpin in the region just upstream from the actual 3' end of the released RNA. Experiments incorporating IMP in the transcript and blocking potential hairpin formation with DNA oligomers support a direct role for an RNA hairpin in triggering the release reaction. Changes in the 3'-proximal DNA sequences generally have little effect on the presence or rate of the release reaction, although there are significant exceptions. The results suggest that the presence of certain RNA hairpins in the region six to ten nucleotides upstream from the transcript growing point can trigger a substantial structural transition in the ternary transcription complex, forming a "release mode" complex from which transcript dissociation is facilitated. This release, mode complex may be a central intermediate in RNA chain termination. A final class of complexes (dead-end complexes) appear to be elongating complexes that have entered a state or conformation that is stable, but is blocked in resuming the normal elongation reaction. Such complexes bear active RNA polymerase, and can be restarted after limited pyrophosphorolysis. The structural elements that determine the formation of dead-end complexes are not yet known.
Arndt, K.M., and M.J. Chamberlain (1988) Transcription termination in Escherichia coli. measurement of the rate of enzyme release from rho-independent terminators. J. Mol. Biol. 202:271-285 The termination/release phase of transcription must involve at least three major steps: cessation of elongation; release of the transcript; and release of the RNA polymerase. We have devised a novel method for measuring the rate of Escherichia coli RNA polymerase release during transcription termination. The method is based on a kinetic analysis of the rate of RNA synthesis during steady-state transcription. Using this method with defined transcription units, we have found that RNA polymerase release occurs rapidly from several rho-independent terminators. Enzyme release from the T7 early terminator occurs within 13(+/- 3) seconds of the cessation of elongation. Neither nusA protein nor supercoiling of the DNA template affects the rate of enzyme release. However, addition of excess sigma factor significantly increases the rate of enzyme recycling during the steady state. Since added sigma factor does not alter the rates of initiation and elongation by E. coli RNA polymerase holoenzyme, it appears that sigma factor stimulates one or more steps in the termination/release process and reduces the rate of enzyme release to a few seconds. We present evidence that suggests sigma may be directly involved in catalyzing release of the core RNA polymerase from the DNA template during transcription termination. The rapid rates of enzyme release we measure make it difficult to be certain of the exact pathway of events that occur in the termination/release phase of transcription. The most plausible pathway involves initial release of the RNA transcript followed by release of core RNA polymerase from the DNA. Studies on the properties of core polymerase-RNA complexes indicate that core polymerase and the RNA transcript probably do not dissociate as a complex from the terminator. Furthermore, these core-RNA complexes are too stable to represent significant intermediates in the termination/release pathway, at least in the early steps of the reaction.
Chamberlain, M.J., K.M. Arndt, J.-F. Briat, R.L. Reynolds, and M.C. Schmidt (1987) Prokaryotic factors involved in termination at rho-independent termination sites. Pp 347-356 in RNA Polymerase and the Regulation of Transcription, Reznikoff, W.S., R.R. Burgess, J.E. Dahlberg, C.A. Gross, M.T. Record, and M.P. Wickens, Ed. Elsevior Science Publishing Co, Inc., New York |
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