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We recently discovered that the Warburg effect, defined by the dramatically enhanced metabolism of glucose to pyruvate, even in well-oxygenated cancer cells, can occur as a consequence of mutations that enhance lipid biosynthesis at the expense of respiratory capacity. Specifically, mutations in the E1 subunit of either of two respiratory enzymes, pyruvate dehydrogenase (PDC) or α-ketoglutarate dehydrogenase (KGDC), change substrate specificity from the 3-carbon α-ketoacid pyruvate, or the 5-carbon α-ketoacid α-ketoglutarate, to the 4-carbon α-ketoacid oxaloacetate (OADC). These mutations result in OADC-catalyzed synthesis of malonyl-CoA (MaCoA), the essential precursor of all fatty acids. These mutants arose as spontaneous suppressors of a yeast acc1(cs) cold-sensitive mutation encoding an altered form of AcCoA carboxylase (Acc1) that fails to produce MaCoA at the restrictive temperature (16 °C). Notably, these suppressors are respiratory defective as a result of the same nuclear mutations that suppress acc1(cs). These mutants also suppress sensitivity to Soraphen A, a potent inhibitor of Acc1 activity, at normal temperature (30 °C). To our knowledge, OADC activity has never been identified in eukaryotic cells. Our results offer a novel perspective on the Warburg effect: the reprogramming of energy metabolism in cancer cells as a consequence of mutational impairment of respiration to meet the fatty acid requirements of rapidly proliferating cells. We suggest OADC activity is a common feature of cancer cells and represents a novel target for the development of chemotherapeutics.

TFIIB is essential for transcription initiation by RNA polymerase II. TFIIB also cross-links to terminator regions and is required for gene loops that juxtapose promoter-terminator elements in a transcription-dependent manner. The Saccharomyces cerevisiae sua7-1 mutation encodes an altered form of TFIIB (E62K) that is defective for both start site selection and gene looping. Here we report the isolation of an ssl2 mutant, encoding an altered form of TFIIH, as a suppressor of the cold-sensitive growth defect of the sua7-1 mutation. Ssl2 (Rad25) is orthologous to human XPB and is a member of the SF2 family of ATP-dependent DNA helicases. The ssl2 suppressor allele encodes an arginine replacement of the conserved histidine residue (H508R) located within the DEVH-containing helicase domain. In addition to suppressing the TFIIB E62K growth defect, Ssl2 H508R partially restores both normal start site selection and gene looping. Moreover, Ssl2, like TFIIB, associates with promoter and terminator regions, and the diminished association of TFIIB E62K with the PMA1 terminator is restored by the Ssl2 H508R suppressor. These results define a novel, functional interaction between TFIIB and Ssl2 that affects start site selection and gene looping.

Gene loops are dynamic structures that juxtapose promoter–terminator regions of Pol II-transcribed genes. Although first described in yeast, gene loops have now been identified in yeast and mammalian cells. Looping requires components of the transcription preinitiation complex, the pre-mRNA 30-end processing machinery, and subunits of the nuclear pore complex. Loop formation is transcription-dependent, but neither basal nor activated transcription requires looping. Rather, looping appears to affect cellular memory of recent transcriptional activity, enabling a more rapid response to subsequent stimuli. The nuclear pore has been implicated in both memory and looping. Our working model is that loops are formed and/or maintained at the nuclear pore to facilitate hand-off of Pol II form the terminator to the promoter, thereby bypassing Pol II recruitment as the rate-limiting step in reactivation of transcription. Involvement of the nuclear pore also suggests that looping might facilitate mRNA export to the cytoplasm. The technology now exists to test these ideas.

Molecular dynamics simulation of Thermus thermophilus (Tt) RNA polymerase (RNAP) in a catalytic conformation demonstrates that the active site dNMP-NTP base pair must be substantially dehydrated to support full active site closing and optimum conditions for phosphodiester bond synthesis. In silico mutant beta R428A RNAP, which was designed based on substitutions at the homologous position (Rpb2 R512) of Saccharomyces cerevisiae (Sc) RNAP II, was used as a reference structure to compare to Tt RNAP in simulations. Long range conformational coupling linking a dynamic segment of the bridge alpha-helix, the extended fork loop, the active site, and the trigger loop-trigger helix is apparent and adversely affected in beta R428A RNAP. Furthermore, bridge helix bending is detected in the catalytic structure, indicating that bridge helix dynamics may regulate phosphodiester bond synthesis as well as translocation. An active site "latch" assembly that includes a key trigger helix residue Tt beta' H1242 and highly conserved active site residues beta E445 and R557 appears to help regulate active site hydration/dehydration. The potential relevance of these observations in understanding RNAP and DNAP induced fit and fidelity is discussed.

The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) is a reiterated heptad sequence (Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7) that plays a key role in the transcription cycle, coordinating the exchange of transcription and RNA processing factors. The structure of the CTD is flexible and undergoes conformational changes in response to serine phosphorylation and proline isomerization. Here we report that the Ess1 peptidyl prolyl isomerase functionally interacts with the transcription initiation factor TFIIB and with the Ssu72 CTD phosphatase and Pta1 components of the CPF 3'-end processing complex. The ess1(A144T) and ess1(H164R) mutants, initially described by Hanes and coworkers (Yeast 5:55-72, 1989), accumulate the pSer5 phosphorylated form of Pol II; confer phosphate, galactose, and inositol auxotrophies; and fail to activate PHO5, GAL10, and INO1 reporter genes. These mutants are also defective for transcription termination, but in vitro experiments indicate that this defect is not caused by altering the processing efficiency of the cleavage/polyadenylation machinery. Consistent with a role in initiation and termination, Ess1 associates with the promoter and terminator regions of the PMA1 and PHO5 genes. We propose that Ess1 facilitates pSer5-Pro6 dephosphorylation by generating the CTD structural conformation recognized by the Ssu72 phosphatase and that pSer5 dephosphorylation affects both early and late stages of the transcription cycle.

The synthesis of mature mRNA in eukaryotes and its utilization in the cytoplasm require cotranscriptional modifications of the pre-mRNA by capping at the 5′ end, removal of introns by splicing, and cleavage at the 3′ end followed by the addition of a poly(A) tail (4, 27, 32). mRNA 3′-end formation is an essential step in mRNA biogenesis and acts at many levels to influence gene expression. Its execution prevents readthrough transcription from interfering with DNA elements such as promoters, centromeres, and replication origins. Without a poly(A) tail, mRNA is targeted for degradation by nuclear surveillance mechanisms, is exported inefficiently from the nucleus, and is poorly translated in the cytoplasm. The maturation of mRNA 3′ ends also serves as an important point at which the cell can regulate the type and amount of mRNA derived from a particular gene. Furthermore, 3′-end processing has been linked to other essential processes such as chromosome segregation, DNA repair, and tissue-specific protein expression (23, 26, 46). mRNA 3′-end formation in Saccharomyces cerevisiae requires the concerted action of two multisubunit factors, cleavage factor (CF) I and cleavage/polyadenylation factor (CPF), that recognize processing signals around the poly(A) site. These complexes are phylogenetically conserved and are comparable to mammalian CstF and CPSF, respectively (27). Cleavage requires CF I and CPF, while tail synthesis requires these factors plus the Pab1 or Nab2 poly(A) binding protein (17). CF I is composed of Rna14, Rna15, Pcf11, Clp1, and Hrp1/Nab4 (21, 22, 35). The holo-CPF complex can be separated into core CPF and the APT subcomplex (29). Core CPF includes Pta1, Cft1, Cft2, Mpe1, Pfs2, Fip1, Pap1, the poly(A) polymerase, and Ysh1/Brr5, the putative pre-mRNA endonuclease (10, 12, 15, 28, 29, 36, 45). The APT subcomplex of CPF includes Pta1, Pti1, Ref2, Swd2, Syc1, and the two phosphatases Ssu72 and Glc7 (29). Even though holo-CPF is important for optimal processing, traditional multistep chromatographic fractionation showed that a smaller complex, called CF II, was sufficient for cleavage in combination with CF I and contained only the Cft1, Cft2, Ysh1, and Pta1 subunits (48). The essential Pta1 subunit was initially defined by a conditional growth mutation, pta1-1, that causes the accumulation of unspliced pre-tRNA in vivo (31). Pta1 is important for both cleavage and poly(A) addition (35, 48), and its phosphorylation inhibits the poly(A) addition step (16). Pta1 also has roles in redirecting the machinery to the 3′ end of nonadenylated snoRNA transcripts (29), in regulating the phosphorylated state of the RNA polymerase II (RNAP II) C-terminal domain (CTD) (25), and in the gene looping that juxtaposes the 5′ and 3′ ends of genes (1). Symplekin, the Pta1 homolog in higher eukaryotes, has been proposed to be a scaffold for assembling the mammalian 3′-end-processing complex (43). Symplekin has also been implicated in the formation of the cleaved, unadenylated ends of replication-dependent histone mRNAs (24), in the cytoplasmic polyadenylation of stored maternal mRNAs in preparation for their translation (2), and in the splicing of tRNA precursors (34). Consistent with a scaffold function, the 90-kDa Pta1 interacts physically and/or genetically with the CPF subunits Ysh1 (the candidate nuclease), Pti1 (thought to suppress CPF's polyadenylation activity on snoRNA transcripts), Ssu72 (an RNAP II CTD serine-5 phosphatase and the only factor dedicated to cleavage), Glc7 (a phosphatase needed for the polyadenylation step), and Syc1 (a negative regulator of mRNA 3′-end formation) (9, 15, 16, 29, 49). However, it is unknown whether all of these contacts are made simultaneously; perhaps some occur sequentially during mRNA synthesis or only in certain types of complexes, while others may never happen inside the cell. The range of Pta1 interactions, especially with three of the enzymes of the complex (Ssu72, Ysh1, and Glc7) and functionally with the fourth, Pap1, suggests that Pta1 occupies a central position within the 3′-end processing complex and helps coordinate its various activities. However, little is known about how Pta1 performs this critical function. In this study, we explore how Pta1 might act as a scaffold protein and show that it uses different regions to contact subunits that are essential to the function of CPF in mRNA 3′-end processing, snoRNA termination, CTD Ser5-P dephosphorylation, and gene looping. In addition, we identify new interactions between Pta1 and CF I, indicating that the organizational role of Pta1 includes making cross-factor connections. We also make the surprising observation that a Pta1 derivative that lacks the essential 300 amino acids at the N terminus and is incapable of interacting with and stabilizing Ssu72 is completely functional in cleavage and polyadenylation. These findings support a model in which the primary function of Ssu72 in mRNA 3′-end processing is to block an inhibitory activity of the Pta1 N-terminal domain.

Many eukaryotic genes undergo alternative 3′-end poly(A)-site selection producing transcript isoforms with 3′-UTRs of different lengths and post-transcriptional fates. Gene loops are dynamic structures that juxtapose the 3′-ends of genes with their promoters. Several functions have been attributed to looping, including memory of recent transcriptional activity and polarity of transcription initiation. In this study, we investigated the relationship between gene loops and alternative poly(A)-site. Using the KlCYC1 gene of the yeast Kluyveromyces lactis, which includes a single promoter and two poly(A) sites separated by 394 nucleotides, we demonstrate in two yeast species the formation of alternative gene loops (L1 and L2) that juxtapose the KlCYC1 promoter with either proximal or distal 3′-end processing sites, resulting in the synthesis of short and long forms of KlCYC1 mRNA. Furthermore, synthesis of short and long mRNAs and formation of the L1 and L2 loops are growth phase-dependent. Chromatin immunoprecipitation experiments revealed that the Ssu72 RNA polymerase II carboxyl-terminal domain phosphatase, a critical determinant of looping, peaks in early log phase at the proximal poly(A) site, but as growth phase advances, it extends to the distal site. These results define a cause-and-effect relationship between gene loops and alternative poly(A) site selection that responds to different physiological signals manifested by RNA polymerase II carboxyl-terminal domain phosphorylation status.

Recent studies demonstrated the existence of gene loops that juxtapose the promoter and terminator regions of genes with exceptionally long ORFs in yeast. Here we report that looping is not idiosyncratic to long genes but occurs between the distal ends of genes with ORFs as short as 1 kb. Moreover, looping is dependent upon the general transcription factor TFIIB: the E62K (glutamic acid 62 --> lysine) form of TFIIB adversely affects looping at every gene tested, including BLM10, SAC3, GAL10, SEN1, and HEM3. TFIIB crosslinks to both the promoter and terminator regions of the PMA1 and BLM10 genes, and its association with the terminator, but not the promoter, is adversely affected by E62K and by depletion of the Ssu72 component of the CPF 3' end processing complex, and is independent of TBP. We propose a model suggesting that TFIIB binds RNAP II at the terminator, which in turn associates with the promoter scaffold.

Transcription and translation are coordinated events in all organisms. In prokaryotes, the process that couples these two events is clear: The ribosome begins translation of the nascent mRNA while the DNA template is still being transcribed. Indeed, cotranscriptional protein synthesis underlies key regulatory mechanisms in bacteria, including attenuation, the mechanism that regulates RNA polymerase processivity in response to ribosome movement along the mRNA. But how is transcription coordinated with translation in eukaryotic organisms, where mRNA is synthesized in the nucleus and protein synthesis occurs in the cytoplasm? Although these two events are spatially distinct, separated by the nuclear envelope, efficient control of gene expression necessarily requires that transcription and translation be regulated in a coordinated manner. As an example, dFOXO-mediated transcriptional activation produces both an inhibitor of cap-dependent translation, eukaryotic translation initiation factor 4E (eIF4E)-BP, and a form of the insulin receptor mRNA that is translated by a cap-independent mechanism (Marr et al. 2007). In addition, translation requires a fully and accurately processed mRNA, and has mechanisms to help sense that appropriate processing has occurred. In the absence of physical coupling of transcription and translation, how are these two processes coordinated in eukaryotes? In this issue of Genes & Development, Rother and Straser (2007) report that the Ctk1 kinase, a key enzyme that facilitates passage of RNA polymerase II (Pol II) through specific stages of the transcription cycle, is also found in the cytoplasm associated with ribosomes actively engaged in protein synthesis (Fig. 1). Their work defines a physiological role for Ctk1 in translation by showing that cellular depletion of Ctk1 decreases total protein synthesis as well as the fidelity of translation elongation. These effects are likely to be a direct effect of Ctk1, since they identified the small ribosomal subunit protein rpS2 as the specific target of the kinase. Moreover, site-directed replacement of rpS2 Ser238, which they identified as the target of Ctk1, results in the same translational defects as depletion of Ctk1. Thus, Rother and Straser (2007) propose that Ctk1 piggybacks with either the mRNP particle or with the pre-40S ribosomal subunit to transit the nuclear envelope to regulate protein synthesis in the cytoplasm. Ctk1 was first identified as a subunit of the yeast CTDK-I complex that catalyzes phosphorylation of the Pol II C-terminal domain (CTD) (Sterner et al. 1995), a reiterated heptapeptide sequence (Tyr1–Ser2–Pro3– Thr4–Ser5–Pro6–Ser7) present at the C terminus of Rpb1 (Kobor and Greenblatt 2002). The CTD couples Pol II transcription with RNA processing, apparently forming a platform for the association and exchange of transcription and RNA processing factors (Hirose and Manley 2000; Orphanides and Reinberg 2002; Proudfoot et al. 2002; Bentley 2005; Meinhart et al. 2005). These factors include the 5 -capping enzymes, the splicing machinery, the 3 -end processing complex, and the transcription export complex (TREX) that facilitates mRNA translocation to the cytoplasm. Progression of Pol II through the transcription cycle is accompanied by changes in the phosphorylation status of the CTD. Pol II is recruited to the promoter in an unphosphorylated form (Pol IIA) that becomes extensively phosphorylated at Ser2 and Ser5 during different stages of the transcription cycle. Differential CTD phosphorylation promotes the exchange of initiation and elongation factors at promoter clearance (Pokholok et al. 2002) and the exchange of elongation and 3 -end processing factors at termination (Kim et al. 2004). CTD phosphorylation is catalyzed by C-type cyclindependent kinases (Prelich 2002). The first of these complexes to be identified is Kin28–Ccl1, which functions as a subcomplex of the general transcription factor TFIIH. Kin28 catalyzes Ser5 phosphorylation coincident with transcription initiation and as a prerequisite for capping (Hengartner et al. 1998; Rodriguez et al. 2000). The second complex, CTDK-I, is composed of three subunits, the Ctk1 kinase, Ctk2 cyclin, and Ctk3 accessory protein that forms a regulatory complex with Ctk2 (Sterner et al. 1995; Hautbergue and Goguel 2001). CTDK-I catalyzes Ser2 phosphorylation during elongation, coinciCorresponding author. E-MAIL kinzytg@umdnj.edu; FAX (732) 235-5223. Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1564807.

The ssu71 alleles of the TFG1 gene, which encodes the largest subunit of TFIIF, were isolated as suppressors of a TFIIB defect that affects the accuracy of transcription start site selection in the yeast Saccharomyces cerevisiae. Here we report that ssu71-1 also suppresses the cell growth and start site defects associated with an altered form of the Rpb1 subunit of RNA polymerase II (RNAP II). The ssu71-1 and ssu71-2 alleles were cloned and found to encode single amino acid replacements of glycine-363, either glycine to aspartic acid (G363D) or glycine to arginine (G363R). Two other charged replacements, G363E and G363K, were constructed by site-directed mutagenesis and suppress both TFIIB E62K and Rpb1 N445S, whereas neither G363A nor G363P exhibited any effect. G363 is phylogenetically conserved and its counterpart in human TFIIF (RAP74 G112) is located within the RAP74/RAP30 dimerization domain. We propose that the TFIIF dimerization domain is located in proximity to the B-finger of TFIIB near the active center of RNAP II where the TFIIB-TFIIF-RNAP II interface plays a key role in start site selection.

Phosphorylation of serine-2 (S2) and serine-5 (S5) of the C-terminal domain (CTD) of RNA polymerase II (RNAP II) is a dynamic process that regulates the transcription cycle and coordinates recruitment of RNA processing factors. The Fcp1 CTD phosphatase catalyzes dephosphorylation of S2-P. Here, we report that Ssu72, a component of the yeast cleavage/polyadenylation factor (CPF) complex, is a CTD phosphatase with specificity for S5-P. Ssu72 catalyzes CTD S5-P dephosphorylation in association with the Pta1 component of the CPF complex, although its essential role in 3' end processing is independent of catalytic activity. Depletion of Ssu72 impairs transcription in vitro, and this defect can be rescued by recombinant, catalytically active Ssu72. We propose that Ssu72 has a dual role in transcription, one as a CTD S5-P phosphatase that regenerates the initiation-competent, hypophosphorylated form of RNAP II and the other as a factor necessary for cleavage of pre-mRNA and efficient transcription termination.

The general transcription factor TFIIB is required for accurate initiation, although the mechanism by which RNA polymerase II (RNAP II) identifies initiation sites is not well understood. Here we describe results from genetic and biochemical analyses of an altered form of yeast TFIIB containing an arginine-78 --> cysteine (R78C) replacement in the "B-finger" domain. TFIIB R78C shifts start site selection downstream of normal and confers a cold-sensitive growth defect (Csm(-)). Suppression of the R78C Csm(-) phenotype identified a functional interaction between TFIIB and the Rpb2 subunit of RNAP II and defined a novel role for Rpb2 in start site selection. The rpb2 suppressor encodes a glycine-369 --> serine (G369S) replacement, located in the "lobe" domain of Rpb2 and near the Rpb9 subunit, which was identified previously as an effector of start site selection. The Rpb2-Rpb9 "lobe-jaw" region of RNAP II is downstream of the catalytic center and distal to the site of RNAP II-TFIIB interaction. A TFIIB R78C mutant extract was defective for promoter-specific run-on transcription but yielded an altered pattern of abortive initiation products, indicating that the R78C defect does not preclude initiation. The sua7-3 rpb2-101 double mutant was sensitive to 6-azauracil in vivo and to nucleoside triphosphate substrate depletion in vitro. In the context of the recent X-ray structure of the yeast RNAP II-TFIIB complex, these results define a functional interaction between the B-finger domain of TFIIB and the distal lobe-jaw region of RNAP II and provide insight into the mechanism of start site selection.

Histone lysine methylation plays a key role in the organization of chromatin structure and the regulation of gene expression. Recent studies demonstrated that the yeast Set1 and Set2 histone methyltransferases are recruited to mRNA coding regions by the PAF transcription elongation complex in a manner dependent upon the phosphorylation state of the carboxy-terminal domain of RNA polymerase II. These studies define an unexpected link between transcription elongation and histone methylation.

Transcription and processing of pre-mRNA are coupled events. By using a combination of biochemical, molecular, and genetic methods, we have found that the phylogenetically conserved transcription factor Ssu72 is a component of the cleavage/polyadenylation factor (CPF) ofSaccharomyces cerevisiae. Our results demonstrate that Ssu72 is required for 3′ end cleavage of pre-mRNA but is dispensable for poly(A) addition and RNAP II termination. The in vitro cleavage defect caused by depletion of Ssu72 from cells can be rescued by addition of recombinant Ssu72. Ssu72 interacts physically and genetically with the Pta1 subunit of CPF. Overexpression ofPTA1causes synthetic lethality in anssu72-3mutant. Moreover, Sub1, which has been implicated in transcription initiation and termination, also interacts with Pta1, and overexpression ofSUB1suppresses the growth and processing defect of apta1mutation. Physical interactions of Ssu72 and Sub1 with Pta1 are mutually exclusive. Based on the interactions of Ssu72 and Sub1 with both the Pta1 of CPF and the TFIIB component of the initiation complex, we present a model describing how these novel connections between the transcription and 3′ end processing machineries might facilitate transitions in the RNAP II transcription cycle.

Essential components of the eukaryotic transcription apparatus include RNA polymerase II, a common set of initiation factors, and a Mediator complex that transmits regulatory information to the enzyme. Insights into mechanisms of transcription have been gained by three-dimensional structures for many of these factors and their complexes, especially for yeast RNA polymerase II at atomic resolution.

Transitions between the different stages of the RNAPII transcription cycle involve the recruitment and exchange of factors, including mRNA capping enzymes, elongation factors, splicing factors, 3'-end-processing complexes, and termination factors. These transitions are coordinated by the dynamic phosphorylation of the C-terminal domain (CTD) of the largest subunit of RNAPII (Rpb1). The CTD is composed of reiterated heptapeptide repeats (Y(1)S(2)P(3)T(4)S(5)P(6)S(7)) that undergo phosphorylation and dephosphorylation as RNAPII transitions through the transcription cycle. An essential phosphatase in this process is Ssu72, which exhibits catalytic specificity for Ser(P)(5) and Ser(P)(7). Ssu72 is unique in that it is specific for Ser(P)(5) in one orientation of the CTD and for Ser(P)(7) when bound in the opposite orientation. Moreover, Ssu72 interacts with components of the initiation machinery and affects start site selection yet is an integral component of the CPF 3'-end-processing complex. Here we provide a comprehensive view of the effects of Ssu72 with respect to its Ser(P)(5) phosphatase activity. We demonstrate that Ssu72 dephosphorylates Ser(P)(5) at the initiation-elongation transition. Furthermore, Ssu72 indirectly affects the levels of Ser(P)(2) during the elongation stage of transcription but does so independent of its catalytic activity.

The bacterial final sigma(54) protein associates with core RNA polymerase to form a holoenzyme complex that renders cognate promoters enhancer-dependent. Although unusual in bacteria, enhancer-dependent transcription is the paradigm in eukaryotes. Here we report that a fragment of Escherichia coli final sigma(54) encompassing amino acid residues 29-177 functions as a potent transcriptional activator in yeast when fused to a Gal4 DNA binding domain. Activation by Gal4-final sigma(54) is TATA-dependent and requires the SAGA coactivator complex, suggesting that Gal4-final sigma(54) functions by a normal mechanism of transcriptional activation. Surprisingly, deletion of the AHC1 gene, which encodes a polypeptide unique to the ADA coactivator complex, stimulates Gal4-final sigma(54)-mediated activation and enhances the toxicity of Gal4-final sigma(54). Accordingly, the SAGA and ADA complexes, both of which include Gcn5 as their histone acetyltransferase subunit, exert opposite effects on transcriptional activation by Gal4-final sigma(54). Gal4-final sigma(54) activation and toxicity are also dependent upon specific final sigma(54) residues that are required for activator-responsive promoter melting by final sigma(54) in bacteria, implying that activation is a consequence of final sigma(54)-specific features rather than a structurally fortuitous polypeptide fragment. As such, Gal4-final sigma(54) represents a novel tool with the potential to provide insight into the mechanism by which natural activators function in eukaryotic cells.

Eukaryotic RNA polymerase II (RNAP II) is a multisubunit enzyme that is responsible for transcription of all protein-encoding genes. Saccharomyces cerevisiae RNAP II is a 12-subunit complex encoded by the genes RPB1 to RPB12 (reviewed in references 2, 53, and 55). The two largest subunits, Rpb1 and Rpb2, are homologous to the β′ and β subunits of bacterial RNA polymerase (RNAP), respectively (23, 49). The yeast counterpart of the bacterial α subunit appears to be shared between the Rpb3 and Rpb11 subunits (26, 57). Accordingly, Rpb1, Rpb2, Rpb3, and Rpb11 comprise the functional equivalent of the bacterial α2ββ′ core RNAP. The functions of the remaining RNAP II subunits are less clear, although five subunits, Rpb5, Rpb6, Rpb8, Rpb10, and Rpb12, are shared among all three forms of RNAP and therefore function in transcription of all genes. With the exception of Rpb4 and Rpb9, all subunits are essential for cell viability. Rpb4 forms a subcomplex with Rpb7 that is required for promoter-specific initiation but is dispensable for elongation (13). RNAP II is unable to initiate promoter-specific transcription on its own. Promoter recognition requires the general transcription factors (GTFs), which include the TATA binding protein (TBP), TFIIB, TFIIE, TFIIF, and TFIIH (reviewed in references 17 and 33). TBP nucleates assembly of a transcription preinitiation complex by binding the TATA element and inducing a sharp bend in the DNA template. TFIIB binds the TATA-TBP complex and is responsible for defining the polarity of transcription by binding asymmetrically to the BRE element, located immediately upstream of the TATA box (28, 50). RNAP II, in association with TFIIF, binds the TATA-TBP-TFIIB ternary complex, followed by TFIIE and TFIIH. TFIIH is a multisubunit complex with catalytic activities responsible for phosphorylation of the carboxy-terminal repeat domain of the Rpb1 subunit of RNAP II, promoter melting and promoter clearance (25). The GTFs were discovered based on their requirement for accurate initiation in vitro (29, 52) and are highly conserved among eukaryotic organisms (reviewed in references 17, 30, 33, and 38). Furthermore, the TBP and TFIIB requirements for transcription predate the divergence of Eukarya and Archaea (reviewed in reference 5). Nonetheless, the RNAP II requirement for GTFs is not universal but is instead dictated by promoter sequence and architecture (16, 35, 51). Indeed, genome-wide expression analysis revealed that most components of the RNAP II transcriptional machinery are dispensable for expression of subsets of genes (21). Conversely, since only a limited number of promoters have been analyzed in vitro, it is conceivable that additional GTFs might be identified based on their requirement for accurate initiation from specific promoters. Other factors might be identified based on their ability to stimulate activator-independent transcription. For example, the yeast Tsp1 (Sub1) protein was identified as a positive effector of in vitro transcription in the absence of a sequence-specific activator (20). Tsp1 interacts physically and genetically with TFIIB and is homologous to the human transcriptional cofactor PC4 (20, 27). Additional basal factors are likely to be identified, either by their requirement for promoter-specific initiation, by their ability to stimulate activator-independent transcription, or by genetic interactions with GTFs or RNAP II subunits. The yeast gene encoding TFIIB (SUA7) was identified as an effector of transcriptional accuracy (36). Replacements at either of two phylogenetically invariant residues, glutamate-62 (E62) or arginine-78 (R78), caused a marked start site shift downstream of the normal site at the CYC1 and ADH1 promoters (37). Nearly identical effects on transcriptional accuracy are conferred by the sua8 alleles of RPB1, suggesting a functional interaction between TFIIB and Rpb1 during start site selection (6). In an effort to identify other factors that affect initiation, we isolated suppressors of the sua7-1 (E62K) mutation. The ssu71 and ssu73 suppressors encode altered forms of the largest subunit of TFIIF (Tfg1) and the Rpb9 subunit of RNAPII, respectively (46, 47). In contrast, ssu72 was identified as an enhancer of the TFIIB E62K defect: the ssu72-1 allele confers a heat-sensitive (Ts−) growth defect and a dramatic downstream start site shift, with both effects being dependent upon the sua7-1 allele (48). The SSU72 gene is essential for cell viability and encodes a novel protein of undefined function (48). To further characterize Ssu72, we generated a new ssu72 allele that confers a tight Ts− phenotype, independent of a TFIIB defect. We have taken advantage of the ssu72 Ts− phenotype to isolate a new rpb2 mutation encoding an altered from of the Rpb2 subunit of RNAP II. The results presented here demonstrate that Ssu72 is a transcription factor that interacts with the core RNAP II machinery both in vivo and in vitro.

A transcriptional repressor complex encoded by two essential genes, YDR1 and BUR6, was isolated from Saccharomyces cerevisiae and shown to be the functional counterpart of the human repressor complex Dr1-DRAP1. To elucidate the mechanism of repression by this complex, altered forms of Ydr1 and Bur6 were studied in vitro and in vivo. Deletion of the C-terminal 41 amino acids of Ydr1 resulted in loss of repressor activity and a growth defect, suggesting that the C-terminal domain of Ydr1 functions as a potent transcriptional repressor. A screen for extragenic suppressors of a cold-sensitive ydr1 (ydr1(cs)) mutant led to the identification of recessive mutations in the SIN4 gene, which encodes a component of the SRB-MED complex. The sin4 alleles suppressed not only ydr1(cs) mutations but also bur6(cs) mutations. In contrast, deletion of the gal11 gene, whose product is also a member of the SRB-MED complex, failed to suppress ydr1(cs) and bur6(cs) mutations, indicating that suppression is not due to general defects in the SRB-MED complex. Moreover, one of the sin4 alleles, but not the sin4 deletion, was found to specifically suppress the inviability of a ydr1 deletion, demonstrating that the essential function of Ydr1 becomes dispensable in a sin4 mutant background. Biochemical analysis of the SRB-MED complex from the sin4 suppressor strain revealed a structurally distinct form of the SRB-MED complex that lacks a subset of mediator subunits. These results define a delicate balance between positive and negative regulators of transcription operating through the Ydr1-Bur6 repressor complex.

A yeast mutant was isolated encoding a single amino acid substitution [serine-53 → proline (S53P)] in transcription factor TFIIB that impairs activation of the PHO5 gene in response to phosphate starvation. This effect is activation-specific because S53P did not affect the uninduced level of PHO5 expression, yet is not specific to PHO5 because Adr1-mediated activation of the ADH2 gene also was impaired by S53P. Pho4, the principal activator of PHO5 , directly interacted with TFIIB in vitro , and this interaction was impaired by the S53P replacement. Furthermore, Pho4 induced a conformational change in TFIIB, detected by enhanced sensitivity to V8 protease. The S53P replacement also impaired activation of a lexA (op) -lacZ reporter by a LexA fusion protein to the activation domain of Adr1, thereby indicating that the transcriptional effect on ADH2 expression is specific to the activation function of Adr1. These results define an activation-specific role for TFIIB in vivo and suggest that certain activators induce a conformational change in TFIIB as part of their mechanism of transcriptional stimulation.

SUMMARY Transcription initiation by RNA polymerase II (RNA pol II) requires interaction between cis-acting promoter elements and trans-acting factors. The eukaryotic promoter consists of core elements, which include the TATA box and other DNA sequences that define transcription start sites, and regulatory elements, which either enhance or repress transcription in a gene-specific manner. The core promoter is the site for assembly of the transcription preinitiation complex, which includes RNA pol II and the general transcription fctors TBP, TFIIB, TFIIE, TFIIF, and TFIIH. Regulatory elements bind gene-specific factors, which affect the rate of transcription by interacting, either directly or indirectly, with components of the general transcriptional machinery. A third class of transcription factors, termed coactivators, is not required for basal transcription in vitro but often mediates activation by a broad spectrum of activators. Accordingly, coactivators are neither gene-specific nor general transcription factors, although gene-specific coactivators have been described in metazoan systems. Transcriptional repressors include both gene-specific and general factors. Similar to coactivators, general transcriptional repressors affect the expression of a broad spectrum of genes yet do not repress all genes. General repressors either act through the core transcriptional machinery or are histone related and presumably affect chromatin function. This review focuses on the global effectors of RNA polymerase II transcription in yeast, including the general transcription factors, the coactivators, and the general repressors. Emphasis is placed on the role that yeast genetics has played in identifying these factors and their associated functions.

The Kluyveromyces lactis HIS4 gene was cloned by complementation of a Saccharomyces cerevisiae his4 mutant. Sequence analysis revealed a 2388 bp open reading frame encoding a single polypeptide predicted to encompass three distinct enzymatic activities (phosphoribosyl-AMP cyclohydrolase, phosphoribosyl-ATP pyrophosphohydrolase and histidinol dehydrogenase). This structural organization is strikingly similar to that of the His4 proteins from S. cerevisiae and Pichia pastoris. Transcript analysis detected a single mRNA species of 2.5 kb. The EMBL accession number of this gene is Y09503. © 1998 John Wiley & Sons, Ltd.

Yeast RNA polymerase II has been proposed to 'scan' template DNA for transcription start sites. A new study mapping promoter DNA trajectory through the preinitiation complex suggests a mechanism for how this occurs.

We describe a modified 3C ("chromosome conformation capture") protocol for detection of transient, short-range chromatin interactions in the yeast Saccharomyces cerevisiae. 3C was initially described by Job Dekker and involves formaldehyde cross-linking to stabilize transient chromatin interactions, followed by restriction digestion, ligation, and locus-specific PCR. As such, 3C reveals complex three-dimensional interactions between distal genetic elements within intact cells at high resolution. Using a modified version of Dekker's protocol, we are able to detect gene loops that juxtapose promoter and terminator regions of yeast genes with ORFs as short as 1 kb. We are using this technique to define the cis- and trans-acting requirements for the formation and maintenance of gene loops, and to elucidate their physiological consequences. We anticipate that this method will be generally applicable to detect dynamic, short-range chromatin interactions, not limited to gene loops.

A general repressor extensively studied in vitro is the human Dr1/DRAP1 heterodimeric complex. To elucidate the function of Dr1 and DRAP1 in vivo , the yeast Saccharomyces cerevisiae Dr1/DRAP1 repressor complex was identified. The repressor complex is encoded by two essential genes, designated YDR1 and BUR6 . The inviability associated with deletion of the yeast genes can be overcome by expressing the human genes. However, the human corepressor DRAP1 functions in yeast only when human Dr1 is coexpressed. The yDr1/Bur6 complex represses transcription in vitro in a reconstituted RNA polymerase II transcription system. Repression of transcription could be overcome by increasing the concentration of TATA-element binding protein (TBP). Consistent with the in vitro results, overexpression of YDR1 in vivo resulted in decreased mRNA accumulation. Furthermore, YDR1 overexpression impaired cell growth, an effect that could be rescued by overexpression of TBP. In agreement with our previous studies in vitro , we found that overexpression of Dr1 in vivo also affected the accumulation of RNA polymerase III transcripts, but not of RNA polymerase I transcripts. Our results demonstrate that Dr1 functions as a repressor of transcription in vivo and, moreover, directly targets TBP, a global regulator of transcription.

ATP-dependent chromatin remodeling enzymes are highly abundant and play pivotal roles regulating DNA-dependent processes. The mechanisms by which they are targeted to specific loci have not been well understood on a genome-wide scale. Here we present evidence that a major targeting mechanism for the Isw2 chromatin remodeling enzyme to specific genomic loci is through sequence-specific transcription factor (TF)-dependent recruitment. Unexpectedly, Isw2 is recruited in a TF-dependent fashion to a large number of loci without TF binding sites. Using the 3C assay, we show that Isw2 can be targeted by Ume6- and TFIIB-dependent DNA looping. These results identify DNA looping as a previously unknown mechanism for the recruitment of a chromatin remodeling enzyme and defines a novel function for DNA looping. We also present evidence suggesting that Ume6-dependent DNA looping is involved in chromatin remodeling and transcriptional repression, revealing a mechanism by which the three-dimensional folding of chromatin affects DNA-dependent processes.

Sequence analysis of the newly defined SSU81 gene revealed an adjacent open reading frame (ORF) encoding a protein whose deduced amino acid sequence is identical to that of ribosomal protein L17. The DNA sequence of this region is different from that of the RPL17A gene and therefore represents a duplicate gene encoding L17. We have designated this gene RPL17B. The RPL17B coding region is split by an intron that occurs in the same position (codons 14/15) as the intron in RPL17A. The RPL17B promoter region includes two TATA boxes, a canonical UASRPG motif, and several pyrimidine-rich tracts. RPL17B was mapped by CHEF and lambda clone grid hybridization blots to the right arm of chromosome V, linked to the TRP2 and RAD51 genes. A partial ORF was identified adjacent to RPL17B and SSU81 that is homologous to an ORF (designated A509) physically linked to RPL17A. This observation, and the identical position of the introns within the RPL17 genes, suggest that one RPL17 locus arose by duplication and translocation of the other. The complete 3·8 kbp DNA sequence encompassing RPL17B has been entered in the GenBank data library under Accession Number U15653.

What is it? The Mediator complex is an enormous, multi-subunit protein complex that regulates gene expression in eukaryotic cells. It acts as a molecular circuit board that integrates physiological and developmental signals and transmits them to RNA polymerase II (Pol II), the enzyme charged with transcription of all protein-encoding genes. Although initially discovered as a cofactor required for activator-dependent transcription, Mediator also stimulates activator-independent (basal) transcription. Furthermore, Mediator can act as a co-repressor that facilitates transcriptional silencing. Mediator activity is not limited to its effects on transcription initiation, but it can also affect elongation and perhaps all stages of the transcription cycle.How was Mediator discovered? First, a little background information. The 12-subunit Pol II complex (Rpb1–Rpb12) is all that is required for its catalytic activity, i.e. DNA template-directed RNA synthesis. However, Pol II is unable to recognize promoter DNA on its own and instead requires five general transcription factors (GTFs) — TFIIB, TFIID, TFIIE, TFIIF and TFIIH. The GTFs and Pol II assemble in a defined order on promoter DNA to form a pre-initiation complex (PIC). The PIC is sufficient for accurate start-site selection, but does not respond to transcriptional activators. Indeed, as the GTFs were purified to homogeneity, the Pol II response to activators became increasingly weaker. This provided a biochemical assay that led to the discovery of Mediator as a component of a yeast cell extract that dramatically stimulated activator-dependent transcription. Accordingly, Mediator is a key link in the pathway leading from enhancer-bound activators to promoter-bound Pol II.Purification of Mediator revealed that several of its subunits had been identified previously in genetic screens for regulators of gene expression in yeast. In fact, a single genetic selection for suppressors of a growth defect associated with truncation of the carboxy-terminal domain (CTD) of the Rpb1 subunit of Pol II identified nine different Mediator components (SRB genes). Additional subunits had also been identified in other screens for mutants that either activated or repressed gene expression. Thus, the synergy of biochemistry and genetics led to the discovery of Mediator as an essential regulator of Pol II transcription. The identification of more than a third of its components as the products of SRB genes suggested that Mediator affects gene expression, at least in part, through the Pol II CTD. More on that in a moment...Is Mediator found in organisms other than yeast? Mediator appears to be a universal requirement for Pol II transcription. Several Mediator-like complexes were identified in transcription systems derived from fly, worm, mouse, rat and human cells. Each complex was named according to how it was discovered: TRAP (thyroid hormone receptor-associated proteins), SMCC (SRB/Mediator co-activator complex), ARC (activator-recruited complex), DRIP (vitamin D receptor-interacting proteins), CRSP (cofactor required for Sp1 activation) and PC2 (positive cofactor 2). In each case, these complexes were required for the response to transcriptional regulators in vitro. However, their relationship to each other and to Mediator was not immediately apparent due to subunit heterogeneity and to amino acid sequence divergence among orthologous subunits. Further characterization of these complexes established that all are metazoan counterparts of Mediator with a common modular structure that includes orthologs of all yeast subunits (Figure 1Figure 1). Metazoan Mediator complexes often include additional subunits not found in yeast Mediator and exist in multiple forms with variable subunit composition, reflecting more complex transcriptional regulatory programs in these organisms.Figure 1The Mediator complex.(A)The 25-subunit yeast Mediator complex consists of four structurally distinct modules: head, middle/arm, tail and kinase. Although a uniform nomenclature has been assigned to orthologous Mediator subunits from eukaryotic organisms, nine of the yeast subunits were identified in a genetic selection for suppressors of truncation of the Pol II carboxy-terminal domain (CTD); these genes were designated SRBs, including SRB8 to SRB11. (B) A ribbon model of the yeast Mediator head module, composed of 7 subunits, as seen from different angles. Med17 (cyan), Med11 (purple), Med22 (yellow), Med6 (green), Med8 (orange), Med18 (blue), and Med20 (red). This image was generously provided by Dr. Yuichiro Takagi (Indiana University School of Medicine).View Large Image | View Hi-Res Image | Download PowerPoint SlideIs Mediator required for expression of all genes? It seems so. Inactivation of the Med17 (SRB4 gene) subunit of yeast Mediator has the same deleterious effect on transcription as inactivation of the Rpb1 subunit of Pol II — essentially abolishing mRNA synthesis. Furthermore, a technique called chromatin immunoprecipitation (ChIP), which queries the association of specific proteins at any site in the genome, detected Mediator at the promoters of nearly all genes. As mentioned above, Mediator also stimulates activator-independent transcription. Indeed, in vitro transcription is so inefficient in the absence of Mediator that Mediator might be more appropriately viewed as a GTF, essential for all Pol II transcription.What does Mediator look like? Yeast Mediator consists of 25 subunits with a molecular mass of ∼1 million Daltons. Electron microscopy defined a modular structure consisting of four domains: head, middle/arm, tail and kinase (Figure 1Figure 1). The head module can stimulate activator-independent transcription, but does not respond to activators. Conversely, the tail module is the direct target of specific regulatory proteins and appears to be responsible for recruitment of Mediator to DNA. The middle/arm module is a highly flexible structure that orchestrates extensive conformational changes upon Mediator binding to Pol II.The hallmark of the kinase module is the Cdk8–cyclin C pair (Srb10–Srb11), which assembles with Med12 and Med13 (Figure 1Figure 1). All four of these subunits were discovered as SRB genes, implicating the kinase module in CTD function. This interaction might account, in part, for the co-repressor function of Mediator: Pol II is recruited to the promoter in hypo-phosphorylated form such that Cdk8-catalyzed CTD phosphorylation prevents Pol II assembly into the PIC. In human cells, a very different function for the Cdk8–cyclin C module has been reported: epigenetic silencing of neuronal genes via covalent histone modification. Furthermore, stimulatory effects on transcription have also been demonstrated for Cdk8 in yeast and metazoan cells, underscoring the enigmatic nature of the kinase module.What does the structure of Mediator reveal about its function? Just as high-resolution images of Pol II and the ribosome are unraveling the mechanisms of transcription and protein synthesis, structural studies of Mediator are providing insight into how this complex machine regulates transcription. The architecture of the head module was recently solved to high resolution, defining three structural domains — the neck, fixed jaw and movable jaw. These domains are built around a bundle of ten α-helices derived from five different subunits (Figure 1Figure 1). Structural and biochemical information suggests that the head module positions the Pol II CTD in proximity to TFIIH, where the TFIIH kinase (Cdk7) stimulates CTD phosphorylation, in this case (as opposed to Cdk8; see above) promoting dissociation of Pol II from Mediator, coinciding with the transition from initiation to elongation. These structures also provide an informative framework for interpreting the earlier SRB and other MED mutations, including secondary mutations that suppress primary mutations. For example, a Med17 defect is suppressed by a Med6 mutation, apparently by creating a stabilizing salt-bridge between the two subunits.Recent cryo-EM images of the head module in association with a minimal, functional PIC have also illuminated the mechanism of promoter binding. The jaws bind the Rpb4–Rpb7 subunits of Pol II, along with the ‘clamp’ that closes over the active site cleft. In the Pol II closed conformation, the cleft is large enough to accommodate singlestranded DNA, but not double-stranded DNA. Either the two strands of promoter DNA must melt prior to PIC assembly, or Pol II must undergo a conformational change that opens the cleft. The cryo-EM map suggests the latter: head interactions with Pol II cause repositioning of Rpb4–Rpb7 and a large rearrangement of the clamp, thereby opening the active site cleft. This model could account for the stimulatory effect of Mediator on basal transcription and suggests that clamp opening is a fundamental aspect of Mediator function.Does Mediator play a role in human disease? Mediator appears to play a role in diverse pathological states, including tumorigenesis, cardiovascular disease, neurological disease, and metabolic disorders. For instance, Med19 is upregulated in a variety of cancers, such as bladder cancer, breast cancer, lung tumors, and osteosarcoma. In addition, the X-linked Lujan and Opitz-Kaveggia mental retardation syndromes are caused by mutations in Med12, while mutations in Med13 have been found in patients with congenital heart defects. The fact that Mediator is a core component of the gene expression machinery indicates that defects in the transcriptional program are at the heart of these disorders.

The SUA7 gene of Saccharomyces cerevisiae encodes the general transcription factor TFIIB. SUA7 was identified based on the ability of mutations at this locus to shift transcription start site selection at the cyc1 gene downstream of normal. Here, we report the nature of these mutations; the sua7-1 and sua7-2 alleles encode identical E62K replacements, and sua7-3 encodes an R78C replacement. Both Glu-62 and Arg-78 are phylogenetically invariant and occur within the most highly conserved region of TFIIB, immediately distal to a zinc finger motif. A double E62K,R78C mutant was constructed and exhibited the same phenotypes associated with the single mutants, including cold sensitivity and altered start site selection, suggesting that Glu-62 and Arg-78 are functionally related. This observation, and the opposite charge of the 2 residues, suggested that Glu-62 and Arg-78 might interact to form a salt bridge. This was tested by constructing reciprocal E62R and R78E replacements. The E62R mutant is phenotypically identical to the E62K mutant, whereas the R78E mutant is inviable. However, an E62R,R78E double mutant was not only viable but is phenotypically similar to the single mutants. These results define the highly conserved sequence adjacent to the zinc finger of TFIIB as a critical determinant of start site selection and suggest that an Glu-62-Arg-78 salt bridge is an important structural element of that domain.

Mutations in the Saccharomyces cerevisiae sua8 gene were found to be suppressors of an aberrant ATG translation initiation codon in the leader region of the cyc1 gene. Analysis of cyc1 transcripts from sua8 mutants revealed that suppression is a consequence of diminished transcription initiation at the normal start sites in favor of initiation at downstream sites, including a site between the aberrant and normal ATG start codons. This effect is not cyc1 gene specific since initiation at other genes, including ADH1, CYC7, and HIS4, was similarly affected, although initiation at HIS3 and SPT15 was unaffected. The SUA8 gene was cloned and partially sequenced, revealing identity to RPB1, which encodes the largest subunit of RNA polymerase II. The sua8 suppressors are the result of single amino acid replacements of highly conserved residues. Three replacements were found either within or immediately preceding homology block D, and a fourth was found adjacent to homology block H, indicating that these regions play a role in defining start sites in vivo. Nearly identical effects on start site selection were observed for sua7 suppressors, which encode altered forms of TFIIB. Synthetic lethality was associated with double sua7 sua8 suppressor mutations, and recessive sua7 mutants failed to fully complement recessive sua8 mutants in heterozygous diploids (nonallelic noncomplementation). These data indicate that the largest subunit of RNA polymerase II and TFIIB are important determinants of transcription start site selection in S. cerevisiae and suggest that this function might be conferred by interaction between these two proteins.

Histone methylation has emerged as a significant regulator of chromatin structure and function. Two different classes of histone methyltransferase (HMT) have been described, which target either lysine or arginine residues in the histone N-terminal tails. A flurry of recent papers now describe a third class of HMT that affects chromatin silencing indirectly, not by methylation of histone tails, but instead by targeting a conserved lysine residue in the core domain of the nucleosome.

Polyadenylation of eukaryotic mRNAs contributes to stability, transport and translation, and is catalyzed by a large complex of conserved proteins. The Pcf11 subunit of the yeast CF IA factor functions as a scaffold for the processing machinery during the termination and polyadenylation of transcripts. Its partner, Clp1, is needed for mRNA processing, but its precise molecular role has remained enigmatic. We show that Clp1 interacts with the Cleavage-Polyadenylation Factor (CPF) through its N-terminal and central domains, and thus provides cross-factor connections within the processing complex. Clp1 is known to bind ATP, consistent with the reported RNA kinase activity of human Clp1. However, substitution of conserved amino acids in the ATP-binding site did not affect cell growth, suggesting that the essential function of yeast Clp1 does not involve ATP hydrolysis. Surprisingly, non-viable mutations predicted to displace ATP did not affect ATP binding but disturbed the Clp1-Pcf11 interaction. In support of the importance of this interaction, a mutation in Pcf11 that disrupts the Clp1 contact caused defects in growth, 3'-end processing and transcription termination. These results define Clp1 as a bridge between CF IA and CPF and indicate that the Clp1-Pcf11 interaction is modulated by amino acids in the conserved ATP-binding site of Clp1.

Mutations in the Saccharomyces cerevisiae SUA7 gene were isolated as suppressors of an aberrant ATG translation initiation codon in the leader region of the cyc1 gene. Molecular and genetic analysis of the cloned SUA7 gene demonstrated that SUA7 is a single copy, essential gene encoding a basic protein (calculated M r of 38, 142) that is homologous to human transcription factor TFIIB. Analysis of cyc1 transcripts from sua7 strains revealed that suppression is a consequence of diminished transcription initiation at the normal start sites in favor of initiation at downstream sites, including a major site between the aberrant and normal ATG start codons. A similar effect was found at the ADH1 locus, establishing that this effect is not cyc1 gene-specific. Thus, SUA7 encodes a yeast TFIIB homolog and functions in transcription start site selection.

DNA loops that juxtapose the promoter and terminator regions of RNA polymerase II-transcribed genes have been identified in yeast and mammalian cells. Loop formation is transcription-dependent and requires components of the pre-mRNA 3′-end processing machinery. Here we report that looping at the yeast GAL10 gene persists following a cycle of transcriptional activation and repression. Moreover, GAL10 and a GAL1p-SEN1 reporter undergo rapid reactivation kinetics following a cycle of activation and repression—a phenomenon defined as “transcriptional memory”—and this effect correlates with the persistence of looping. We propose that gene loops facilitate transcriptional memory in yeast.

Why such complexity in Pol II transcription? The 60 protein requirement for Pol II transcription does not reflect the complexity of RNA synthesis. After all, the chemistry of the transcription reaction in prokaryotic and eukaryotic organisms is, in essence, identical. Yet bacterial transcription is catalyzed by a five subunit core complex — α2ββ′ω, comparable to eukaryotic Rpb1,2,3,6,11 — with promoter recognition conferred by one of only a handful of different σ subunits that associate with the core enzyme. Instead, the complexity of the eukaryotic Pol II machinery is a consequence of both the organization of the eukaryotic genome, including the packaging of DNA into chromatin, and the myriad regulatory parameters that control gene expression.The challenge that lies ahead is to unravel the physical interactions that occur among and within the different transcription complexes and how these interactions occur in response to different stimuli. High-resolution three-dimensional images have provided remarkable insight into the structural basis of Pol II function. However, the size, complexity and dynamic nature of the TFIID, TFIIH and MED complexes make it unlikely that high-resolution images of these intact complexes will be forthcoming. Instead, new technologies, including novel methods for crosslinking protein–protein and protein–DNA interactions, are likely to illuminate the structural basis of transcription in small, but highly informative increments.

The cycl-362 allele contains a point mutation that generates an aberrant AUG codon upstream of the normal CYC1 translation initiation codon. Mutants containing this allele express only about 2% of normal iso-1-cytochrome c, presumably due to translation initiation at the upstream AUG, termination at a UAA sequence six codons downstream, and failure to reinitiate at the normal AUG codon two nucleotides later. Both intragenic and extragenic revertants of cycl-362, expressing elevated levels of iso-1-cytochrome c, have been isolated simply by selecting for growth on lactate medium. Here we describe an improved method for isolating and readily distinguishing cis- from trans-acting suppressors of the upstream AUG. Eight different genes, designated sua1-sua8, are represented in our current collection of extragenic suppressors; all are recessive and enhance iso-1-cytochrome c levels to 10-60% of normal. None of the sua genes is allelic to SUI2 or sui3, which encode eIF-2 alpha and eIF-2 beta, respectively, or to SUI1. Many of the suppressors exhibit pleiotropic phenotypes, including slow growth, cold (16 degrees C) and heat (37 degrees C) sensitivity. These phenotypes have been exploited to clone the SUA5, SUA7 and SUA8 genes, which are presently being characterized. The structure of cyc1-362 and the number of sua genes already uncovered suggest that the SUA genes are likely to encode factors affecting several different cellular processes, including translation initiation, mRNA stability and possibly transcription start site selection.

“Chromosome conformation capture” (3C) is a powerful method to detect physical interaction between any two genomic loci. 3C involves formaldehyde crosslinking to stabilize transient interactions, followed by restriction digestion, ligation and locus-specific PCR. Accordingly, 3C reveals complex three-dimensional interactions between distal genetic elements within intact cells at high resolution. Here, we describe a modified 3C protocol designed for detection of transient chromatin interactions in the yeast Saccharomyces cerevisiae. Using this protocol, we are able to detect juxtaposition of promoter and terminator regions of genes with ORFs as short as 1 kb in length. We anticipate that this method will be generally applicable to detect dynamic, short-range chromatin interactions and will facilitate the characterization of gene loops and their functional consequences.

In an effort to identify novel components of the PHO regulon in Saccharomyces cerevisiae, we have isolated and characterized suppressors of the Pho(-) phenotype associated with deletion of the Pho4 transcriptional activator. Here we report that either a defective form of the Rsp5 E3 ubiquitin ligase or deletion of the End3 component of the endocytic pathway restores growth of the pho4 Delta mutant in the presence of limiting inorganic phosphate (P i). The spa1-1 suppressor allele of RSP5 encodes a phenylalanine-to-valine replacement at position 748 (F748V) within the catalytic HECT domain of Rsp5. Consistent with suppression due to impaired ubiquitin ligase activity, the heat-sensitive growth defect of the spa1-1 mutant is suppressed either by overexpression of ubiquitin or by osmotic stabilization. Western blot analyses revealed that the cellular levels of the Pho87 and Pho91 low affinity P i are markedly increased in the spa1-1 mutant, yet Pho84 high affinity P i transporter levels are unaffected. Furthermore, Pho87 and Pho91 are ubiquitinated in vivo in an Rsp5-dependent manner, and the Pho+ phenotype of the spa1-1 suppressor is dependent upon Pho87 and Pho91. We conclude that turnover of the low affinity P i transporters is initiated by Rsp5-mediated ubiquitination followed by internalization and degradation by the endocytic pathway.

Translation is a highly complex process that is regulated by a multitude of factors. Here, we show that the conserved kinase Ctk1 functions in translation by enhancing decoding fidelity. Ctk1 associates with translating ribosomes in vivo and is needed for efficient translation. Ctk1 phosphorylates Rps2, a protein of the small ribosomal subunit, on Ser 238. Importantly, Ctk1-depleted as well as rps2-S238A mutant cells show a defect in translation elongation through an increase in the frequency of miscoding. The role of Ctk1 in translation may be conserved as the mammalian homolog of Ctk1, CDK9, also associates with polysomes. Since Ctk1 interacts with the TREX (transcription and mRNA export) complex, which couples transcription to mRNA export, Ctk1/CDK9 might bind to correctly processed mRNPs during transcription and accompany the mRNP to the ribosomes in the cytoplasm, where Ctk1 enhances efficient and accurate translation of the mRNA.

The RNA polymerase II (RNAP II) transcription cycle is accompanied by changes in the phosphorylation status of the C-terminal domain (CTD), a reiterated heptapeptide sequence (Y(1)S(2)P(3)T(4)S(5)P(6)S(7)) present at the C terminus of the largest RNAP II subunit. One of the enzymes involved in this process is Ssu72, a CTD phosphatase with specificity for serine-5-P. Here we report that the ssu72-2-encoded Ssu72-R129A protein is catalytically impaired in vitro and that the ssu72-2 mutant accumulates the serine-5-P form of RNAP II in vivo. An in vitro transcription system derived from the ssu72-2 mutant exhibits impaired elongation efficiency. Mutations in RPB1 and RPB2, the genes encoding the two largest subunits of RNAP II, were identified as suppressors of ssu72-2. The rpb1-1001 suppressor encodes an R1281A replacement, whereas rpb2-1001 encodes an R983G replacement. This information led us to identify the previously defined rpb2-4 and rpb2-10 alleles, which encode catalytically slow forms of RNAP II, as additional suppressors of ssu72-2. Furthermore, deletion of SPT4, which encodes a subunit of the Spt4-Spt5 early elongation complex, also suppresses ssu72-2, whereas the spt5-242 allele is suppressed by rpb2-1001. These results define Ssu72 as a transcription elongation factor. We propose a model in which Ssu72 catalyzes serine-5-P dephosphorylation subsequent to addition of the 7-methylguanosine cap on pre-mRNA in a manner that facilitates the RNAP II transition into the elongation stage of the transcription cycle.

The TAFs are transcription factors associated with the TATA-binding protein, and until recently they were assumed to link specific activators to the general transcription machinery. Recent results suggest that the essential functions of TAFs are not as coactivators of transcription but as determinants of promoter selectivity.

The prevailing view of the RNA polymerase II (RNAP II) transcription cycle is that RNAP II is recruited to the promoter, transcribes a linear DNA template, then terminates transcription and dissociates from the template. Subsequent rounds of transcription are thought to require de novo recruitment of RNAP II to the promoter. Several recent findings, including physical interaction of 3′-end processing factors with both promoter and terminator regions, challenge this concept. Here we report a physical association of promoter and terminator regions of the yeast BUD3 and SEN1 genes. These interactions are transcription-dependent, require the Ssu72 and Pta1 components of the CPF 3′-end processing complex, and require the phosphatase activity of Ssu72. We propose a model for RNAP II transcription in which promoter and terminator regions are juxtaposed, and that the resulting gene loops facilitate transcription reinitiation by the same molecule of RNAP II in a manner dependent upon Ssu72-mediated CTD dephosphorylation.

Negative regulatory elements (NREs) are deoxyribonucleic acid (DNA) sequences that repress gene expression. NREs can act locally to repress expression of individual genes, or globally to repress expression of an expansive chromosomal domain. In either case, NREs act as binding sites for specific proteins, which in turn interact with other factors either to block recruitment of ribonucleic acid (RNA) polymerase or to facilitate formation of heterochromatin. Keywords: chromatin; repression; silencing; transcription

The phosphorylated carboxyl-terminal domain (CTD) of RNA polymerase II, consisting of ((1)YSPTSPS(7))(n) heptad repeats, encodes information about the state of the transcriptional apparatus that can be conveyed to factors that regulate mRNA synthesis and processing. Here we describe how the CTD code is read by two classes of protein phosphatases, plant CPLs and yeast Ssu72, that specifically dephosphorylate Ser(5) in vitro. The CPLs and Ssu72 recognize entirely different positional cues in the CTD primary structure. Whereas the CPLs rely on Tyr(1) and Pro(3) located on the upstream side of the Ser(5)-PO(4) target site, Ssu72 recognizes Thr(4) and Pro(6) flanking the target Ser(5)-PO(4) plus the downstream Tyr(1) residue of the adjacent heptad. We surmise that the reading of the CTD code does not obey uniform rules with respect to the location and phasing of specificity determinants. Thus, CTD code, like the CTD structure, is plastic.

We used site-specific protein-DNA photo-cross-linking to define contact points between Saccharomyces cerevisiae RNA polymerase II (RNAP II) and the general transcription factors TBP, TFIIB, and TFIIF on promoter DNA. We present three key findings: (i) the overall pattern of cross-link sites is remarkably similar between the yeast and the previously described human system, even though transcription initiates downstream of the DNA-TBP-TFIIB-RNAP II-TFIIF complex in the S. cerevisiae system; (ii) the yeast Rpb7 subunit of RNAP II forms strong and reproducible cross-links to both strands of promoter DNA; and (iii) a TFIIB arginine-78 to cysteine replacement (R78C), which shifts start site selection downstream of normal, does not affect TFIIB-DNA cross-links prior to promoter melting but instead affects downstream TFIIF-DNA interactions. These results support the premise that the overall structure of the RNAP II preinitiation complex is similar in all eukaryotes and imply that yeast RNAP II is able to scan template DNA downstream of the preinitiation complex for acceptable start sites. The novel Rpb7-DNA contact sites imply that either promoter DNA does not follow a straight path from TATA to the initiation site or the topology of Rpb7 within the DNA-TBP-TFIIB-RNAP II-TFIIF complex is different from that defined in the 12-subunit RNAP II X-ray structure. We discuss the implications of these results for the mechanism of preinitiation complex assembly and promoter melting.

Histone lysine methylation plays a key role in the organization of chromatin structure and the regulation of gene expression. Recent studies demonstrated that the yeast Set1 and Set2 histone methyltransferases are recruited to mRNA coding regions by the PAF transcription elongation complex in a manner dependent upon the phosphorylation state of the carboxy-terminal domain of RNA polymerase II. These studies define an unexpected link between transcription elongation and histone methylation.

The textbook illustration of a gene depicts a linear structure, with flanking regulatory sequences—a promoter on the left and a terminator on the right, to start and stop transcription, respectively. However, recent analyses of chromatin architecture have revealed that the promoter and terminator elements are not necessarily separate in three-dimensional space, but can be juxtaposed to form “gene loops” ( 1 , 2 ). Gene loops are not static structures; they form transiently in a transcription-dependent manner. But what function do they serve? On page 671 of this issue, Tan-Wong et al. report that gene loops restrict divergent transcription from inherently bidirectional promoters, repressing the synthesis of noncoding RNA ( 3 ).