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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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

In 1993, the isolation of the gene (HD) responsible for Huntington's disease offered hope for understanding the molecular basis of this dreadful neurodegenerative disease. But, the amino acid sequence of the encoded protein, huntingtin, offered few clues to its function, and expression of HD appeared to be ubiquitous, rather than limited to the central nervous system. Instead, the key to Huntington's disease seemed to lie in the sequence of HD alleles: all disease alleles include a CAG trinucleotide repeat expansion, encoding a polyglutamine (polyQ) tract within the mature protein. CAG repeat expansions have now been identified in seven other genes, all associated with fatal neurodegenerative diseases.Expanded polyQ tracts form insoluble aggregates resembling amyloid fibrils and can cause neurotoxic effects in divergent organisms, independent of protein context. But what are the cellular targets of polyQ, and how does this sequence cause neuronal dysfunction? An early clue was that polyQ-rich sequences are common among transcription factors. Furthermore, polyQ tracts interact directly with components of the transcriptional machinery, including factors involved in histone modification. In recent studies using mouse models, proteins with expanded polyQ tracts induced transcriptional dysregulation before the onset of neurological symptoms, suggesting a cause-and-effect relationship. Moreover, many of the affected genes are relevant to neuronal function. Could transcriptional dysregulation cause polyQ pathogenesis?To examine whether expanded polyQ tracts interfere with gene expression, Hughes et al. [1xAltered transcription in yeast expressing expanded polyglutamine. Hughes, R.E. et al. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13201–13206Crossref | PubMed | Scopus (99)See all References][1] performed DNA microarray analyses in yeast strains expressing nuclear-targeted, polyQ tracts. Transcriptional alterations were identified, including, most prominently, repression of a small set of genes, several of which are specific for phosphate metabolism. These changes are remarkably similar to the altered gene expression profiles associated with deletion of components of the SAGA histone acetyltransferase complex. Consistent with an effect of polyQ on histone acetylation, the promoter from one affected gene (PHO84) was repressed by polyQ, and this effect was offset by trichostatin A, a histone deacetylase inhibitor. These results not only support transcriptional dysregulation as a pathogenic mechanism of polyQ diseases, but substantiate the growing body of evidence that polyQ targets the phylogenetically conserved histone acetylation machinery.An important concern regarding polyQ-mediated transcriptional dysregulation as a cause of neuronal dysfunction is the issue of disease specificity. Although the CAG repeat expansion diseases are related, each exhibits distinct clinical and pathological differences. How does transcriptional dysregulation by expanded polyQ tracts account for these differences? Despite this caveat, the discovery that polyQ-mediated transcriptional defects seen in animal models might occur in yeast, and be mediated through conserved components of the transcriptional machinery, offers a powerful experimental system to address the mechanisms underlying polyQ-mediated neuronal dysfunction.

Aneuploidy refers to an increase or decrease in the normal chromosome complement by one or more chromosomes. A well-known case of aneuploidy in humans is trisomy for chromosome 21, the cause of Down syndrome. Changes in chromosome copy number are widely recognized as a class of mutation in human cells and the mechanisms leading to genome instability are the subject of intense investigation. The yeast Saccharomyces cerevisiae has provided especially fertile ground for studying mechanisms affecting chromosome stability. Nonetheless, aneuploidy is generally overlooked as a class of mutation in yeast. This is certain to change with publication of the recent paper by Hughes et al.1xWidespread aneuploidy revealed by DNA microarray expression profiling. Hughes, T.R. et al. Nat. Genet. 2000; 25: 333–337Crossref | PubMed | Scopus (332)See all References1, demonstrating that aneuploidy is more widespread than commonly assumed or even appreciated.Using whole-genome expression data from nearly 300 Saccharomyces cerevisiae deletion mutants, Hughes et al. discovered an unexpected similarity in transcript profiles for unrelated mutants. In itself, this result is not surprising; indeed, similar expression profiles from genome-wide analyses have been used to group genes of similar function. However, the shared regulation between two of the mutants in this study corresponded to enhanced expression of genes located specifically on chromosome VII. Closer inspection revealed that this effect could be explained by gene dosage, a consequence of aneuploidy for chromosome VII. This turns out to be neither an isolated incident nor due to genome instability mutations. Rather, 22 of 290 mutants were found to be aneuploid for one or more chromosomes and additional examples of chromosome-wide expression biases were found among independent profiling data. Remarkably, the extra chromosome in several cases included a paralog of the gene that had been deleted in the mutant, suggesting that aneuploidy was selected based on suppression of a growth defect associated with the deletion. Consistent with this idea, expression bias for an rpl20a mutant correlated with an increase in genomic DNA corresponding precisely to a segment of chromosome XV that is flanked by retrotransposon LTRs and includes the RPL20B paralog of RPL20A.The significance of this paper is far-reaching. First, it warns those performing whole-genome expression profiles to beware of spurious correlations due to aneuploidy. By the same token, these results underscore the potential of microarray analyses to readily detect DNA copy number variations, which are common among transformed human cells. Second, the observation that transcript abundance is altered for nearly every gene on the trisomic or monosomic chromosome implies that yeast has no dosage-compensation mechanism to normalize gene expression. Finally, this paper warns the yeast community about the potential for aneuploidy to either mask or suppress mutations. Widespread aneuploidy and its potential to confound mutant analyses will certainly cause many geneticists to take pause.

The RNA polymerase II transcription factor TFIID comprises the TATA-binding protein (TBP) and a dozen or so TBP-associated factors known as TAFs. TBP performs an essential and well-defined role in transcription, binding core promoter DNA and nucleating assembly of the initiation complex. By contrast, TAFs appear to facilitate transcription in a gene-specific manner, although this conclusion is controversial and the function of most TAFs remains elusive. The most complex of the TFIID subunits, denoted TAF250 in mammalian cells, has histone acetyltransferase and protein kinase activities. TAF250 also includes two bromodomains, a common structural feature among transcription and chromatin-remodeling factors. Interestingly, Taf145p, the yeast homolog of TAF250, has histone acetyltransferase activity, but lacks the protein kinase and bromodomains. This structural distinction suggested that yeast TFIID might lack specific functions associated with mammalian TFIID.Matangkasombut et al.1xBromodomain factor 1 corresponds to a missing piece of yeast TFIID. Matangkasombut, O. et al. Genes Dev. 2000; 14: 951–962PubMedSee all References now report that the yeast Bdf1p protein corresponds to the segment of TAF250 missing from Taf145p. Using Taf67p as the bait in a two-hybrid screen for interacting proteins, Bdf1p and its homolog Bdf2p were identified. Like TAF250, Bdf1p has protein kinase activity and includes two bromodomains. Thus, the yeast counterpart of human TAF250 appears to be split between Taf145p and Bdf1p. Consistent with this conclusion, Bdf1p is recruited to promoter DNA and is required for the expression of many, but not all, genes. Bdf1p interacts directly with Taf67p and coimmunoprecipitates with TFIID. Nonetheless, purified preparations of yeast TFIID have not been reported to include Bdf1p, implying that Bdf1p is not a bona fide TFIID component. Matangkasombut et al.1xBromodomain factor 1 corresponds to a missing piece of yeast TFIID. Matangkasombut, O. et al. Genes Dev. 2000; 14: 951–962PubMedSee all References speculate that acetylated histones might interact with the bromodomain of Bdf1p, which would, in turn, stimulate transcription by recruiting TFIID to the promoter.

Since the discovery in 1996 that histone acetylation and deacetylation are catalysed by transcription factors, the transcription and chromatin fields have merged. Indeed, the mechanisms and consequences of covalent histone modifications have taken center stage. Acetylation of the N-terminal tails of histones H3 and H4 has occupied the limelight, although N-terminal phosphorylation of H3 and its effect on chromosome dynamics has assumed a prominent role. Much less attention has been directed toward ubiquitination, ADP-ribosylation and methylation, histone modifications the functional consequences of which have remained obscure. This is about to change with recent reports of two novel histone modifications in yeast. Robzyk et al.1xRad6-dependent ubiquitination of histone H2B in yeast. Robzyk, K. et al. Science. 2000; 287: 501–504Crossref | PubMed | Scopus (450)See all References used a sensitive immunoassay to reinvestigate a decade-old report that yeast histones are not ubiquitinated. They report that histone H2B is in fact ubiquitinated, representing as much as 10% of total cellular H2B. A single molecule of ubiquitin is attached to a lysine residue (K123) near the C-terminus of H2B, catalysed by the ubiquitin-conjugating enzyme Rad6. A substitution of K123 eliminated H2B ubiquitination and resulted in mitotic and meiotic growth defects, demonstrating the functional relevance of histone ubiquitination in vivo. In the other paper, Tanny et al.2xAn enzymatic activity in the yeast Sir2 protein that is essential for gene silencing. Tanny, J.C. et al. Cell. 1999; 99: 735–745Abstract | Full Text | Full Text PDF | PubMedSee all References identified an enzymatic activity for Sir2, a protein essential for silencing in yeast. Remarkably, Sir2 was found to be an NAD-dependent ADP-ribosyltransferase, able to modify itself, as well as histones, in vitro. The enzymatic activity of Sir2 is required for silencing in vivo, but not for the association of Sir2 with chromatin. These results define a chromatin-associated enzymatic activity that is essential for silencing and raise the interesting possibility that silencing might be regulated by the cellular redox state. The histone substrate specificity of Sir2 and whether the Sir2 requirement for silencing is actually a consequence of histone ADP-ribosylation remain to be determined. Regardless, both papers highlight the diversity of covalent histone modifications and expand the repertoire of mechanisms controlling chromosome function in eukaryotic cells.

Understanding the mechanism and regulation of RNA synthesis is a key objective in molecular biology. In recent years a plethora of factors that regulate transcription has been identified. Most of these factors either facilitate or impair access of RNA polymerases to template DNA. Crosslinking and mutational studies have provided key information regarding the mechanism of RNA synthesis. Nonetheless, the structural basis of RNA synthesis has remained elusive. Until now, structural information for multisubunit RNA polymerases was limited to low-resolution electron crystallography of yeast and bacterial enzymes.We now enter a new era with high-resolution X-ray crystal structures for eukaryotic and bacterial RNA polymerases. Poglitsch et al.1xElectron crystal structure of an RNA polymerase II transcription elongation complex. Poglitsch, C.L. et al. Cell. 1999; 98: 791–798Abstract | Full Text | Full Text PDF | PubMed | Scopus (55)See all References1 have extended the low-resolution structure of yeast RNA polymerase II to an active transcription complex that includes template DNA and product RNA. In addition to identifying the catalytic region of the enzyme, this structure defines a novel path for the DNA template across one face of the enzyme. Furthermore, Fu et al.2xYeast RNA polymerase II at 5 A resolution. Fu, J. et al. Cell. 1999; 98: 799–810Abstract | Full Text | Full Text PDF | PubMed | Scopus (104)See all References2 have solved the X-ray structure of yeast RNA polymerase II in the absence of nucleic acid at 5 A resolution. Comparison of this structure with the transcriptionally active complex identified two flexible domains as potential DNA and RNA clamps. Zhang et al.3xCrystal structure of Thermus aquaticus core RNA polymerase at 3.3 A resolution. Zhang, G. et al. Cell. 1999; 98: 811–824Abstract | Full Text | Full Text PDF | PubMed | Scopus (548)See all References3 treat us to an even clearer view of the enzyme with the crystal structure of bacterial core RNA polymerase at 3.3 A resolution. This is truly a landmark achievement that defines the overall architecture of RNA polymerase, subunit structures and interactions, and the active center of the enzyme. The results from DNA and RNA crosslinking studies could also be mapped onto this structure to define components involved in DNA and RNA binding. These X-ray structures are consistent with the earlier electron crystallographic structures and most features are conserved between the bacterial and yeast enzymes. We can now anticipate seeing more refined structures of RNA polymerases and their complexes trapped at different stages of the transcription cycle. A detailed mechanistic understanding of this most fundamental process in gene expression is rapidly coming into focus.