Yeast RNA Polymerase Research Articles

Mapping RNA exit channel on transcribing RNA polymerase II by FRET analysis

A simple genetic tag-based labeling method that permits specific attachment of a fluorescence probe near the C terminus of virtually any subunit of a protein complex is implemented. Its immediate application to yeast RNA polymerase II (pol II) enables us to test various hypotheses of RNA exit channel by using fluorescence resonance energy transfer (FRET) analysis. The donor dye is labeled on a site near subunit Rpb3 or Rpb4, and the acceptor dye is attached to the 5' end of RNA transcript in the pol II elongation complex. Both in-gel and single-molecule FRET analysis show that the growing RNA is leading toward Rpb4, not Rpb3, supporting the notion that RNA exits through the proposed channel 1. Distance constraints derived from our FRET results, in conjunction with triangulation, reveal the exit track of RNA transcript on core pol II by identifying amino acids in the vicinity of the 5' end of RNA and show that the extending RNA forms contacts with the Rpb7 subunit. The significance of RNA exit route in promoter escape and that in cotranscriptional mRNA processing is discussed.

The Rat1p 5′ to 3′ Exonuclease Degrades Telomeric Repeat-Containing RNA and Promotes Telomere Elongation in Saccharomyces cerevisiae

Vertebrate telomeres are transcribed into telomeric repeat-containing RNA (TERRA) that associates with telomeres and may be important for telomere function. Here, we demonstrate that telomeres are also transcribed in Saccharomyces cerevisiae by RNA polymerase II (RNAPII). Yeast TERRA is polyadenylated and stabilized by Pap1p and regulated by the 5' to 3' exonuclease, Rat1p. rat1-1 mutant cells accumulate TERRA and harbor short telomeres because of defects in telomerase-mediated telomere elongation. Overexpression of RNaseH overcomes telomere elongation defects in rat1-1 cells, indicating that RNA/DNA hybrids inhibit telomerase function at chromosome ends in these mutants. Thus, telomeric transcription combined with Rat1p-dependent TERRA degradation is important for regulating telomerase in yeast. Telomere transcription is conserved in different kingdoms of the eukaryotic domain.

Histone N-terminal Tails Interfere with Nucleosome Traversal by RNA Polymerase II

We determined the effect of the N-terminal histone tails on nucleosome traversal by yeast and human RNA polymerase II (pol II). Removal of H2A/H2B tails, H3/H4 tails, or all tails increased complete traversal of the nucleosome by human pol II, although the increase varied considerably depending on the template and on which tails were removed. Human pol II achieved >80% traversal of one nucleosomal template lacking the H2A/H2B tails, but even in those reactions, the transcript elongation rate was lower than the rate on pure DNA templates. For yeast pol II, transcription proceeded much farther into the nucleosome in the absence of tails, but complete read-through was not substantially increased by tail removal. Transcription factor IIS provided roughly the same level of read-through stimulation for transcript elongation in the presence or absence of tails. FACT also stimulated elongation on nucleosomal templates, and this effect was similar regardless of the presence of tails. For both polymerases, removal of the H2A/H2B tails reduced pausing throughout the nucleosome, suggesting that histone tails affect a common step at most points during nucleosome traversal. We conclude that histone tails provide a significant part of the nucleosomal barrier to pol II transcript elongation.

A nano-positioning system for macromolecular structural analysis

Very often, the positions of flexible domains within macromolecules as well as within macromolecular complexes cannot be determined by standard structural biology methods. To overcome this problem, we developed a method that uses probabilistic data analysis to combine single-molecule measurements with X-ray crystallography data. The method determines not only the most likely position of a fluorescent dye molecule attached to the domain but also the complete three-dimensional probability distribution depicting the experimental uncertainty. With this approach, single-pair fluorescence resonance energy transfer measurements can now be used as a quantitative tool for investigating the position and dynamics of flexible domains within macromolecular complexes. We applied this method to find the position of the 5' end of the nascent RNA exiting transcription elongation complexes of yeast (Saccharomyces cerevisiae) RNA polymerase II and studied the influence of transcription factor IIB on the position of the RNA.

Genome-associated RNA Polymerase II Includes the Dissociable Rpb4/7 Subcomplex

Yeast RNA polymerase (Pol) II consists of a 10-subunit core enzyme and the Rpb4/7 subcomplex, which is dispensable for catalytic activity and dissociates in vitro. To investigate whether Rpb4/7 is an integral part of DNA-associated Pol II in vivo, we used chromatin immunoprecipitation coupled to high resolution tiling microarray analysis. We show that the genome-wide occupancy profiles for Rpb7 and the core subunit Rpb3 are essentially identical. Thus, the complete Pol II associates with DNA in vivo, consistent with functional roles of Rpb4/7 throughout the transcription cycle.

Jamming the ratchet of transcription

The exact mechanism by which cellular RNA polymerases translocate and maintain exceptionally high fidelity during transcription remains an important unresolved issue. Two recent structural studies of yeast RNA polymerase II in complex with its potent inhibitor, the fungal toxin α-amanitin, address this matter by describing crucial and surprising details about the dynamic organization of the enzyme catalytic center.

Epigenetic Control of rDNA Loci in Response to Intracellular Energy Status

Intracellular energy balance is important for cell survival. In eukaryotic cells, the most energy-consuming process is ribosome biosynthesis, which adapts to changes in intracellular energy status. However, the mechanism that links energy status and ribosome biosynthesis is largely unknown. Here, we describe eNoSC, a protein complex that senses energy status and controls rRNA transcription. eNoSC contains Nucleomethylin, which binds histone H3 dimethylated Lys9 in the rDNA locus, in a complex with SIRT1 and SUV39H1. Both SIRT1 and SUV39H1 are required for energy-dependent transcriptional repression, suggesting that a change in the NAD(+)/NADH ratio induced by reduction of energy status could activate SIRT1, leading to deacetylation of histone H3 and dimethylation at Lys9 by SUV39H1, thus establishing silent chromatin in the rDNA locus. Furthermore, eNoSC promotes restoration of energy balance by limiting rRNA transcription, thus protecting cells from energy deprivation-dependent apoptosis. These findings provide key insight into the mechanisms of energy homeostasis in cells.

Genomewide Recruitment Analysis of Rpb4, a Subunit of Polymerase II in Saccharomyces cerevisiae , Reveals Its Involvement in Transcription Elongation

The Rpb4/Rpb7 subcomplex of yeast RNA polymerase II (Pol II) has counterparts in all multisubunit RNA polymerases from archaebacteria to higher eukaryotes. The Rpb4/7 subcomplex in Saccharomyces cerevisiae is unique in that it easily dissociates from the core, unlike the case in other organisms. The relative levels of Rpb4 and Rpb7 in yeasts affect the differential gene expression and stress response. Rpb4 is nonessential in S. cerevisiae and affects expression of a small number of genes under normal growth conditions. Here, using a chromatin immunoprecipitation ("ChIP on-chip") technique, we compared genomewide binding of Rpb4 to that of a core Pol II subunit, Rpb3. Our results showed that in spite of being nonessential for survival, Rpb4 was recruited on coding regions of most transcriptionally active genes, similar to the case with the core Pol II subunit, Rpb3, albeit to a lesser extent. The extent of Rpb4 recruitment increased with increasing gene length. We also observed Pol II lacking Rpb4 to be defective in transcribing long, GC-rich transcription units, suggesting a role for Rpb4 in transcription elongation. This role in transcription elongation was supported by the observed 6-azauracil (6AU) sensitivity of the rpb4Delta mutant. Unlike most phenotypes of rpb4Delta, the 6AU sensitivity of the rpb4Delta strain was not rescued by overexpression of RPB7. This report provides the first instance of a distinct role for Rpb4 in transcription, which is independent of its interacting partner, Rpb7.

Multiplexed Proteomics Mapping of Yeast RNA Polymerase II and III Allows Near-Complete Sequence Coverage and Reveals Several Novel Phosphorylation Sites

The multisubunit RNA polymerases (Pols) II and III synthesize mainly eukaryotic mRNAs and tRNAs, respectively. Pol II and Pol III are protein complexes consisting of 12 and 17 subunits. Here we analyzed both yeast Pol II and Pol III by multiplexed mass spectrometric analysis using various proteases and both collision induced and electron transfer dissociation. The cumulative data obtained from using the various proteases (trypsin, chymotrypsin, Glu-C and Lys-C) and the two peptide fragmentation approaches allowed us to map nearly the complete sequences of all constituents of both Pol II and III. Notably, chymotrypsin behaved equally well as and in certain circumstances better than trypsin in the context of protein coverage. Although the available high resolution structures have exposed extensive mechanistic insights into transcription, the role of post-translational modification in these processes has been addressed to a lesser extent. In our analysis of Pol II and III we detected 19 phosphorylation sites, of which 12 have not been previously reported. Identified phosphosites were mapped on the Pol II structure which provided indications that they might play a role in regulating the conformation of the clamp region and, as a consequence, interaction of Pol II with nucleic acids. The described multiplexed proteomics approach is generic and reveals that it is possible to map a protein complex to near completion while applying less than 5 mug (approximately 10 pmol) of total starting material.

Budding yeast RNA polymerases I and II employ parallel mechanisms of transcriptional termination

Both RNA polymerase I and II (Pol I and Pol II) in budding yeast employ a functionally homologous "torpedo-like" mechanism to promote transcriptional termination. For two well-defined Pol II-transcribed genes, CYC1 and PMA1, we demonstrate that both Rat1p exonuclease and Sen1p helicase are required for efficient termination by promoting degradation of the nascent transcript associated with Pol II, following mRNA 3' end processing. Similarly, Pol I termination relies on prior Rnt1p cleavage at the 3' end of the pre-rRNA 35S transcript. This is followed by the combined actions of Rat1p and Sen1p to degrade the Pol I-associated nascent transcript that consequently promote termination in the downstream rDNA spacer sequence. Our data suggest that the previously defined in vitro Pol I termination mechanism involving the action of the Reb1p DNA-binding factor to "road-block" Pol I transcription close to the termination region may have overlooked more complex in vivo molecular processes.

Characterization of the active site of yeast RNA polymerase II by DFT and ReaxFF calculations

Most known DNA-dependent RNA polymerases (RNAPs) share a universal heptapeptide, called the NAD- FDGD motif. The crystal structures of RNAPs indicate that in all cases this motif forms a loop with an embedded triad of aspartic acid residues. This conserved loop is the key part of the active site. Based on the crystal structures of the yeast RNAP II, we have studied this common active site for three cases: (1) single RNAP, (2) pre-translocation elon- gation complex, and (3) post-translocation elongation com- plex. Here we have applied two different modeling methods,

Site specific phosphorylation of yeast RNA polymerase I

All nuclear RNA polymerases are phosphoprotein complexes. Yeast RNA polymerase I (Pol I) contains approximately 15 phosphate groups, distributed to 5 of the 14 subunits. Information about the function of the single phosphosites and their position in the primary, secondary and tertiary structure is lacking. We used a rapid and efficient way to purify yeast RNA Pol I to determine 13 phosphoserines and –threonines. Seven of these phosphoresidues could be located in the 3D-homology model for Pol I, five of them are more at the surface. The single phosphorylated residues were systematically mutated and the resulting strains and Pol I preparations were analyzed in cellular growth, Pol I composition, stability and genetic interaction with non-essential components of the transcription machinery. Surprisingly, all Pol I phosphorylations analyzed were found to be non-essential post-translational modifications. However, one mutation (subunit A190 S685D) led to higher growth rates in the presence of 6AU or under environmental stress conditions, and was synthetically lethal with a deletion of the Pol I subunit A12.2, suggesting a role in RNA cleavage/elongation or termination. Our results suggest that individual major or constitutively phosphorylated residues contribute to non-essential Pol I-functions.

Structure–function analysis of the RNA polymerase cleft loops elucidates initial transcription, DNA unwinding and RNA displacement

The active center clefts of RNA polymerase (RNAP) from the archaeon Pyrococcus furiosus (Pfu) and of yeast RNAP II are nearly identical, including four protruding loops, the lid, rudder, fork 1 and fork 2. Here we present a structure–function analysis of recombinant Pfu RNAP variants lacking these cleft loops, and analyze the function of each loop at different stages of the transcription cycle. All cleft loops except fork 1 were required for promoter-directed transcription and efficient elongation. Unprimed de novo transcription required fork 2, the lid was necessary for primed initial transcription. Analysis of templates containing a pre-melted bubble showed that rewinding of upstream DNA drives RNA separation from the template. During elongation, downstream DNA strand separation required template strand binding to an invariant arginine in switch 2, and apparently interaction of an invariant arginine in fork 2 with the non-template strand.

RNA Polymerase I: A Multifunctional Molecular Machine

In this issue, Kuhn et al. (2007) report the complete structure of the 14-subunit yeast RNA polymerase (Pol) I enzyme at 12 A resolution using cryo-electron microscopy (cryo-EM). Their study reveals that three subunits of Pol I perform functions in transcription elongation that are outsourced to the transcription factors TFIIF and TFIIS in the analogous Pol II transcription system.

The Rpb7p subunit of yeast RNA polymerase II plays roles in the two major cytoplasmic mRNA decay mechanisms

The steady-state level of mRNAs is determined by the balance between their synthesis by RNA polymerase II (Pol II) and their decay. In the cytoplasm, mRNAs are degraded by two major pathways; one requires decapping and 5′ to 3′ exonuclease activity and the other involves 3′ to 5′ degradation. Rpb7p is a Pol II subunit that shuttles between the nucleus and the cytoplasm. Here, we show that Rpb7p is involved in the two mRNA decay pathways and possibly couples them. Rpb7p stimulates the deadenylation stage required for execution of both pathways. Additionally, Rpb7p is both an active component of the P bodies, where decapping and 5′ to 3′ degradation occur, and is capable of affecting the P bodies function. Moreover, Rpb7p interacts with the decapping regulator Pat1p in a manner important for the mRNA decay machinery. Rpb7p is also involved in the second pathway, as it stimulates 3′ to 5′ degradation. Our genetic analyses suggest that Rpb7p plays two distinct roles in mRNA decay, which can both be uncoupled from Rpb7p's role in transcription. Thus, Rpb7p plays pivotal roles in determining mRNA levels.

Human RNA polymerase II-associated factor complex: dysregulation in cancer

Genetic instabilities are believed to be one of the major causes of developing a cancer phenotype in humans. During the progression of cancer, aberrant expression of proteins, either owing to genetic (amplification, mutation and deletion) or epigenetic modifications (DNA methylation and histone deacetylation), contributes in different ways to the development of cancer. By differential screening analysis, an amplification of the 19q13 locus containing a novel pancreatic differentiation 2 (PD2) gene was identified. PD2 is the human homolog of the yeast RNA polymerase II-associated factor 1 (yPaf1) and is part of the human RNA polymerase II-associated factor (hPAF) complex. hPAF is comprised of five subunits that include PD2/hPaf1, parafibromin, hLeo1, hCtr9 and hSki8. This multifaceted complex was first identified in yeast (yPAF) and subsequently in Drosophila and human. Recent advances in the study on PAF have revealed various functions of the complex in human, which are similar to yPAF, including efficient transcription elongation, mRNA quality control and cell-cycle regulation. Although the precise function of this complex in cancer is not clearly known, some of its subunits have been linked to a malignant phenotype. Its core subunit, PD2/hPaf1, is amplified and overexpressed in many cancers. Further, an overexpression of PD2/hPaf1 results in the induction of a transformed phenotype, suggesting its possible involvement in tumorigenesis. The parafibromin subunit of the hPAF complex is a product of the HRPT-2 (hereditary hyperparathyroidism type 2) tumor suppressor gene, which is mutated in the germ line of hyperparathyroidism-jaw tumor patients. This review focuses on the functions of the PAF complex and its individual subunits, the interaction of the subunits with each other and/or with other molecules, and dysregulation of the complex, providing an insight into its potential involvement in the development of cancer.

Insights into Transcription Initiation and Termination from the Electron Microscopy Structure of Yeast RNA Polymerase III

RNA polymerase III (RNAPIII) synthesizes tRNA, 5S RNA, U6 snRNA, and other small RNAs. The structure of yeast RNAPIII, determined at 17 A resolution by cryo-electron microscopy and single-particle analysis, reveals a hand-like shape typical of RNA polymerases. Compared to RNAPII, RNAPIII is characterized by a bulkier stalk and by prominent features extending from the DNA binding cleft. We attribute the latter primarily to five RNAPIII-specific subunits, present as two distinct subcomplexes (C82/C34/C31 and C53/C37). Antibody labeling experiments localize the C82/C34/C31 subcomplex to the clamp side of the DNA binding cleft, consistent with its known role in transcription initiation. The C53/C37 subcomplex appears to be situated across the cleft, near the presumed location of downstream DNA, accounting for its role in transcription termination. Our structure rationalizes available mutagenesis and biochemical data and provides insights into RNAPIII-mediated transcription.

Identification and Characterization of a Schizosaccharomyces pombe RNA Polymerase II Elongation Factor with Similarity to the Metazoan Transcription Factor ELL

ELL family transcription factors activate the rate of transcript elongation by suppressing transient pausing by RNA polymerase II at many sites along the DNA. ELL-associated factors 1 and 2 (EAF1 and EAF2) bind stably to ELL family members and act as strong positive regulators of their transcription activities. Orthologs of ELL and EAF have been identified in metazoa, but it has been unclear whether such RNA polymerase II elongation factors are utilized in lower eukaryotes. Using bioinformatic and biochemical approaches, we have identified a new Schizosaccharomyces pombe RNA polymerase II elongation factor that is composed of two subunits designated SpELL and SpEAF, which share weak sequence similarity with members of the metazoan ELL and EAF families. Like mammalian ELL-EAF, SpELL-SpEAF stimulates RNA polymerase II transcription elongation and pyrophosphorolysis. In addition, like many yeast RNA polymerase II elongation factors, deletion of the SpELL gene renders S. pombe sensitive to the drug 6-azauracil. Finally, phylogenetic analyses suggest that the SpELL and SpEAF proteins are evolutionarily conserved in many fungi but not in Saccharomyces cerevisiae.

Functional organization of the Rpb5 subunit shared by the three yeast RNA polymerases.

Rpb5, a subunit shared by the three yeast RNA polymerases, combines a eukaryotic N-terminal module with a globular C-end conserved in all non-bacterial enzymes. Conditional and lethal mutants of the moderately conserved eukaryotic module showed that its large N-terminal helix and a short motif at the end of the module are critical in vivo. Lethal or conditional mutants of the C-terminal globe altered the binding of Rpb5 to Rpb1-β25/26 (prolonging the Bridge helix) and Rpb1-α44/47 (ahead of the Switch 1 loop and binding Rpb5 in a two-hybrid assay). The large intervening segment of Rpb1 is held across the DNA Cleft by Rpb9, consistent with the synergy observed for rpb5 mutants and rpb9Δ or its RNA polymerase I rpa12Δ counterpart. Rpb1-β25/26, Rpb1-α44/45 and the Switch 1 loop were only found in Rpb5-containing polymerases, but the Bridge and Rpb1-α46/47 helix bundle were universally conserved. We conclude that the main function of the dual Rpb5–Rpb1 binding and the Rpb9–Rpb1 interaction is to hold the Bridge helix, the Rpb1-α44/47 helix bundle and the Switch 1 loop into a closely packed DNA-binding fold around the transcription bubble, in an organization shared by the two other nuclear RNA polymerases and by the archaeal and viral enzymes.

Genome-Wide Distribution of Yeast RNA Polymerase II and Its Control by Sen1 Helicase

Functional engagement of RNA polymerase II (Pol II) with eukaryotic chromosomes is a fundamental and highly regulated biological process. Here we present a high-resolution map of Pol II occupancy across the entire yeast genome. We compared a wild-type strain with a strain bearing a substitution in the Sen1 helicase, which is a Pol II termination factor for noncoding RNA genes. The wild-type pattern of Pol II distribution provides unexpected insights into the mechanisms by which genes are repressed or silenced. Remarkably, a single amino acid substitution that compromises Sen1 function causes profound changes in Pol II distribution over both noncoding and protein-coding genes, establishing an important function of Sen1 in the regulation of transcription. Given the strong similarity of the yeast and human Sen1 proteins, our results suggest that progressive neurological disorders caused by substitutions in the human Sen1 homolog Senataxin may be due to misregulation of transcription.