Saccharomyces Cerevisiae RNA Research Articles

Characterization of RNA polymerase II trigger loop mutations using molecular dynamics simulations and machine learning.

Catalysis and fidelity of multisubunit RNA polymerases rely on a highly conserved active site domain called the trigger loop (TL), which achieves roles in transcription through conformational changes and interaction with NTP substrates. The mutations of TL residues cause distinct effects on catalysis including hypo- and hyperactivity and altered fidelity. We applied molecular dynamics simulation (MD) and machine learning (ML) techniques to characterize TL mutations in the Saccharomyces cerevisiae RNA Polymerase II (Pol II) system. We did so to determine relationships between individual mutations and phenotypes and to associate phenotypes with MD simulated structural alterations. Using fitness values of mutants under various stress conditions, we modeled phenotypes along a spectrum of continual values. We found that ML could predict the phenotypes with 0.68 R2 correlation from amino acid sequences alone. It was more difficult to incorporate MD data to improve predictions from machine learning, presumably because MD data is too noisy and possibly incomplete to directly infer functional phenotypes. However, a variational auto-encoder model based on the MD data allowed the clustering of mutants with different phenotypes based on structural details. Overall, we found that a subset of loss-of-function (LOF) and lethal mutations tended to increase distances of TL residues to the NTP substrate, while another subset of LOF and lethal substitutions tended to confer an increase in distances between TL and bridge helix (BH). In contrast, some of the gain-of-function (GOF) mutants appear to cause disruption of hydrophobic contacts among TL and nearby helices.

A novel narnavirus is widespread in Saccharomyces cerevisiae and impacts multiple host phenotypes.

RNA viruses are a widespread, biologically diverse group that includes the narnaviridiae, a family of unencapsidated RNA viruses containing a single ORF that encodes an RNA-dependent RNA polymerase. In the yeast Saccharomyces cerevisiae, the 20S and 23S RNA viruses are well-studied members of the narnaviridiae, which are present at low intracellular copy numbers, unless induced by stress or unfavorable growth conditions (Wickneret al. 2013), and are not known to affect host fitness. In this study, we describe a new Saccharomyces cerevisiae narnavirus that we designate as N1199. We show that N1199 is uniquely present as a double-stranded RNA at a high level relative to other known members of this family in one strain background, YJM1199, and is present as a single-stranded RNA at lower levels in 98 of the remaining 100-genomes strains. Further, we see a strong association between the presence of high level N1199 and host phenotype defects, including greatly reduced sporulation efficiency and growth on multiple carbon sources. Finally, we describe associations between N1199 abundance and host phenotype defects, including autophagy.

Saccharomyces cerevisiae RNA lariat debranching enzyme, Dbr1p, is required for completion of reverse transcription by the retrovirus-like element Ty1 and cleaves branched Ty1 RNAs.

RNA debranching enzymes are 2'-5' phosphodiesterases found in all eukaryotes. Their main role is cleavage of intron RNA lariat branch points, promoting RNA turnover via exonucleases. Consistent with this role, cells with reduced RNA debranching enzyme activity accumulate intron RNA lariats. The Saccharomyces cerevisiae RNA debranching enzyme Dbr1p is also a host factor for the yeast long terminal repeat (LTR) retrotransposon Ty1, a model for many aspects of retroviral replication. Fittingly, the human RNA debranching enzyme Dbr1 is a host factor for the human immunodeficiency virus, HIV-1. The yeast and human RNA debranching enzymes act at the reverse transcription stages for Ty1 and HIV-1, respectively. Although efficient production of full-length Ty1 cDNA requires Dbr1p, the findings reported here indicate that production of the earliest distinct cDNA product, minus strand strong stop DNA (-sssDNA), is equivalent in wild type and dbr1∆ mutant cells. Several branched Ty1 RNAs are shown to accumulate in dbr1∆ cells during retrotransposition. These data are consistent with creation of Ty1 RNA branches prior to Ty1 reverse transcription and their removal by Dbr1p to allow efficient extension of early cDNA products. The data support the possibility that RNA branch formation and cleavage play broadly shared, but unknown roles in retroviral and LTR retrotransposon reverse transcription.

The RNA binding protein Npl3 promotes resection of DNA double-strand breaks by regulating the levels of Exo1.

Eukaryotic cells preserve genome integrity upon DNA damage by activating a signaling network that promotes DNA repair and controls cell cycle progression. One of the most severe DNA damage is the DNA double-strand break (DSB), whose 5΄ ends can be nucleolitically resected by multiple nucleases to create 3΄-ended single-stranded DNA tails that trigger DSB repair by homologous recombination. Here, we identify the Saccharomyces cerevisiae RNA binding protein Npl3 as a new player in DSB resection. Npl3 is related to both the metazoan serine-arginine-rich and the heterogeneous nuclear ribonucleo-proteins. NPL3 deletion impairs the generation of long ssDNA tails at the DSB ends, whereas it does not exacerbate the resection defect of exo1Δ cells. Furthermore, either the lack of Npl3 or the inactivation of its RNA-binding domains causes decrease of the exonuclease Exo1 protein levels as well as generation of unusual and extended EXO1 RNA species. These findings, together with the observation that EXO1 overexpression partially suppresses the resection defect of npl3Δ cells, indicate that Npl3 participates in DSB resection by promoting the proper biogenesis of EXO1 mRNA.

High-Resolution Phenotypic Landscape of the RNA Polymerase II Trigger Loop.

The active sites of multisubunit RNA polymerases have a “trigger loop” (TL) that multitasks in substrate selection, catalysis, and translocation. To dissect the Saccharomyces cerevisiae RNA polymerase II TL at individual-residue resolution, we quantitatively phenotyped nearly all TL single variants en masse. Three mutant classes, revealed by phenotypes linked to transcription defects or various stresses, have distinct distributions among TL residues. We find that mutations disrupting an intra-TL hydrophobic pocket, proposed to provide a mechanism for substrate-triggered TL folding through destabilization of a catalytically inactive TL state, confer phenotypes consistent with pocket disruption and increased catalysis. Furthermore, allele-specific genetic interactions among TL and TL-proximal domain residues support the contribution of the funnel and bridge helices (BH) to TL dynamics. Our structural genetics approach incorporates structural and phenotypic data for high-resolution dissection of transcription mechanisms and their evolution, and is readily applicable to other essential yeast proteins.

Transcription Start Site Scanning and the Requirement for ATP during Transcription Initiation by RNA Polymerase II

Saccharomyces cerevisiae RNA polymerase (Pol) II locates transcription start sites (TSS) at TATA-containing promoters by scanning sequences downstream from the site of preinitiation complex formation, a process that involves the translocation of downstream promoter DNA toward Pol II. To investigate a potential role of yeast Pol II transcription in TSS scanning, HIS4 promoter derivatives were generated that limited transcripts in the 30-bp scanned region to two nucleotides in length. Although we found that TSS scanning does not require RNA synthesis, our results revealed that transcription in the purified yeast basal system is largely ATP-independent despite a requirement for the TFIIH DNA translocase subunit Ssl2. This result is rationalized by our finding that, although they are poorer substrates, UTP and GTP can also be utilized by Ssl2. ATPγS is a strong inhibitor of rNTP-fueled translocation, and high concentrations of ATPγS make transcription completely dependent on added dATP. Limiting Pol II function with low ATP concentrations shifted the TSS position downstream. Combined with prior work, our results show that Pol II transcription plays an important role in TSS selection but is not required for the scanning reaction.

A long and winding road to the RNA world.

A long and winding road to the RNA world.

RNA-processing proteins regulate Mec1/ATR activation by promoting generation of RPA-coated ssDNA.

Eukaryotic cells respond to DNA double-strand breaks (DSBs) by activating a checkpoint that depends on the protein kinases Tel1/ATM and Mec1/ATR. Mec1/ATR is activated by RPA-coated single-stranded DNA (ssDNA), which arises upon nucleolytic degradation (resection) of the DSB. Emerging evidences indicate that RNA-processing factors play critical, yet poorly understood, roles in genomic stability. Here, we provide evidence that the Saccharomyces cerevisiae RNA decay factors Xrn1, Rrp6 and Trf4 regulate Mec1/ATR activation by promoting generation of RPA-coated ssDNA. The lack of Xrn1 inhibits ssDNA generation at the DSB by preventing the loading of the MRX complex. By contrast, DSB resection is not affected in the absence of Rrp6 or Trf4, but their lack impairs the recruitment of RPA, and therefore of Mec1, to the DSB. Rrp6 and Trf4 inactivation affects neither Rad51/Rad52 association nor DSB repair by homologous recombination (HR), suggesting that full Mec1 activation requires higher amount of RPA-coated ssDNA than HR-mediated repair. Noteworthy, deep transcriptome analyses do not identify common misregulated gene expression that could explain the observed phenotypes. Our results provide a novel link between RNA processing and genome stability.

Refined secondary-structure models of the core of yeast and human telomerase RNAs directed by SHAPE.

Telomerase catalyzes the addition of nucleotides to the ends of chromosomes to complete genomic DNA replication in eukaryotes and is implicated in multiple diseases, including most cancers. The core enzyme is composed of a reverse transcriptase and an RNA subunit, which provides the template for DNA synthesis. Despite extensive divergence at the sequence level, telomerase RNAs share several structural features within the catalytic core, suggesting a conserved enzyme mechanism. We have investigated the structure of the core of the human and yeast telomerase RNAs using SHAPE, which interrogates flexibility of each nucleotide. We present improved secondary-structure models, refined by addition of five base triples within the yeast pseudoknot and an alternate pairing within the human-specific element J2a.1 in the human pseudoknot, both of which have implications for thermodynamic stability. We also identified a potentially structured CCC region within the template that may facilitate substrate binding and enzyme mechanism. Overall, the SHAPE findings reveal multiple similarities between the Saccharomyces cerevisiae and Homo sapiens telomerase RNA cores.

Increased transcription of RPL40A and RPL40B is important for the improvement of RNA production in Saccharomyces cerevisiae

Yeast (Saccharomyces cerevisiae) RNA is an important source of 5'-ribonucleotides that is used in both the food and pharmaceutical industries. Efficient transcription of rDNA is very important to construct yeast strains with high RNA content. The gene RRN10, which encodes, a component of the upstream activation factor, is essential to promote high-level transcription of rDNA. In our previous study, we isolated SupE strain as a dominant suppressor, which showed the ability to restore the severe growth defects and reduced RNA content caused by disruption of the RRN10 gene. SupE strain has multiple mutations which we designated collectively as SUPE. Further analysis on SUPE mutation indicated that RPL40A was responsible for suppression of defect caused by rrn10 disruption. However, there were no base changes in this gene as compared with the parental Δrrn10 strain, thus suggesting that an additional copy of RPL40A suppress the defects caused by Δrrn10 disruption, and that, in SupE strain, these defects are suppressed by increased transcription of RPL40A whose copy is doubled. When multiple copies of RPL40A were combined with SUPE mutation on an RRN10⁺ background, the resultant SupE strain had significantly higher RNA content than wild-type strain. In addition, increased transcription of RPL40B also showed significant effect to restore the growth defect and reduced RNA content caused by Δrrn10 disruption. We propose a model to explain how SUPE mutation increases the transcription of ribosomal protein genes such as RPL40A and RPL40B in SupE strain, resulting in an increase in RNA content.

Optimization of yeast (Saccharomyces cerevisiae) RNA isolation method for real-time quantitative PCR and microarray analysis

Quality of the starting RNA is indispensably important for obtaining highly reproduciblequantitative polymerase chain reaction (qPCR) and microarray results for all organisms as well as S. cerevisiae. Isolating RNA from yeast cells with a maximum quality was especially critical since these cells were rich in polysaccharides and proteins. The method has been optimized through modification pretreatment applications for the isolation of S. cerevisiae RNA for qPCR and microarray analysis. Two extraction assay a TRIzol reagent-based method with three pretreatment applications and a commercially available kit with own pretreatment application, were compared for this purpose. Furthermore, the concentration yeast cells and enzyme were controlled in the range of 2 × 106 to 6 × 107 cells and 0.5 to 5 mg/ml, respectively to prevent RNA yields decrease and RNA degradation. Results of RNA isolation of the middle scales of yeast cells and enzyme concentrations which obtained fluxional deduct were not discussed here. Concentration and integrity of RNA samples were determined by µL spectrophotometer and Bioanalyzer, respectively. Quality of cDNA prepared from RNA samples was inspected with amplification using 18SrRNA primers in qPCR reactions. Furthermore, quality of RNA samples were evaluated using quality control parameters associated with performance of the assay and hybridization in microarray experiments. The highest yield and quality of RNA, which was appropriate for reverse transcription, cDNA library construction, qPCR reactions and microarray hybridizations without further processing was obtained by using Protocol C which used highest yeast cell and enzyme concentrations during the pretreatment application. Key words: RNA isolation, Saccharomyces cerevisiae, quantitative PCR, microarray.

Study of the essentiality of the Aspergillus fumigatus triA gene, encoding RNA triphosphatase, using the heterokaryon rescue technique and the conditional gene expression driven by the alcA and niiA promoters

The identification of essential genes represents a critical step in the discovery of novel therapeutic targets in Aspergillus fumigatus. Structural analyses of the Saccharomyces cerevisiae RNA triphosphatase pointed out this enzyme as an attractive therapeutic target for fungal infections. In addition, demonstration of the essentiality of the S. cerevisiae RNA triphosphatase encoding gene enhanced the value of this potential therapeutic target. Nevertheless, consideration of a fungal RNA triphosphatase as an ideal therapeutic target needs confirmation of the essentiality of the respective gene in a fungal pathogen. In this work, we analyzed the essentiality of the A. fumigatus triA gene, encoding RNA triphosphatase, by conditional gene expression and heterokaryon deletion. Using the conditional gene expression driven by the alcA promoter ( alcA P), we found that TriA depletion causes morphological abnormalities that result in a very strong growth inhibition. Nevertheless, since a strict terminal phenotype was not observed, the essentiality of the triA gene could not be ensured. Accordingly, the essentiality of this gene was analyzed by the heterokaryon rescue technique. Results obtained unequivocally demonstrated the essentiality of the A. fumigatus triA gene, indicating the suitability of the RNA triphosphatase as an ideal therapeutic target to treat A. fumigatus infections. Besides, a second conditional gene expression system, based on the niiA promoter ( niiA P), was utilized in this work. Although the niiA P-mediated repression of triA was less severe than that driven by the alcA P, a strong growth inhibition was also found in niiA P- triA strains. Finally, E-tests performed to determine whether triA down-regulated cells became more sensitive to antifungals suggest a synergic effect between amphotericin B and another antifungal inhibiting the A. fumigatus RNA triphosphatase activity.

Direct RNA sequencing

Our understanding of human biology and disease is ultimately dependent on a complete understanding of the genome and its functions. The recent application of microarray and sequencing technologies to transcriptomics has changed the simplistic view of transcriptomes to a more complicated view of genome-wide transcription where a large fraction of transcripts emanates from unannotated parts of genomes, and underlined our limited knowledge of the dynamic state of transcription. Most of this broad body of knowledge was obtained indirectly because current transcriptome analysis methods typically require RNA to be converted to complementary DNA (cDNA) before measurements, even though the cDNA synthesis step introduces multiple biases and artefacts that interfere with both the proper characterization and quantification of transcripts. Furthermore, cDNA synthesis is not particularly suitable for the analysis of short, degraded and/or small quantity RNA samples. Here we report direct single molecule RNA sequencing without prior conversion of RNA to cDNA. We applied this technology to sequence femtomole quantities of poly(A)(+) Saccharomyces cerevisiae RNA using a surface coated with poly(dT) oligonucleotides to capture the RNAs at their natural poly(A) tails and initiate sequencing by synthesis. We observed transcript 3' end heterogeneity and polyadenylated small nucleolar RNAs. This study provides a path to high-throughput and low-cost direct RNA sequencing and achieving the ultimate goal of a comprehensive and bias-free understanding of transcriptomes.

RNA Sequence and Two-dimensional Structure Features Required for Efficient Substrate Modification by the Saccharomyces cerevisiae RNA:Ψ-Synthase Pus7p

The RNA:pseudouridine (Psi) synthase Pus7p of Saccharomyces cerevisiae is a multisite-specific enzyme that is able to modify U(13) in several yeast tRNAs, U(35) in the pre-tRNA(Tyr) (GPsiA), U(35) in U2 small nuclear RNA, and U(50) in 5 S rRNA. Pus7p belongs to the universally conserved TruD-like family of RNA:Psi-synthases found in bacteria, archaea, and eukarya. Although several RNA substrates for yeast Pus7p have been identified, specificity of their recognition and modification has not been studied. However, conservation of a 7-nt-long sequence, including the modified U residue, in all natural Pus7p substrates suggested the importance of these nucleotides for Pus7p recognition and/or catalysis. Using site-directed mutagenesis, we designed a set of RNA variants derived from the yeast tRNA(Asp)(GUC), pre-tRNA(Tyr)(GPsiA), and U2 small nuclear RNA and tested their ability to be modified by Pus7p in vitro. We demonstrated that the highly conserved U(-2) and A(+1) residues (nucleotide numbers refer to target U(0)) are crucial identity elements for efficient modification by Pus7p. Nucleotide substitutions at other surrounding positions (-4, -3, +2, +3) have only a moderate effect. Surprisingly, the identity of the nucleotide immediately 5' to the target U(0) residue (position -1) is not important for efficient modification. Alteration of tRNA three-dimensional structure had no detectable effect on Pus7p activity at position 13. However, our results suggest that the presence of at least one stem-loop structure including or close to the target U nucleotide is required for Pus7p-catalyzed modification.

Polyphosphatase Activity of CthTTM, a Bacterial Triphosphate Tunnel Metalloenzyme

Triphosphate tunnel metalloenzymes (TTMs) are a superfamily of phosphotransferases with a distinctive active site located within an eight-stranded beta barrel. The best understood family members are the eukaryal RNA triphosphatases, which catalyze the initial step in mRNA capping. The RNA triphosphatases characteristically hydrolyze nucleoside 5'-triphosphates in the presence of manganese and are inept at cleaving inorganic tripolyphosphate. We recently identified a TTM protein from the bacterium Clostridium thermocellum (CthTTM) with the opposite substrate preference. Here we report that CthTTM catalyzes hydrolysis of guanosine 5'-tetraphosphate to yield GTP and P(i) (K(m) = 70 microm, k(cat) = 170 s(-1)) much more effectively than it converts GTP to GDP and P(i) (K(m) = 70 microm, k(cat) = 0.3 s(-1)), implying that a nucleoside interferes when positioned too close to the tunnel entrance. CthTTM is capable of quantitatively cleaving diadenosine hexaphosphate but has feeble activity with shorter derivatives diadenosine tetraphosphate and diadenosine pentaphosphate. We propose that the tunnel opens to accommodate the dumbbell-shaped diadenosine hexaphosphate and then closes around it to perform catalysis. We find that CthTTM can exhaustively hydrolyze a long-chain inorganic polyphosphate, a molecule that plays important roles in bacterial physiology. CthTTM differs from other known polyphosphatases in that it yields a approximately 2:1 mixture of P(i) and PP(i) end products. Bacterial/archaeal TTMs have a C-terminal helix located near the tunnel entrance. Deletion of this helix from CthTTM exerts pleiotropic effects. (i) It suppresses hydrolysis of guanosine 5'-tetraphosphate and inorganic PPP(i); (ii) it stimulates NTP hydrolysis; and (iii) it biases the outcome of the long-chain polyphosphatase reaction more strongly in favor of P(i) production. We discuss models for substrate binding in the triphosphate tunnel.

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.

The RNA Polymerase II Trigger Loop Functions in Substrate Selection and Is Directly Targeted by α-Amanitin

Structural, biochemical, and genetic studies have led to proposals that a mobile element of multisubunit RNA polymerases, the Trigger Loop (TL), plays a critical role in catalysis and can be targeted by antibiotic inhibitors. Here we present evidence that the Saccharomyces cerevisiae RNA Polymerase II (Pol II) TL participates in substrate selection. Amino acid substitutions within the Pol II TL preferentially alter substrate usage and enzyme fidelity, as does inhibition of transcription by alpha-amanitin. Finally, substitution of His1085 in the TL specifically renders Pol II highly resistant to alpha-amanitin, indicating a functional interaction between His1085 and alpha-amanitin that is supported by rerefinement of an alpha-amanitin-Pol II crystal structure. We propose that alpha-amanitin-inhibited Pol II elongation, which is slow and exhibits reduced substrate selectivity, results from direct alpha-amanitin interference with the TL.

The Saccharomyces cerevisiae RNA polymerase III recruitment factor subunits Brf1 and Bdp1 impose a strict sequence preference for the downstream half of the TATA box

Association of the TATA-binding protein (TBP) with its cognate site within eukaryotic promoters is key to accurate and efficient transcriptional initiation. To achieve recruitment of Saccharomyces cerevisiae RNA polymerase III, TBP is associated with two additional factors, Brf1 and Bdp1, to form the initiation factor TFIIIB. Previous data have suggested that the structure or dynamics of the TBP–DNA complex may be altered upon entry of Brf1 and Bdp1 into the complex. We show here, using the altered specificity TBP mutant TBPm3 and an iterative in vitro selection assay, that entry of Brf1 and Bdp1 into the complex imposes a strict sequence preference for the downstream half of the TATA box. Notably, the selected sequence (TGTAAATA) is a perfect match to the TATA box of the RNA polymerase III-transcribed U6 small nuclear RNA (SNR6) gene. We suggest that the selected T•A base pair step at the downstream end of the 8 bp TBP site may provide a DNA flexure that promotes TFIIIB-DNA complex formation.

Interaction of RNA polymerase II general transcription complex proteins with oligoribonucleotides

Saccharomyces cerevisiae RNA polymerase II general transcription complex proteins (RNA polymerase II, TBP, TFIIB, TFIIF, TFIIE, and TFIIH) were tested for interaction with oligoribonucleotides. Electrophoretic mobility shift assays showed that 32P-labeled CU-rich oligonucleotide p-17 (5′-ACUCUCUUCCG-CAUCGC- 3′) interacted with the proteins to yield three types of complexes, corresponding to three retardation bands. All complexes were RNA-specific, because [32P]p-17 was displaced by an excess of nonlabeled S. cerevisiae RNA and was not displaced by a 500-fold molar excess of a nonlabeled oligodeoxyribonucleotide. The complexes corresponding to the middle retardation band were specific for p-17, because [32P]p-17 was displaced almost completely by a 100-fold molar excess of nonlabeled p-17 and was not displaced by a 500-fold molar excess of an AU-rich oligoribonucleotide. The complexes corresponding to the upper retardation band were RNA-specific, and those corresponding to the lower band were assumed to result from nonspecific sorption of [32P]p-17 on a protein. Oligoribonucleotides and oligodeoxyribonucleotides did not compete for the binding with RNA polymerase II general transcription complex proteins.

High-resolution protein-DNA contacts for the yeast RNA polymerase II general transcription machinery.

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.