Yeast RNA Polymerase Research Articles

BioEssays 7∕2014

BioEssaysVolume 36, Issue 7 Cover PictureFree Access BioEssays 7∕2014 First published: 05 June 2014 https://doi.org/10.1002/bies.201470071AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Graphical Abstract The pE-MAP technology allows for functional dissection of macromolecular assemblies by genetic interrogation of point mutants. Application to yeast RNA polymerase II has enabled functional assignment of its regions and uncovered details related to its catalytic activity. On pages 706–713 of this issue, Braberg et al. discuss the key applications of pE-MAP, as well as its potential use for personalized medicine when extended to human cells. Shown on the cover is RNA polymerase II with mutated residues highlighted as magenta colored spheres and a pE-MAP in place of the growing RNA strand. Cover by Hannes Braberg et al. Volume36, Issue7July 2014 RelatedInformation

BioEssays 7∕2014

BioEssaysVolume 36, Issue 7 Cover PictureFree Access BioEssays 7∕2014 First published: 05 June 2014 https://doi.org/10.1002/bies.201470071AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract The pE-MAP technology allows for functional dissection of macromolecular assemblies by genetic interrogation of point mutants. Application to yeast RNA polymerase II has enabled functional assignment of its regions and uncovered details related to its catalytic activity. On pages 706–713 of this issue, Braberg et al. discuss the key applications of pE-MAP, as well as its potential use for personalized medicine when extended to human cells. Shown on the cover is RNA polymerase II with mutated residues highlighted as magenta colored spheres and a pE-MAP in place of the growing RNA strand. Cover by Hannes Braberg et al. Volume36, Issue7July 2014 RelatedInformation

Unveiling translocation intermediates of RNA polymerase.

Translocation of RNA polymerase (RNAP) is a robust target for regulation of gene expression in prokaryotes and eukaryotes (1⇓–3). During elongation, RNAP frequently encounters a broad range of DNA/RNA conformations (RNA hairpins, curved and cruciform DNA), DNA-binding proteins, DNA lesions, and misincorporation events at the 3′ ends of the RNA. These encounters impede forward translocation, leading to RNAP pausing (3). There are many protein factors that strengthen or weaken pausing by targeting translocation, such as archaeal/eukaryotic Spt5 and bacterial NusG/RfaH (4) or N/Nun proteins of lambdoid phages (3). In metazoa, RNAP II pausing in promoter-proximal regions plays a role in having polymerases in place for a rapid transcription response to environmental stimuli, such as heat-shock or cell differentiation, and maintaining a basal level of gene expression (5, 6). Translocation blocks also initiate RNAP backtracking (7). Backtracking events disengage the 3′ RNA end from the RNAP catalytic site, which stabilizes pausing; this type of event broadly controls gene transcription in eukaryotes (8). Forward translocation of RNAP along DNA has long been regarded as the movement of the RNA–DNA hybrid through the catalytic cleft, which vacates the active center, termed i+1 site, for an NTP binding. In this sense, the mechanism of translocation has been primarily focused on the hybrid movement, with only limited emphasis on the DNA sequences surrounding the hybrid (1, 2, 9). However, several reports indicate that the DNA sequences immediately upstream and downstream from the hybrid regulate translocation (7, 10⇓–12). In PNAS, Silva et al. (13) identify an important component of the translocation mechanism using millisecond molecular dynamics (MD) simulation of translocation of yeast RNAP II: Translocation of the hybrid occurs before entry of the template DNA base to the i+1 site, where it can pair …

Understanding the functional interplay between DNA demethylation and transcription (561.1)

Dynamic regulation and faithful maintenance of proper DNA methylation patterns are essential for many cellular functions. 5‐Hydroxylmethylcytosine (5hmC), 5‐formylcytosine (5fC) and 5‐carboxylcytosine, newly discovered oxidized forms of 5‐methylcytosine (5mC) are involved in active DNA demethylation process. The roles of these oxidized species of 5mC in epigenetic and transcription regulation have been an area of intensive study recently. Here we report a systematic study of the effects of five different forms of cytosine in DNA on mammalian and yeast RNA polymerase II transcription elongation, providing new insights into potential functional interplay between cytosine methylation status and transcription. In addition, we will discuss our recent effort in developing new chemical tools that target the formylcytosine sites and modulate 5fC‐related biological processes.Grant Funding Source: NIH GM085136, NIH GM102362

RNAs nonspecifically inhibit RNA polymerase II by preventing binding to the DNA template

Many RNAs are known to act as regulators of transcription in eukaryotes, including certain small RNAs that directly inhibit RNA polymerases both in prokaryotes and eukaryotes. We have examined the potential for a variety of RNAs to directly inhibit transcription by yeast RNA polymerase II (Pol II) and find that unstructured RNAs are potent inhibitors of purified yeast Pol II. Inhibition by RNA is achieved by blocking binding of the DNA template and requires binding of the RNA to Pol II prior to open complex formation. RNA is not able to displace a DNA template that is already stably bound to Pol II, nor can RNA inhibit elongating Pol II. Unstructured RNAs are more potent inhibitors than highly structured RNAs and can also block specific transcription initiation in the presence of basal transcription factors. Crosslinking studies with ultraviolet light show that unstructured RNA is most closely associated with the two large subunits of Pol II that comprise the template binding cleft, but the RNA has contacts in a basic residue channel behind the back wall of the active site. These results are distinct from previous observations of specific inhibition by small, structured RNAs in that they demonstrate a sensitivity of the holoenzyme to inhibition by unstructured RNA products that bind to a surface outside the DNA cleft. These results are discussed in terms of the need to prevent inhibition by RNAs, either though sequestration of nascent RNA or preemptive interaction of Pol II with the DNA template.

Activation of a Chimeric Rpb5/RpoH Subunit Using Library Selection

Rpb5 is a general subunit of all eukaryotic RNA polymerases which consists of a N-terminal and a C-terminal domain. The corresponding archaeal subunit RpoH contains only the conserved C-terminal domain without any N-terminal extensions. A chimeric construct, termed rp5H, which encodes the N-terminal yeast domain and the C-terminal domain from Pyrococcus furiosus is unable to complement the lethal phenotype of a yeast rpb5 deletion strain (Δrpb5). By applying a random mutagenesis approach we found that the amino acid exchange E197K in the C-terminal domain of the chimeric Rp5H, either alone or with additional exchanges in the N-terminal domain, leads to heterospecific complementation of the growth deficiency of Δrpb5. Moreover, using a recently described genetic system for Pyrococcus we could demonstrate that the corresponding exchange E62K in the archaeal RpoH subunit alone without the eukaryotic N-terminal extension was stable, and growth experiments indicated no obvious impairment in vivo. In vitro transcription experiments with purified RNA polymerases showed an identical activity of the wild type and the mutant Pyrococcus RNA polymerase. A multiple alignment of RpoH sequences demonstrated that E62 is present in only a few archaeal species, whereas the great majority of sequences within archaea and eukarya contain a positively charged amino acid at this position. The crystal structures of the Sulfolobus and yeast RNA polymerases show that the positively charged arginine residues in subunits RpoH and Rpb5 most likely form salt bridges with negatively charged residues from subunit RpoK and Rpb1, respectively. A similar salt bridge might stabilize the interaction of Rp5H-E197K with a neighboring subunit of yeast RNA polymerase and thus lead to complementation of Δrpb5.

Zernike phase contrast cryo-electron microscopy reveals 100 kDa component in a protein complex

Cryo-electron microscopy (cryo-EM) has become a powerful technique for obtaining near atomic structures for large protein assemblies or large virus particles, but the application to protein particles smaller than 200–300 kDa has been hampered by the feeble phase contrast obtained for such small samples and the limited number of electrons tolerated by them without incurring excessive radiation damage. By implementing a thin-film quarter-wave phase plate to a cryo-EM, Nagayama, one of the present authors, has recently restored the long-lost very low spatial frequencies, generating in-focus phase contrast superior to that of conventional defocusing phase contrast, and successfully applied the so-called Zernike phase-plate cryo-EM to target various biological samples in native state. Nevertheless, the sought-after goal of using enhanced phase contrast to reveal a native protein as small as 100 kDa waits to be realized. Here, we report a study in which 200 kV Zernike phase-plate cryo-EM with a plate cut-on periodicity of 36 nm was applied to visualize 100 kDa components of various protein complexes, including the small domains on the surface of an icosahedral particle of ∼38 nm derived from the dragon grouper nervous necrosis virus (DGNNV) and the labile sub-complex dissociated from yeast RNA polymerase III of 17 nm. In the former case, we observed a phase contrast reversal phenomenon at the centre of the icosahedral particle and traced its root cause to the near matching of the cut-on size and the particle size. In summary, our work has demonstrated that Zernike phase-plate implementation can indeed expand the size range of proteins that can be successfully investigated by cryo-EM, opening the door for countless proteins. Finally, we briefly discuss the possibility of using a transfer lens system to enlarge the cut-on periodicity without further miniaturizing the plate pinhole.

Complete dissection of transcription elongation reveals slow translocation of RNA polymerase II in a linear ratchet mechanism

During transcription elongation, RNA polymerase has been assumed to attain equilibrium between pre- and post-translocated states rapidly relative to the subsequent catalysis. Under this assumption, recent single-molecule studies proposed a branched Brownian ratchet mechanism that necessitates a putative secondary nucleotide binding site on the enzyme. By challenging individual yeast RNA polymerase II with a nucleosomal barrier, we separately measured the forward and reverse translocation rates. Surprisingly, we found that the forward translocation rate is comparable to the catalysis rate. This finding reveals a linear, non-branched ratchet mechanism for the nucleotide addition cycle in which translocation is one of the rate-limiting steps. We further determined all the major on- and off-pathway kinetic parameters in the elongation cycle. The resulting translocation energy landscape shows that the off-pathway states are favored thermodynamically but not kinetically over the on-pathway states, conferring the enzyme its propensity to pause and furnishing the physical basis for transcriptional regulation. DOI:http://dx.doi.org/10.7554/eLife.00971.001.

126 Replication impairment as a source of transcription- and R-loop-associated recombination

Coordination of DNA replication with DNA-damage sensing, repair, and cell cycle progression ensures with high probability genome integrity during cell divisions, thus preventing mutations and DNA rearrangements. Such events are usually associated with pathological disorders, including premature aging, various cancer predispositions, and inherited diseases (Aguilera & Gómez-González, 2008). One important type of genome instability is that associated with transcription. Transcription of a DNA sequence increases its frequency of recombination, a phenomenon referred to as transcription-associated recombination (TAR). We will provide new data on the analysis of protein factors involved in transcription and RNA export that provide evidence that TAR is mediated by replication impairment and that it can be further enhanced by dysfunction of replication and repair factors in yeast and human cells via R-loops. Notably, our study provides a first connection between RNA processing, R-loops, and chromatin structure that will be discussed. In addition, we have analyzed a number of mutants of the yeast RNA polymerase II that are dependent on double-strand repair functions for their viability. We show that in these mutants, replication fork progression and DNA-damage response are impaired by the presence of strong transcription-dependent obstacles. Our data provide evidence that specific alterations of mRNP biogenesis/export machinery lead to different ways by which chromatin structure and progression of replication can be compromised leading to genome instability.

Isolation and Characterization of RNA Polymerase rpoB Mutations That Alter Transcription Slippage during Elongation in Escherichia coli

Transcription fidelity is critical for maintaining the accurate flow of genetic information. The study of transcription fidelity has been limited because the intrinsic error rate of transcription is obscured by the higher error rate of translation, making identification of phenotypes associated with transcription infidelity challenging. Slippage of elongating RNA polymerase (RNAP) on homopolymeric A/T tracts in DNA represents a special type of transcription error leading to disruption of open reading frames in Escherichia coli mRNA. However, the regions in RNAP involved in elongation slippage and its molecular mechanism are unknown. We constructed an A/T tract that is out of frame relative to a downstream lacZ gene on the chromosome to examine transcriptional slippage during elongation. Further, we developed a genetic system that enabled us for the first time to isolate and characterize E. coli RNAP mutants with altered transcriptional slippage in vivo. We identified several amino acid residues in the β subunit of RNAP that affect slippage in vivo and in vitro. Interestingly, these highly clustered residues are located near the RNA strand of the RNA-DNA hybrid in the elongation complex. Our E. coli study complements an accompanying study of slippage by yeast RNAP II and provides the basis for future studies on the mechanism of transcription fidelity.

Maf1, a general negative regulator of RNA polymerase III in yeast

tRNA synthesis by yeast RNA polymerase III (Pol III) is down-regulated under growth-limiting conditions. This control is mediated by Maf1, a global negative regulator of Pol III transcription. Conserved from yeast to man, Maf1 was originally discovered in Saccharomyces cerevisiae by a genetic approach. Details regarding the molecular basis of Pol III repression by Maf1 are now emerging from the recently reported structural and biochemical data on Pol III and Maf1. The phosphorylation status of Maf1 determines its nuclear localization and interaction with the Pol III complex and several Maf1 kinases have been identified to be involved in Pol III control. Moreover, Maf1 indirectly affects tRNA maturation and decay. Here I discuss the current understanding of the mechanisms that oversee the Maf1-mediated regulation of Pol III activity and the role of Maf1 in the control of tRNA biosynthesis in yeast. This article is part of a Special Issue entitled: Transcription by Odd Pols.

Characterization of the Interactions of Mammalian RNA Polymerase I Associated Proteins PAF53 and PAF49

Masami Muramatsu's laboratory demonstrated the critical role of RNA polymerase I (Pol I)-associated factor PAF53 in mammalian rRNA transcription. They have also identified a second polymerase associated factor, PAF49. Both PAF49 and PAF53 copurify with that fraction of the RNA polymerase I molecules that can function in transcription initiation in vitro. PAF49 and PAF53 are the mammalian homologues of two subunits of yeast RNA polymerase I, A34.5 and A49, that form a TFIIF-related subcomplex in yeast RNA polymerase I. In light of those publications, we investigated the interactions between various deletion and substitution mutants of mammalian PAF49 and PAF53 with the purpose of identifying those domains of the mammalian proteins that interact. Comparison of our results with structural studies on yeast A34.5 and A49 demonstrates that the yeast and mammalian proteins may in fact share structural similarities. In fact, the deletion mutagenesis data confirmed and extended the structural studies. For example, amino acids 41-86 of PAF49 were sufficient to provide the basis for heterodimerization. In silico structural analysis predicted that this region could assume a structure similar to the homologous region of yeast A34.5. Those similarities are insufficient, by themselves, for the proteins to form interspecific heterodimers. However, substitution of amino acids 52-98 of yeast A34.5 with amino acids 41-86 of mammalian PAF49 resulted in a protein that could heterodimerize with mouse PAF53.

A theoretical study of the mechanism of the nucleotidyl transfer reaction catalyzed by yeast RNA polymerase II

The mechanism of the nucleotidyl transfer reaction catalyzed by yeast RNA polymerase II has been investigated using molecular mechanics and quantum mechanics methods. Molecular dynamics (MD) simulations were carried out using the TIP3 water model and generalized solvent boundary potential (GSBP) by CHARMM based on the X-ray crystal structure. Two models of the ternary elongation complex were constructed based on CHARMM MD calculations. All the species including reactants, transition states, intermediates, and products were optimized using the DFT-PBE method coupled with the basis set DZVP and the auxiliary basis set GEN-A2. Three pathways were explored using the DFT method. The most favorable reaction pathway involves indirect proton migration from the RNA primer 3′-OH to the oxygen atom of α-phosphate via a solvent water molecule, proton rotation from the oxygen atom of α-phosphate to the α-phosphate side, the RNA primer 3′-O nucleophilic attack on the α-phosphorus atom, and P-O bond breakage. The corresponding reaction potential profile was obtained. The rate limiting step, with a barrier height of 21.5 kcal/mol, is the RNA primer 3′-O nucleophilic attack, rather than the commonly considered proton transfer process. A high-resolution crystal structure including crystallographic water molecules is required for further studies.

5-formylcytosine and 5-carboxylcytosine reduce the rate and substrate specificity of RNA polymerase II transcription

While the roles of 5-methyl-cytosine and 5-hydroxymethyl-cytosine in epigenetic regulation of gene expression are well-established, the functional effects of 5-formyl-cytosine and 5-carboxyl-cytosine in the genome on transcription are not clear. Here we report the first systematic study of the effects of five different forms of cytosine in DNA on mammalian and yeast RNA polymerase II transcription, providing new insights into potential functional interplay between cytosine methylation status and transcription.

Architecture of the RNA polymerase II preinitiation complex and mechanism of ATP-dependent promoter opening

SummaryYeast RNA polymerase (Pol) II general factor TFIIE and the TFIIH subunit Ssl2/XPB function in transition of the preinitiation complex (PIC) to the open complex. We find that the three TFIIE winged helix (WH) domains form a heterodimer, with the Tfa1/TFIIEα WH binding the Pol II clamp and the Tfa2/TFIIEβ tandem WH domain encircling promoter DNA that becomes single stranded in the open complex. Ssl2 lies adjacent to TFIIE, enclosing downstream promoter DNA. In contrast to previous proposals, comparison of the PIC and open complex models strongly suggests that Ssl2 promotes DNA opening by functioning as a double stranded DNA translocase, feeding 15 bp of double stranded DNA into the Pol II cleft. Right-handed threading of DNA through the Ssl2 binding groove, combined with the fixed position of upstream promoter DNA, will lead to DNA unwinding and the open state.

Crystal Structure of Elongator Subcomplex Elp4–6

Elongator is a multiprotein complex composed of two subcomplexes, Elp1-3 and Elp4-6. Elongator is highly conserved between yeast and humans and plays an important role in RNA polymerase II-mediated transcriptional elongation and many other processes, including cytoskeleton organization, exocytosis, and tRNA modification. Here, we determined the crystal structure of the Elp4-6 subcomplex of yeast. The overall structure of Elp4-6 revealed that Elp6 acts as a bridge to assemble Elp4 and Elp5. Detailed structural and sequence analyses revealed that each subunit in the Elp4-6 subcomplex forms a RecA-ATPase-like fold, although it lacks the key sequence signature of ATPases. Site-directed mutagenesis and biochemical analyses indicated that the Elp4-6 subcomplex can assemble into a hexameric ring-shaped structure in vitro and in vivo. Furthermore, GST pulldown assays showed that the ring-shaped assembly of the Elp4-6 subcomplex is important for its specific histone H3 binding. Our results may shed light on the substrate recognition and assembly of the holo-Elongator complex.

Architecture of the Yeast RNA Polymerase II Open Complex and Regulation of Activity by TFIIF

To investigate the function and architecture of the open complex state of RNA polymerase II (Pol II), Saccharomyces cerevisiae minimal open complexes were assembled by using a series of heteroduplex HIS4 promoters, TATA binding protein (TBP), TFIIB, and Pol II. The yeast system demonstrates great flexibility in the position of active open complexes, spanning 30 to 80 bp downstream from TATA, consistent with the transcription start site scanning behavior of yeast Pol II. TFIIF unexpectedly modulates the activity of the open complexes, either repressing or stimulating initiation. The response to TFIIF was dependent on the sequence of the template strand within the single-stranded bubble. Mutations in the TFIIB reader and linker region, which were inactive on duplex DNA, were suppressed by the heteroduplex templates, showing that a major function of the TFIIB reader and linker is in the initiation or stabilization of single-stranded DNA. Probing of the architecture of the minimal open complexes with TFIIB-FeBABE [TFIIB-p-bromoacetamidobenzyl-EDTA-iron(III)] derivatives showed that the TFIIB core domain is surprisingly positioned away from Pol II, and the addition of TFIIF repositions the TFIIB core domain to the Pol II wall domain. Together, our results show an unexpected architecture of minimal open complexes and the regulation of activity by TFIIF and the TFIIB core domain.

Structural basis of transcription by bacterial and eukaryotic RNA polymerases

DNA-dependent RNA polymerase (RNAP) is responsible for cellular gene transcription. Although crystallographic studies on prokaryotic and eukaryotic RNAPs have elucidated the basic RNAP architectures, the structural details of many essential events during transcription initiation, elongation, and termination are still largely unknown. Recent crystallographic studies on a bacterial RNAP and yeast RNAP II have revealed different RNAP structural states from that of the normal transcribing complex, as well as the basis of transcription factor functions, advancing our understanding of transcription. These studies have highlighted unexpected similarities in many fundamental aspects of transcription mechanisms between the bacterial and eukaryotic transcription machineries. Remarkable differences also exist between the bacterial and eukaryotic transcription systems, suggesting directions for future studies.

The elongation rate of RNA polymerase determines the fate of transcribed nucleosomes

Upon transcription, histones can either detach from DNA or transfer behind the polymerase through a process believed to involve template looping. The details governing nucleosomal fate during transcription are not well understood. Our atomic force microscopy images of RNA polymerase II-nucleosome complexes confirm the presence of looped transcriptional intermediates and provide mechanistic insight into the histone-transfer process via the distribution of transcribed nucleosome positions. Significantly, we find that a fraction of the transcribed nucleosomes are remodeled to hexasomes, and that this fraction depends on the transcription elongation rate. A simple model involving the kinetic competition between transcription elongation, histone transfer, and histone-histone dissociation quantitatively rationalizes our observations and unifies results obtained with other polymerases. Factors affecting the relative magnitude of these processes provide the physical basis for nucleosomal fate during transcription and, therefore, for the regulation of gene expression.

Rpa43 and its partners in the yeast RNA polymerase I transcription complex

An Rpa43/Rpa14 stalk protrudes from RNA polymerase I (RNAPI), with homology to Rpb7/Rpb4 (RNAPII), Rpc25/Rpc17 (RNAPIII) and RpoE/RpoF (archaea). In fungi and vertebrates, Rpa43 contains hydrophilic domains forming about half of its size, but these domains lack in Schizosaccharomyces pombe and most other eukaryote lineages. In Saccharomyces cerevisiae, they can be lost with little or no growth effect, as shown by deletion mapping and by domain swapping with fission yeast, but genetically interact with rpa12Δ, rpa34Δ or rpa49Δ, lacking non-essential subunits important for transcript elongation. Two-hybrid data and other genetic evidence suggest that Rpa43 directly bind Spt5, an RNAPI elongation factor also acting in RNAPII-dependent transcription, and may also interact with the nucleosomal chaperone Spt6. Structured summary of protein interactionsRPA43physically interactswithSPT6bytwo hybrid(View interaction)HMO1andSPT6colocalizebyfluorescence microscopy(View interaction)RPA43physically interactswithRPA14bytwo hybrid(View interaction)RPA43physically interactswithSPT5bytwo hybrid(View interaction)HMO1andSPT5colocalizebyfluorescence microscopy(View interaction)