Structure and function of the Si3 insertion integrated into the trigger loop/helix of cyanobacterial RNA polymerase.

Cyanobacteria and evolutionarily related chloroplasts of algae and plants possess unique RNA polymerases (RNAPs) with characteristics that distinguish them from canonical bacterial RNAPs. The largest subunit of cyanobacterial RNAP (cyRNAP) is divided into two polypeptides, ß'1 and ß'2, and contains the largest known lineage-specific insertion domain, Si3, located in the middle of the trigger loop and spanning approximately half of the ß'2 subunit. In this study, we present the X-ray crystal structure of Si3 and the cryo-EM structures of the cyRNAP transcription elongation complex plus the NusG factor with and without incoming nucleoside triphosphate (iNTP) bound at the active site. Si3 has a well-ordered and elongated shape that exceeds the length of the main body of cyRNAP, fits into cavities of cyRNAP in the absence of iNTP bound at the active site and shields the binding site of secondary channel-binding proteins such as Gre and DksA. A small transition from the trigger loop to the trigger helix upon iNTP binding results in a large swing motion of Si3; however, this transition does not affect the catalytic activity of cyRNAP due to its minimal contact with cyRNAP, NusG, or DNA. This study provides a structural framework for understanding the evolutionary significance of these features unique to cyRNAP and chloroplast RNAP and may provide insights into the molecular mechanism of transcription in specific environment of photosynthetic organisms and organelle.

Structure and function of the Si3 insertion integrated into the trigger loop/helix of cyanobacterial RNA polymerase.

Cyanobacteria and evolutionarily related chloroplasts of algae and plants possess unique RNA polymerases (RNAPs) with characteristics that distinguish from canonical bacterial RNAPs. The largest subunit of cyanobacterial RNAP (cyRNAP) is divided into two polypeptides, ß'1 and ß'2, and contains the largest known lineage-specific insertion domain, Si3, located in the middle of the trigger loop and spans approximately half of the ß'2 subunit. In this study, we present the X-ray crystal structure of Si3 and the cryo-EM structures of the cyRNAP transcription elongation complex plus the NusG factor with and without incoming nucleoside triphosphate (iNTP) bound at the active site. Si3 has a well-ordered and elongated shape that exceeds the length of the main body of cyRNAP, fits into cavities of cyRNAP and shields the binding site of secondary channel-binding proteins such as Gre and DksA. A small transition from the trigger loop to the trigger helix upon iNTP binding at the active site results in a large swing motion of Si3; however, this transition does not affect the catalytic activity of cyRNAP due to its minimal contact with cyRNAP, NusG or DNA. This study provides a structural framework for understanding the evolutionary significance of these features unique to cyRNAP and chloroplast RNAP and may provide insights into the molecular mechanism of transcription in specific environment of photosynthetic organisms.

Nonthermal acceleration of protein hydration by sub-terahertz irradiation.

The collective intermolecular dynamics of protein and water molecules, which overlap in the sub-terahertz (THz) frequency region, are relevant for expressing protein functions but remain largely unknown. This study used dielectric relaxation (DR) measurements to investigate how externally applied sub-THz electromagnetic fields perturb the rapid collective dynamics and influence the considerably slower chemical processes in protein-water systems. We analyzed an aqueous lysozyme solution, whose hydration is not thermally equilibrated. By detecting time-lapse differences in microwave DR, we demonstrated that sub-THz irradiation gradually decreases the dielectric permittivity of the lysozyme solution by reducing the orientational polarization of water molecules. Comprehensive analysis combining THz and nuclear magnetic resonance spectroscopies suggested that the gradual decrease in the dielectric permittivity is not induced by heating but is due to a slow shift toward the hydrophobic hydration structure in lysozyme. Our findings can be used to investigate hydration-mediated protein functions based on sub-THz irradiation.

Repetitive DNA symmetry elements negatively regulate gene expression in embryonic stem cells.

Transcription factor (TF) binding to genomic DNA elements constitutes one of the key mechanisms that regulates gene expression program in cells. Both consensus and nonconsensus DNA sequence elements influence the recognition specificity of TFs. Based on the analysis of experimentally determined c-Myc binding preferences to genomic DNA, here we statistically predict that certain repetitive, nonconsensus DNA symmetry elements can relatively reduce TF-DNA binding preferences. This is in contrast to a different set of repetitive, nonconsensus symmetry elements that can increase the strength of TF-DNA binding. Using c-Myc enhancer reporter system containing consensus motif flanked by nonconsensus sequences in embryonic stem cells, we directly demonstrate that the enrichment in such negatively regulating repetitive symmetry elements is sufficient to reduce the gene expression level compared with native genomic sequences. Negatively regulating repetitive symmetry elements around consensus c-Myc motif and DNA sequences containing consensus c-Myc motif flanked by entirely randomized sequences show similar expression baseline. A possible explanation for this observation is that rather than complete repression, negatively regulating repetitive symmetry elements play a regulatory role in fine-tuning the reduction of gene expression, most probably by binding TFs other than c-Myc.

Nonthermal excitation effects mediated by sub-terahertz radiation on hydrogen exchange in ubiquitin.

Water dynamics in the hydration layers of biomolecules play crucial roles in a wide range of biological functions. A hydrated protein contains multiple components of diffusional and vibrational dynamics of water and protein, which may be coupled at ∼0.1-THz frequency (10-ps timescale) at room temperature. However, the microscopic description of biomolecular functions based on various modes of protein-water-coupled motions remains elusive. A novel approach for perturbing the hydration dynamics in the subterahertz frequency range and probing them at the atomic level is therefore warranted. In this study, we investigated the effect of klystron-based, intense 0.1-THz excitation on the slow dynamics of ubiquitin using NMR-based measurements of hydrogen-deuterium exchange. We demonstrated that the subterahertz irradiation accelerated the hydrogen-deuterium exchange of the amides located in the interior of the protein and hydrophobic surfaces while decelerating this exchange in the amides located in the surface loop and short 310 helix regions. This subterahertz-radiation-induced effect was qualitatively contradictory to the increased-temperature-induced effect. Our results suggest that the heterogeneous water dynamics occurring at the protein-water interface include components that are nonthermally excited by the subterahertz radiation. Such subterahertz-excited components may be linked to the slow function-related dynamics of the protein.

Gre factors prevent thermal and mechanical stresses induced by terahertz irradiation during transcription.

During transcription in cells, the transcription complex consisting of RNA polymerase, DNA and nascent RNA is exposed to fluctuating temperature and pressure. However, little is known about the mechanism of transcriptional homeostasis under fluctuating physical parameters. In this study, we generated these fluctuating parameters using pulsed local heating and acoustic waves in the reaction system of transcription by Escherichia coli RNA polymerase, using a terahertz free-electron laser. We demonstrated that transcription processes, including abortive initiation and elongation pausing, and the fidelity of elongation are significantly affected by the laser-based local perturbations. We also found that all these functional alternations in the transcription process are almost completely mitigated by the presence of Gre proteins. It is well known that Gre proteins enhance RNA cleavage of polymerase by binding to the pore structure termed secondary channel. Recently, the chaperone activities have also been proposed for Gre proteins, yet the details directly associated with transcription are largely unknown. Our finding indicates that Gre proteins are necessary for maintaining transcriptional homeostasis under thermal and mechanical stresses.

Control of Transcription Initiation by Biased Thermal Fluctuations on Repetitive Genomic Sequences.

In the process of transcription initiation by RNA polymerase, promoter DNA sequences affect multiple reaction pathways determining the productivity of transcription. However, the question of how the molecular mechanism of transcription initiation depends on the sequence properties of promoter DNA remains poorly understood. Here, combining the statistical mechanical approach with high-throughput sequencing results, we characterize abortive transcription and pausing during transcription initiation by Escherichia coli RNA polymerase at a genome-wide level. Our results suggest that initially transcribed sequences, when enriched with thymine bases, contain the signal for inducing abortive transcription, whereas certain repetitive sequence elements embedded in promoter regions constitute the signal for inducing pausing. Both signals decrease the productivity of transcription initiation. Based on solution NMR and in vitro transcription measurements, we suggest that repetitive sequence elements within the promoter DNA modulate the nonlocal base pair stability of its double-stranded form. This stability profoundly influences the reaction coordinates of the productive initiation via pausing.

Transcription Factor Binding in Embryonic Stem Cells Is Constrained by DNA Sequence Repeat Symmetry.

Transcription factor (TF) recognition is dictated by the underlying DNA motif sequence specific for each TF. Here, we reveal that DNA sequence repeat symmetry plays a central role in defining TF-DNA-binding preferences. In particular, we find that different TFs bind similar symmetry patterns in the context of different developmental layers. Most TFs possess dominant preferences for similar DNA repeat symmetry types. However, in some cases, preferences of specific TFs are changed during differentiation, suggesting the importance of information encoded outside of known motif regions. Histone modifications also exhibit strong preferences for similar DNA repeat symmetry patterns unique to each type of modification. Next, using an in vivo reporter assay, we show that gene expression in embryonic stem cells can be positively modulated by the presence of genomic and computationally designed DNA oligonucleotides containing identified nonconsensus-repetitive sequence elements. This supports the hypothesis that certain nonconsensus-repetitive patterns possess a functional ability to regulate gene expression. We also performed a solution NMR experiment to probe the stability of double-stranded DNA via imino proton resonances for several double-stranded DNA sequences characterized by different repetitive patterns. We suggest that such local stability might play a key role in determining TF-DNA binding preferences. Overall, our findings show that despite the enormous sequence complexity of the TF-DNA binding landscape in differentiating embryonic stem cells, this landscape can be quantitatively characterized in simple terms using the notion of DNA sequence repeat symmetry.

RNA Polymerase and Transcription Mechanisms: The Forefront of Physicochemical Studies of Chemical Reactions.

The study of transcription and its regulation is an interdisciplinary field that is closely connected with genetics, structural biology, and reaction theory. Among these, although less attention has been paid to reaction theory, it is becoming increasingly useful for research on transcription. Rate equations are commonly used to describe reactions involved in transcription, but they tend to be used unaware of the timescales of relevant physical processes. In this review, we discuss the limitation of rate equation for describing three-dimensional diffusion and one-dimensional diffusion along DNA. We then introduce the chemical ratchet mechanism recently proposed for explaining the antenna effect, an enhancement of the binding affinity to a specific site on longer DNA, which deviates from a thermodynamic rule. We show that chemical ratchet cannot be described with a single set of rate equations but alternative sets of rate equations that temporally switch no faster than the binding reaction.

The Role of Pyrophosphorolysis in the Initiation-to-Elongation Transition by E. coli RNA Polymerase.

RNA polymerase can cleave a phosphodiester bond at the 3' end of a nascent RNA in the presence of pyrophosphate producing NTP. Pyrophosphorolysis has been characterized during elongation steps of transcription where its rate is significantly slower than the forward rate of NMP addition. In contrast, we report here that pyrophosphorolysis can occur in a millisecond time scale during the transition of Escherichia coli RNA polymerase from initiation to elongation at the psbA2 promoter. This rapid pyrophosphorolysis occurs during productive RNA synthesis as opposed to abortive RNA synthesis. Dissociation of σ70 or RNA extension beyond nine nucleotides dramatically reduces the rate of pyrophosphorolysis. We argue that the rapid pyrophosphorolysis allows iterative cycles of cleavage and re-synthesis of the 3' phosphodiester bond by the productive complexes in the early stage of transcription. This iterative process may provide an opportunity for the σ70 to dissociate from the RNA exit channel of the enzyme, enabling RNA to extend through the channel.

Single-round isolation of diverse RNA aptamers from a random sequence pool.

Aptamers are oligonucleotide ligands with specific binding affinity to target molecules. Generally, RNA aptamers are selected from an RNA pool with random sequences, using the technique termed SELEX, in which the target-binding RNA molecules are repeatedly isolated and exponentially amplified. Despite several advantages, SELEX often produces uncertain results during the iterative amplifications of the rare target-binding RNA molecules. Here, we develop a non-repeated, primer-less and target immobilization-free isolation method for generating RNA aptamers, which is robust to experimental noise. Uniquely, this method focuses on finding and removal of non-aptamer sequences from the RNA pool by RNase digestion leaving target-bound aptamer molecules, and thus is independent of aptamer types. The undigested RNA sequences remaining are so few in number that they must be mixed with a large excess of a known sequence for further manipulations and this sequence is then removed by restriction digestion followed by high-throughput sequencing analysis to identify aptamers. Using this method, we generated multiple RNA aptamers targeting α-thrombin and TGFß1 proteins, independently. This method potentially generates thousands of sequences as aptamer candidates, which may enable us to predict a common average sequence or structural property of these aptamers that is different from input RNA.

Transcription pausing: biological significance of thermal fluctuations biased by repetitive genomic sequences.

Transcription of DNA by RNA polymerase (RNAP) takes place in a cell environment dominated by thermal fluctuations. How are transcription reactions including initiation, elongation, and termination on genomic DNA so well-controlled during such fluctuations? A recent statistical mechanical approach using high-throughput sequencing data reveals that repetitive DNA sequence elements embedded into a genomic sequence provide the key mechanism to functionally bias the fluctuations of transcription elongation complexes. In particular, during elongation pausing, such repetitive sequence elements can increase the magnitude of one-dimensional diffusion of the RNAP enzyme on the DNA upstream of the pausing site, generating a large variation in the dwell times of RNAP pausing under the control of these genomic signals.

Control of transcriptional pausing by biased thermal fluctuations on repetitive genomic sequences.

In the process of transcription elongation, RNA polymerase (RNAP) pauses at highly nonrandom positions across genomic DNA, broadly regulating transcription; however, molecular mechanisms responsible for the recognition of such pausing positions remain poorly understood. Here, using a combination of statistical mechanical modeling and high-throughput sequencing and biochemical data, we evaluate the effect of thermal fluctuations on the regulation of RNAP pausing. We demonstrate that diffusive backtracking of RNAP, which is biased by repetitive DNA sequence elements, causes transcriptional pausing. This effect stems from the increased microscopic heterogeneity of an elongation complex, and thus is entropy-dominated. This report shows a linkage between repetitive sequence elements encoded in the genome and regulation of RNAP pausing driven by thermal fluctuations.

Visualizing translocation dynamics and nascent transcript errors in paused RNA polymerases in vivo.

BACKGROUND: Transcription elongation is frequently interrupted by pausing signals in DNA, with downstream effects on gene expression. Transcription errors also induce prolonged pausing, which can lead to a destabilized genome by interfering with DNA replication. Mechanisms of pausing associated with translocation blocks and misincorporation have been characterized in vitro, but not in vivo. RESULTS: We investigate the pausing pattern of RNA polymerase (RNAP) in Escherichia coli by a novel approach, combining native elongating transcript sequencing (NET-seq) with RNase footprinting of the transcripts (RNET-seq). We reveal that the G-dC base pair at the 5' end of the RNA-DNA hybrid interferes with RNAP translocation. The distance between the 5' G-dC base pair and the 3' end of RNA fluctuates over a three-nucleotide width. Thus, the G-dC base pair can induce pausing in post-translocated, pre-translocated, and backtracked states of RNAP. Additionally, a CpG sequence of the template DNA strand spanning the active site of RNAP inhibits elongation and induces G-to-A errors, which leads to backtracking of RNAP. Gre factors efficiently proofread the errors and rescue the backtracked complexes. We also find that pausing events are enriched in the 5' untranslated region and antisense transcription of mRNA genes and are reduced in rRNA genes. CONCLUSIONS: In E. coli, robust transcriptional pausing involves RNAP interaction with G-dC at the upstream end of the RNA-DNA hybrid, which interferes with translocation. CpG DNA sequences induce transcriptional pausing and G-to-A errors.

Transcription elongation. Heterogeneous tracking of RNA polymerase and its biological implications.

Regulation of transcription elongation via pausing of RNA polymerase has multiple physiological roles. The pausing mechanism depends on the sequence heterogeneity of the DNA being transcribed, as well as on certain interactions of polymerase with specific DNA sequences. In order to describe the mechanism of regulation, we introduce the concept of heterogeneity into the previously proposed alternative models of elongation, power stroke and Brownian ratchet. We also discuss molecular origins and physiological significances of the heterogeneity.

Direct assessment of transcription fidelity by high-resolution RNA sequencing.

Cancerous and aging cells have long been thought to be impacted by transcription errors that cause genetic and epigenetic changes. Until now, a lack of methodology for directly assessing such errors hindered evaluation of their impact to the cells. We report a high-resolution Illumina RNA-seq method that can assess noncoded base substitutions in mRNA at 10(-4)-10(-5) per base frequencies in vitro and in vivo. Statistically reliable detection of changes in transcription fidelity through ∼10(3) nt DNA sites assures that the RNA-seq can analyze the fidelity in a large number of the sites where errors occur. A combination of the RNA-seq and biochemical analyses of the positions for the errors revealed two sequence-specific mechanisms that increase transcription fidelity by Escherichia coli RNA polymerase: (i) enhanced suppression of nucleotide misincorporation that improves selectivity for the cognate substrate, and (ii) increased backtracking of the RNA polymerase that decreases a chance of error propagation to the full-length transcript after misincorporation and provides an opportunity to proofread the error. This method is adoptable to a genome-wide assessment of transcription fidelity.

Intrinsic translocation barrier as an initial step in pausing by RNA polymerase II.

Pausing of RNA polymerase II (RNAP II) by backtracking on DNA is a major regulatory mechanism in control of eukaryotic transcription. Backtracking occurs by extrusion of the 3' end of the RNA from the active center after bond formation and before translocation of RNAP II on DNA. In several documented cases, backtracking requires a special signal such as A/T-rich sequences forming an unstable RNA-DNA hybrid in the elongation complex. However, other sequence-dependent backtracking signals and conformations of RNAP II leading to backtracking remain unknown. Here, we demonstrate with S. cerevisiae RNAP II that a cleavage-deficient elongation factor TFIIS (TFIIS(AA)) enhances backtracked pauses during regular transcription. This is due to increased efficiency of formation of an intermediate that leads to backtracking. This intermediate may involve misalignment at the 3' end of the nascent RNA in the active center of the yeast RNAP II, and TFIIS(AA) promotes formation of this intermediate at the DNA sequences, presenting a high-energy barrier to translocation. We proposed a three-step mechanism for RNAP II pausing in which a prolonged dwell time in the pre-translocated state increases the likelihood of the 3' RNA end misalignment facilitating a backtrack pausing. These results demonstrate an important role of the intrinsic blocks to forward translocation in pausing by RNAP II.

CTCF-promoted RNA polymerase II pausing links DNA methylation to splicing.

Alternative splicing of pre-messenger RNA is a key feature of transcriptome expansion in eukaryotic cells, yet its regulation is poorly understood. Spliceosome assembly occurs co-transcriptionally, raising the possibility that DNA structure may directly influence alternative splicing. Supporting such an association, recent reports have identified distinct histone methylation patterns, elevated nucleosome occupancy and enriched DNA methylation at exons relative to introns. Moreover, the rate of transcription elongation has been linked to alternative splicing. Here we provide the first evidence that a DNA-binding protein, CCCTC-binding factor (CTCF), can promote inclusion of weak upstream exons by mediating local RNA polymerase II pausing both in a mammalian model system for alternative splicing, CD45, and genome-wide. We further show that CTCF binding to CD45 exon 5 is inhibited by DNA methylation, leading to reciprocal effects on exon 5 inclusion. These findings provide a mechanistic basis for developmental regulation of splicing outcome through heritable epigenetic marks.