Compatibility of termination mechanisms in bacterial transcription with inference on eukaryotic models.

Transcription termination has evolved to proceed through diverse mechanisms. For several classes of terminators, multiple models have been debatably proposed. Recent single-molecule studies on bacterial terminators have resolved several long-standing controversies. First, termination mode or outcome is twofold rather than single. RNA is released alone before DNA or together with DNA from RNA polymerase (RNAP), i.e. with RNA release for termination, RNAP retains on or dissociates off DNA, respectively. The concomitant release, described in textbooks, results in one-step decomposition of transcription complexes, and this 'decomposing termination' prevails at ρ factor-dependent terminators. Contrastingly, the sequential release was recently discovered abundantly from RNA hairpin-dependent intrinsic terminations. RNA-only release allows RNAP to diffuse on DNA in both directions and recycle for reinitiation. This 'recycling termination' enables one-dimensional reinitiation, which would be more expeditious than three-dimensional reinitiation by RNAP dissociated at decomposing termination. Second, while both recycling and decomposing terminations occur at a hairpin-dependent terminator, four termination mechanisms compatibly operate at a ρ-dependent terminator with ρ in alternative modes and even intrinsically without ρ. RNA-bound catch-up ρ mediates recycling termination first and decomposing termination later, while RNAP-prebound stand-by ρ invokes only decomposing termination slowly. Without ρ, decomposing termination occurs slightly and sluggishly. These four mechanisms operate on distinct timescales, providing orderly fail-safes. The stand-by mechanism is benefited by terminational pause prolongation and modulated by accompanying riboswitches more greatly than the catch-up mechanisms. Conclusively, any mechanism alone is insufficient to perfect termination, and multiple mechanisms operate compatibly to achieve maximum possible efficiency under separate controls.

Mature microRNA-binding protein QKI promotes microRNA-mediated gene silencing.

Although Argonaute (AGO) proteins have been the focus of microRNA (miRNA) studies, we observed AGO-free mature miRNAs directly interacting with RNA-binding proteins, implying the sophisticated nature of fine-tuning gene regulation by miRNAs. To investigate microRNA-binding proteins (miRBPs) globally, we analyzed PAR-CLIP data sets to identify RBP quaking (QKI) as a novel miRBP for let-7b. Potential existence of AGO-free miRNAs were further verified by measuring miRNA levels in genetically engineered AGO-depleted human and mouse cells. We have shown that QKI regulates miRNA-mediated gene silencing at multiple steps, and collectively serves as an auxiliary factor empowering AGO2/let-7b-mediated gene silencing. Depletion of QKI decreases interaction of AGO2 with let-7b and target mRNA, consequently controlling target mRNA decay. This finding indicates that QKI is a complementary factor in miRNA-mediated mRNA decay. QKI, however, also suppresses the dissociation of let-7b from AGO2, and slows the assembly of AGO2/miRNA/target mRNA complexes at the single-molecule level. We also revealed that QKI overexpression suppresses cMYC expression at post-transcriptional level, and decreases proliferation and migration of HeLa cells, demonstrating that QKI is a tumour suppressor gene by in part augmenting let-7b activity. Our data show that QKI is a new type of RBP implicated in the versatile regulation of miRNA-mediated gene silencing.

Fast, sensitive, and specific multiplexed single-molecule detection of circulating tumor DNA.

Circulating tumor DNA (ctDNA) analysis has emerged as a highly promising non-invasive assay for detection and monitoring of cancer. However, identification of multiple point-mutant ctDNAs, particularly at extremely low frequencies in early cancer stages, remains a significant challenge. To address this issue, we present a multiplexed ctDNA detection technique, SIMUL (single-molecule detection of multiple low-frequency mutations). SIMUL involves an unbiased preamplification of both wild-type and mutant DNAs, followed by the detection of mutant DNAs through single-molecule multicolor imaging. SIMUL enables highly sensitive and specific detection of multiple single-nucleotide mutations in a short span of time, even in the presence of 10,000-fold excess of wild-type DNA. Importantly, SIMUL can accurately measure mutant fractions due to its linear correlation between the number of single-molecule spots and the variant allele frequency. This breakthrough technique holds immense potential for clinical applications, offering significant improvements for example in early cancer detection and accurate evaluation of anticancer treatment responses.

Translocating RNA polymerase generates R-loops at DNA double-strand breaks without any additional factors.

The R-loops forming around DNA double-strand breaks (DSBs) within actively transcribed genes play a critical role in the DSB repair process. However, the mechanisms underlying R-loop formation at DSBs remain poorly understood, with diverse proposed models involving protein factors associated with RNA polymerase (RNAP) loading, pausing/backtracking or preexisting transcript RNA invasion. In this single-molecule study using Escherichia coli RNAP, we discovered that transcribing RNAP alone acts as a highly effective DSB sensor, responsible for generation of R-loops upon encountering downstream DSBs, without requiring any additional factors. The R-loop formation efficiency is greatly influenced by DNA end structures, ranging here from 2.8% to 73%, and notably higher on sticky ends with 3' or 5' single-stranded overhangs compared to blunt ends without any overhangs. The R-loops extend unidirectionally upstream from the DSB sites and can reach the transcription start site, interfering with ongoing-round transcription. Furthermore, the extended R-loops can persist and maintain their structures, effectively preventing the efficient initiation of subsequent transcription rounds. Our results are consistent with the bubble extension model rather than the 5'-end invasion model or the middle insertion model. These discoveries provide valuable insights into the initiation of DSB repair on transcription templates across bacteria, archaea and eukaryotes.

Transcriptional pause extension benefits the stand-by rather than catch-up Rho-dependent termination.

Transcriptional pause is essential for all types of termination. In this single-molecule study on bacterial Rho factor-dependent terminators, we confirm that the three Rho-dependent termination routes operate compatibly together in a single terminator, and discover that their termination efficiencies depend on the terminational pauses in unexpected ways. Evidently, the most abundant route is that Rho binds nascent RNA first and catches up with paused RNA polymerase (RNAP) and this catch-up Rho mediates simultaneous releases of transcript RNA and template DNA from RNAP. The fastest route is that the catch-up Rho effects RNA-only release and leads to 1D recycling of RNAP on DNA. The slowest route is that the RNAP-prebound stand-by Rho facilitates only the simultaneous rather than sequential releases. Among the three routes, only the stand-by Rho's termination efficiency positively correlates with pause duration, contrary to a long-standing speculation, invariably in the absence or presence of NusA/NusG factors, competitor RNAs or a crowding agent. Accordingly, the essential terminational pause does not need to be long for the catch-up Rho's terminations, and long pauses benefit only the stand-by Rho's terminations. Furthermore, the Rho-dependent termination of mgtA and ribB riboswitches is controlled mainly by modulation of the stand-by rather than catch-up termination.

Werner syndrome protein works as a dimer for unwinding and replication fork regression.

The determination of the oligomeric state of functional enzymes is essential for the mechanistic understanding of their catalytic activities. RecQ helicases have diverse biochemical activities, but it is still unclear how their activities are related to their oligomeric states. We use single-molecule multi-color fluorescence imaging to determine the oligomeric states of Werner syndrome protein (WRN) during its unwinding and replication fork regression activities. We reveal that WRN binds to a forked DNA as a dimer, and unwinds it without any change of its oligomeric state. In contrast, WRN binds to a replication fork as a tetramer, and is dimerized during activation of replication fork regression. By selectively inhibiting the helicase activity of WRN on specific strands, we reveal how the active dimers of WRN distinctly use the energy of ATP hydrolysis for repetitive unwinding and replication fork regression.

Rapid quantification of miRNAs using dynamic FRET-FISH.

MicroRNAs (miRNAs) are short regulatory RNAs that control gene expression at the post-transcriptional level. Various miRNAs playing important roles in cancer development are emerging as promising diagnostic biomarkers for early cancer detection. Accurate miRNA detection, however, remains challenging because they are small and highly homologous. Recently developed miRNA detection techniques based on single-molecule imaging enabled highly specific miRNA quantification without amplification, but the time required for these techniques to detect a single miRNA was larger than 10 minutes, making rapid profiling of numerous miRNAs impractical. Here we report a rapid miRNA detection technique, dynamic FRET-FISH, in which single-molecule imaging at high probe concentrations and thus high-speed miRNA detection is possible. Dynamic FRET-FISH can detect miRNAs in 10 s at 1.2 µM probe concentration while maintaining the high-specificity of single-nucleotide discrimination. We expect dynamic FRET-FISH will be utilized for early detection of cancers by profiling hundreds of cancer biomarkers in an hour.

Rho-dependent transcription termination proceeds via three routes.

Rho is a general transcription termination factor in bacteria, but many aspects of its mechanism of action are unclear. Diverse models have been proposed for the initial interaction between the RNA polymerase (RNAP) and Rho (catch-up and stand-by pre-terminational models); for the terminational release of the RNA transcript (RNA shearing, RNAP hyper-translocation or displacing, and allosteric models); and for the post-terminational outcome (whether the RNAP dissociates or remains bound to the DNA). Here, we use single-molecule fluorescence assays to study those three steps in transcription termination mediated by E. coli Rho. We find that different mechanisms previously proposed for each step co-exist, but apparently occur on various timescales and tend to lead to specific outcomes. Our results indicate that three kinetically distinct routes take place: (1) the catch-up mode leads first to RNA shearing for RNAP recycling on DNA, and (2) later to RNAP displacement for decomposition of the transcriptional complex; (3) the last termination usually follows the stand-by mode with displacing for decomposing. This three-route model would help reconcile current controversies on the mechanisms.

Single-molecule Fluorescence Technique to Monitor the Co-transcriptional Formation of G-quadruplex and R-loop Structures.

G-quadruplexes (GQ) and R-loops are non-canonical nucleic acid structures related to gene regulation and genome instability that can be formed during transcription; however, their formation mechanisms remain elusive. To address this question, we developed a single-molecule fluorescence technique to monitor the formation of G-quadruplex and R-loop structures during transcription. Using this technique, we found that R-loop formation precedes GQ formation and that there exists a positive feedback loop between G-quadruplex and R-loop formation.

A Novel N-terminal Region to Chromodomain in CHD7 is Required for the Efficient Remodeling Activity.

Chromodomain-Helicase DNA binding protein 7 (CHD7) is an ATP dependent chromatin remodeler involved in maintaining open chromatin structure. Mutations of CHD7 gene causes multiple developmental disorders, notably CHARGE syndrome. However, there is not much known about the molecular mechanism by which CHD7 remodels nucleosomes. Here, we performed biochemical and biophysical analysis on CHD7 chromatin remodeler and uncover that N-terminal to the Chromodomain (N-CRD) interacts with nucleosome and contains a high conserved arginine stretch, which is reminiscent of arginine anchor. Importantly, this region is required for efficient ATPase stimulation and nucleosome remodeling activity of CHD7. Furthermore, smFRET analysis shows the mutations in the N-CRD causes the defects in remodeling activity. Collectively, our results uncover the functional importance of a previously unidentified N-terminal region in CHD7 and implicate that the multiple domains in chromatin remodelers are involved in regulating their activities.

Hopping and Flipping of RNA Polymerase on DNA during Recycling for Reinitiation after Intrinsic Termination in Bacterial Transcription.

Two different molecular mechanisms, sliding and hopping, are employed by DNA-binding proteins for their one-dimensional facilitated diffusion on nonspecific DNA regions until reaching their specific target sequences. While it has been controversial whether RNA polymerases (RNAPs) use one-dimensional diffusion in targeting their promoters for transcription initiation, two recent single-molecule studies discovered that post-terminational RNAPs use one-dimensional diffusion for their reinitiation on the same DNA molecules. Escherichia coli RNAP, after synthesizing and releasing product RNA at intrinsic termination, mostly remains bound on DNA and diffuses in both forward and backward directions for recycling, which facilitates reinitiation on nearby promoters. However, it has remained unsolved which mechanism of one-dimensional diffusion is employed by recycling RNAP between termination and reinitiation. Single-molecule fluorescence measurements in this study reveal that post-terminational RNAPs undergo hopping diffusion during recycling on DNA, as their one-dimensional diffusion coefficients increase with rising salt concentrations. We additionally find that reinitiation can occur on promoters positioned in sense and antisense orientations with comparable efficiencies, so reinitiation efficiency depends primarily on distance rather than direction of recycling diffusion. This additional finding confirms that orientation change or flipping of RNAP with respect to DNA efficiently occurs as expected from hopping diffusion.

FRET-based dynamic structural biology: Challenges, perspectives and an appeal for open-science practices.

Single-molecule FRET (smFRET) has become a mainstream technique for studying biomolecular structural dynamics. The rapid and wide adoption of smFRET experiments by an ever-increasing number of groups has generated significant progress in sample preparation, measurement procedures, data analysis, algorithms and documentation. Several labs that employ smFRET approaches have joined forces to inform the smFRET community about streamlining how to perform experiments and analyze results for obtaining quantitative information on biomolecular structure and dynamics. The recent efforts include blind tests to assess the accuracy and the precision of smFRET experiments among different labs using various procedures. These multi-lab studies have led to the development of smFRET procedures and documentation, which are important when submitting entries into the archiving system for integrative structure models, PDB-Dev. This position paper describes the current 'state of the art' from different perspectives, points to unresolved methodological issues for quantitative structural studies, provides a set of 'soft recommendations' about which an emerging consensus exists, and lists openly available resources for newcomers and seasoned practitioners. To make further progress, we strongly encourage 'open science' practices.

Yeast Chd1p Unwraps the Exit Side DNA upon ATP Binding to Facilitate the Nucleosome Translocation Occurring upon ATP Hydrolysis.

Chromodomain-helicase-DNA-binding protein 1 (CHD1) remodels chromatin by translocating nucleosomes along DNA, but its mechanism remains poorly understood. We use single-molecule fluorescence experiments to clarify the mechanism by which yeast CHD1 (Chd1p) remodels nucleosomes. We find that binding of ATP to Chd1p induces transient unwrapping of the DNA on the exit side of the nucleosome, facilitating nucleosome translocation. ATP hydrolysis is required to induce nucleosome translocation. The unwrapped DNA after translocation is then rewrapped after the release of the hydrolyzed nucleotide and phosphate, revealing that each step of the ATP hydrolysis cycle is responsible for a distinct step of nucleosome remodeling. These results show that Chd1p remodels nucleosomes via a mechanism that is unique among the other ATP-dependent chromatin remodelers.

Quantification of purified endogenous miRNAs with high sensitivity and specificity.

MicroRNAs (miRNAs) are short (19-24 nt) non-coding RNAs that suppress the expression of protein coding genes at the post-transcriptional level. Differential expression profiles of miRNAs across a range of diseases have emerged as powerful biomarkers, making a reliable yet rapid profiling technique for miRNAs potentially essential in clinics. Here, we report an amplification-free multi-color single-molecule imaging technique that can profile purified endogenous miRNAs with high sensitivity, specificity, and reliability. Compared to previously reported techniques, our technique can discriminate single base mismatches and single-nucleotide 3'-tailing with low false positive rates regardless of their positions on miRNA. By preloading probes in Thermus thermophilus Argonaute (TtAgo), miRNAs detection speed is accelerated by more than 20 times. Finally, by utilizing the well-conserved linearity between single-molecule spot numbers and the target miRNA concentrations, the absolute average copy numbers of endogenous miRNA species in a single cell can be estimated. Thus our technique, Ago-FISH (Argonaute-based Fluorescence In Situ Hybridization), provides a reliable way to accurately profile various endogenous miRNAs on a single miRNA sensing chip.

Single-molecule fluorescence studies on cotranscriptional G-quadruplex formation coupled with R-loop formation.

G-quadruplex (GQ) is formed at various regions of DNA, including telomeres of chromosomes and regulatory regions of oncogenes. Since GQ is important in both gene regulation and genome instability, the biological and medical implications of this abnormal DNA structure have been intensively studied. Its formation mechanisms, however, are not clearly understood yet. We report single-molecule fluorescence experiments to monitor the cotranscriptional GQ formation coupled with R-loop formation using T7 RNA polymerase. The GQ is formed very rarely per single-round transcription. R-loop formation precedes and facilitates GQ formation. Once formed, some GQs are extremely stable, resistant even to RNase H treatment, and accumulate in multiple-round transcription conditions. On the other hand, GQ existing in the non-template strand promotes the R-loop formation in the next rounds of transcription. Our study clearly shows the existence of a positive feedback mechanism of GQ and R-loop formations, which may possibly contribute to gene regulation and genome instability.

Transcription reinitiation by recycling RNA polymerase that diffuses on DNA after releasing terminated RNA.

Despite extensive studies on transcription mechanisms, it is unknown how termination complexes are disassembled, especially in what order the essential components dissociate. Our single-molecule fluorescence study unveils that RNA transcript release precedes RNA polymerase (RNAP) dissociation from the DNA template much more often than their concurrent dissociations in intrinsic termination of bacterial transcription. As termination is defined by the release of product RNA from the transcription complex, the subsequent retention of RNAP on DNA constitutes a previously unidentified stage, termed here as recycling. During the recycling stage, post-terminational RNAPs one-dimensionally diffuse on DNA in downward and upward directions, and can initiate transcription again at the original and nearby promoters in the case of retaining a sigma factor. The efficiency of this event, termed here as reinitiation, increases with supplement of a sigma factor. In summary, after releasing RNA product at intrinsic termination, recycling RNAP diffuses on the DNA template for reinitiation most of the time.

Single-Molecule FRET Assay for Studying Cotranscriptional RNA Folding.

Cotranscriptional RNA folding plays important roles in gene regulation steps such as splicing, transcription termination, and translation initiation. Progression of our understanding of cotranscriptional RNA folding mechanisms is still retarded by the lacking of experimental tools to study the kinetics of cotranscriptional RNA folding properly. In this chapter, we describe fluorescence resonance energy transfer (FRET) assay that enables the study of RNA cotranscriptional folding at the single-molecule level.

ATAD5 promotes replication restart by regulating RAD51 and PCNA in response to replication stress.

Maintaining stability of replication forks is important for genomic integrity. However, it is not clear how replisome proteins contribute to fork stability under replication stress. Here, we report that ATAD5, a PCNA unloader, plays multiple functions at stalled forks including promoting its restart. ATAD5 depletion increases genomic instability upon hydroxyurea treatment in cultured cells and mice. ATAD5 recruits RAD51 to stalled forks in an ATR kinase-dependent manner by hydroxyurea-enhanced protein-protein interactions and timely removes PCNA from stalled forks for RAD51 recruitment. Consistent with the role of RAD51 in fork regression, ATAD5 depletion inhibits slowdown of fork progression and native 5-bromo-2'-deoxyuridine signal induced by hydroxyurea. Single-molecule FRET showed that PCNA itself acts as a mechanical barrier to fork regression. Consequently, DNA breaks required for fork restart are reduced by ATAD5 depletion. Collectively, our results suggest an important role of ATAD5 in maintaining genome integrity during replication stress.

Structural basis of recognition and destabilization of the histone H2B ubiquitinated nucleosome by the DOT1L histone H3 Lys79 methyltransferase.

DOT1L is a histone H3 Lys79 methyltransferase whose activity is stimulated by histone H2B Lys120 ubiquitination, suggesting cross-talk between histone H3 methylation and H2B ubiquitination. Here, we present cryo-EM structures of DOT1L complexes with unmodified or H2B ubiquitinated nucleosomes, showing that DOT1L recognizes H2B ubiquitin and the H2A/H2B acidic patch through a C-terminal hydrophobic helix and an arginine anchor in DOT1L, respectively. Furthermore, the structures combined with single-molecule FRET experiments show that H2B ubiquitination enhances a noncatalytic function of the DOT1L-destabilizing nucleosome. These results establish the molecular basis of the cross-talk between H2B ubiquitination and H3 Lys79 methylation as well as nucleosome destabilization by DOT1L.

Accelerated FRET-PAINT microscopy.

Recent development of FRET-PAINT microscopy significantly improved the imaging speed of DNA-PAINT, the previously reported super-resolution fluorescence microscopy with no photobleaching problem. Here we try to achieve the ultimate speed limit of FRET-PAINT by optimizing the camera speed, dissociation rate of DNA probes, and bleed-through of the donor signal to the acceptor channel, and further increase the imaging speed of FRET-PAINT by 8-fold. Super-resolution imaging of COS-7 microtubules shows that high-quality 40-nm resolution images can be obtained in just tens of seconds.