Macromolecular crowding has opposite effects on two critical sub-steps of transcription initiation.

Transcription initiation, the first step in gene expression, has been studied extensively in dilute buffer, a condition which fails to consider the crowded environment in live cells. Recent reports indicate the kinetics of promoter escape is altered in crowded conditions for a consensus bacterial promoter. Here, we use a real-time fluorescence enhancement assay to study the kinetics of unwound bubble formation and promoter escape for three separate promoters. We find that the effect of crowding on transcription initiation is complex, with lower rates of unwound bubble formation, higher rates of promoter escape, and large variations depending on promoter identity. Based on our results, we suggest that altered conditions of crowding inside a live cell can trigger global changes.

A new twist on PIFE: photoisomerisation-related fluorescence enhancement.

PIFE was first used as an acronym for protein-induced fluorescence enhancement, which refers to the increase in fluorescence observed upon the interaction of a fluorophore, such as a cyanine, with a protein. This fluorescence enhancement is due to changes in the rate ofcis/transphotoisomerisation. It is clear now that this mechanism is generally applicable to interactions with any biomolecule. In this review, we propose that PIFE is thereby renamed according to its fundamental working principle as photoisomerisation-related fluorescence enhancement, keeping the PIFE acronym intact. We discuss the photochemistry of cyanine fluorophores, the mechanism of PIFE, its advantages and limitations, and recent approaches to turning PIFE into a quantitative assay. We provide an overview of its current applications to different biomolecules and discuss potential future uses, including the study of protein-protein interactions, protein-ligand interactions and conformational changes in biomolecules.

Bleaching-resistant, Near-continuous Single-molecule Fluorescence and FRET Based on Fluorogenic and Transient DNA Binding.

Photobleaching of fluorescent probes limits the observation span of typical single-molecule fluorescence measurements and hinders observation of dynamics at long timescales. Here, we present a general strategy to circumvent photobleaching by replenishing fluorescent probes via transient binding of fluorogenic DNAs to complementary DNA strands attached to a target molecule. Our strategy allows observation of near-continuous single-molecule fluorescence for more than an hour, a timescale two orders of magnitude longer than the typical photobleaching time of single fluorophores under our conditions. Using two orthogonal sequences, we show that our method is adaptable to Förster Resonance Energy Transfer (FRET) and that can be used to study the conformational dynamics of dynamic structures, such as DNA Holliday junctions, for extended periods. By adjusting the temporal resolution and observation span, our approach enables capturing the conformational dynamics of proteins and nucleic acids over a wide range of timescales.

A new twist on PIFE: photoisomerisation-related fluorescence enhancement.

PIFE was first used as an acronym for protein-induced fluorescence enhancement, which refers to the increase in fluorescence observed upon the interaction of a fluorophore, such as a cyanine, with a protein. This fluorescence enhancement is due to changes in the rate of cis/trans photoisomerisation. It is clear now that this mechanism is generally applicable to interactions with any biomolecule and, in this review, we propose that PIFE is thereby renamed according to its fundamental working principle as photoisomerisation-related fluorescence enhancement, keeping the PIFE acronym intact. We discuss the photochemistry of cyanine fluorophores, the mechanism of PIFE, its advantages and limitations, and recent approaches to turn PIFE into a quantitative assay. We provide an overview of its current applications to different biomolecules and discuss potential future uses, including the study of protein-protein interactions, protein-ligand interactions and conformational changes in biomolecules.

Genome-wide identification and development of miniature inverted-repeat transposable elements and intron length polymorphic markers in tea plant (Camellia sinensis).

Marker-assisted breeding and tagging of important quantitative trait loci for beneficial traits are two important strategies for the genetic improvement of plants. However, the scarcity of diverse and informative genetic markers covering the entire tea genome limits our ability to achieve such goals. In the present study, we used a comparative genomic approach to mine the tea genomes of Camellia sinensis var. assamica (CSA) and C. sinensis var. sinensis (CSS) to identify the markers to differentiate tea genotypes. In our study, 43 and 60 Camellia sinensis miniature inverted-repeat transposable element (CsMITE) families were identified in these two sequenced tea genomes, with 23,170 and 37,958 putative CsMITE sequences, respectively. In addition, we identified 4912 non-redundant, Camellia sinensis intron length polymorphic (CsILP) markers, 85.8% of which were shared by both the CSS and CSA genomes. To validate, a subset of randomly chosen 10 CsMITE markers and 15 CsILP markers were tested and found to be polymorphic among the 36 highly diverse tea genotypes. These genome-wide markers, which were identified for the first time in tea plants, will be a valuable resource for genetic diversity analysis as well as marker-assisted breeding of tea genotypes for quality improvement.

Transcription initiation at a consensus bacterial promoter proceeds via a 'bind-unwind-load-and-lock' mechanism.

Transcription initiation starts with unwinding of promoter DNA by RNA polymerase (RNAP) to form a catalytically competent RNAP-promoter complex (RPo). Despite extensive study, the mechanism of promoter unwinding has remained unclear, in part due to the transient nature of intermediates on path to RPo. Here, using single-molecule unwinding-induced fluorescence enhancement to monitor promoter unwinding, and single-molecule fluorescence resonance energy transfer to monitor RNAP clamp conformation, we analyse RPo formation at a consensus bacterial core promoter. We find that the RNAP clamp is closed during promoter binding, remains closed during promoter unwinding, and then closes further, locking the unwound DNA in the RNAP active-centre cleft. Our work defines a new, 'bind-unwind-load-and-lock', model for the series of conformational changes occurring during promoter unwinding at a consensus bacterial promoter and provides the tools needed to examine the process in other organisms and at other promoters.

Isolation and characterization of two virulent Aeromonads associated with haemorrhagic septicaemia and tail-rot disease in farmed climbing perch Anabas testudineus.

Diseased Anabas testudineus exhibiting signs of tail-rot and ulcerations on body were collected from a fish farm in Assam, India during the winter season (November 2018 to January 2019). Swabs from the infected body parts were streaked on sterilized nutrient agar. Two dominant bacterial colonies were obtained, which were then isolated and labelled as AM-31 and AM-05. Standard biochemical characterisation and 16S rRNA and rpoB gene sequencing identified AM-31 isolate as Aeromonas hydrophila and AM-05 as Aeromonas jandaei. Symptoms similar to that of natural infection were observed on re-infecting both bacteria to disease-free A. testudineus, which confirmed their virulence. LC50 was determined at 1.3 × 104 (A. hydrophila) and 2.5 × 104 (A. jandaei) CFU per fish in intraperitoneal injection. Further, PCR amplification of specific genes responsible for virulence (aerolysin and enterotoxin) confirmed pathogenicity of both bacteria. Histopathology of kidney and liver in the experimentally-infected fishes revealed haemorrhage, tubular degeneration and vacuolation. Antibiotic profiles were also assessed for both bacteria. To the best of our knowledge, the present work is a first report on the mortality of farmed climbing perch naturally-infected by A. hydrophila as well as A. jandaei, with no records of pathogenicity of the latter in this fish.

RNA polymerase clamp conformational dynamics: long-lived states and modulation by crowding, cations, and nonspecific DNA binding.

The RNA polymerase (RNAP) clamp, a mobile structural element conserved in RNAP from all domains of life, has been proposed to play critical roles at different stages of transcription. In previous work, we demonstrated using single-molecule Förster resonance energy transfer (smFRET) that RNAP clamp interconvert between three short-lived conformational states (lifetimes ∼ 0.3-0.6 s), that the clamp can be locked into any one of these states by small molecules, and that the clamp stays closed during initial transcription and elongation. Here, we extend these studies to obtain a comprehensive understanding of clamp dynamics under conditions RNAP may encounter in living cells. We find that the RNAP clamp can populate long-lived conformational states (lifetimes > 1.0 s) and can switch between these long-lived states and the previously observed short-lived states. In addition, we find that clamp motions are increased in the presence of molecular crowding, are unchanged in the presence of elevated monovalent-cation concentrations, and are reduced in the presence of elevated divalent-cation concentrations. Finally, we find that RNAP bound to non-specific DNA predominantly exhibits a closed clamp conformation. Our results raise the possibility of additional regulatory checkpoints that could affect clamp dynamics and consequently could affect transcription and transcriptional regulation.

Genome-wide association studies using 50 K rice genic SNP chip unveil genetic architecture for anaerobic germination of deep-water rice population of Assam, India.

North Eastern part of India such as Assam is inundated by flood every year where the farmers are forced to grow the traditional tall deep-water rice. Genetic improvement of this type of rice is slow because of insufficient knowledge about their genetic architecture and population structure. In the present investigation, the genetic diversity architecture of 94 deep-water rice genotypes of Assam and association mapping strategy was, for the first time, applied to determine the significant SNPs and genes for deep-water rice. These genotypes are known for their unique elongation ability under deep-water condition. The anaerobic germination (AG) related trait-associated genes identified here can provide affluent resources for rice breeding especially in flood-prone areas. We investigated the genome-wide association studies (GWAS) using 50 K rice genic SNP chip across 94 deep-water rice genotypes collected from different flood-prone districts/villages of Assam. Population structure and diversity analysis revealed that these genotypes were stratified into four sub-populations. Using GWAS approach, 20 significant genes were identified and found to be associated with AG-related traits. Of them, two most relevant genes (OsXDH1and SSXT) have been identified which explain phenotypic variability (R2 > 20%) in the population. These genes were located in Chr 3 (LOC_Os03g31550) which encodes for enzyme xanthine dehydrogenase 1(OsXDH1) and in Chr 12 (LOC_Os12g31350) which encodes for SSXT family protein. Both of these genes were found to be associated with anaerobic response index (increase in the coleoptile length under water in anaerobic condition with respect to control), respectively. Interestingly, OsXDH1is involved in purine catabolism pathway and acts as a scavenger of reactive oxygen species in plants, whereas SSXT is GRF1-interacting factor 3. These two candidate genes associated with AG of deep-water rice have been found to be reported for the first time. Thus, this study provides a greater resource for breeders not only for improvement of deep-water rice, but also for AG tolerant variety useful for direct-seeded rice in flood-affected areas.

Closing and opening of the RNA polymerase trigger loop.

The RNA polymerase (RNAP) trigger loop (TL) is a mobile structural element of the RNAP active center that, based on crystal structures, has been proposed to cycle between an "unfolded"/"open" state that allows an NTP substrate to enter the active center and a "folded"/"closed" state that holds the NTP substrate in the active center. Here, by quantifying single-molecule fluorescence resonance energy transfer between a first fluorescent probe in the TL and a second fluorescent probe elsewhere in RNAP or in DNA, we detect and characterize TL closing and opening in solution. We show that the TL closes and opens on the millisecond timescale; we show that TL closing and opening provides a checkpoint for NTP complementarity, NTP ribo/deoxyribo identity, and NTP tri/di/monophosphate identity, and serves as a target for inhibitors; and we show that one cycle of TL closing and opening typically occurs in each nucleotide addition cycle in transcription elongation.

Transcriptional dynamics of Zn-accumulation in developing kernels of maize reveals important Zn-uptake mechanisms.

In the present study, transcriptomic analysis of 10-days old baby kernels of two contrasting maize genotypes, namely VQL-2 (high kernel Zn accumulator) and CM-145 (low kernel Zn accumulator), under low- and optimum- soil Zn conditions generated 1948 differentially expressed transcripts. Among these, 666 and 437 transcripts were up-regulated and down-regulated respectively in VQL-2; whereas, 437 and 408 transcripts were up-regulated and down-regulated respectively in CM-145. Remarkably, 135 transcription factors and 77 known Zn transporters expressed differentially. By comparing the transcripts differentially expressed between the optimum-Zn and low-Zn libraries of the contrasting genotypes, we identified 21,986 and 26,871 SNPs, respectively. Similarly, 6810 and 8192 InDels were found between optimum- and low-Zn growing conditions, respectively. Further, 21 differentially expressed genes were co-localized with already known QTLs associated with Zn uptake, such as qZn10, CQZnK9-1 and YNZnK6. These findings will be useful to develop high Zn-accumulator maize through marker-assisted breeding in future.

Psilorhynchus nahlongthai, a new fish species (Teleostei: Psilorhynchidae) from the Brahmaputra drainage, northeast India.

Psilorhynchus nahlongthai, a new psilorhynchid fish, is described from the Diyung River, a tributary to the Kopili River (itself a southern tributary of the Brahmaputra drainage) in Assam, northeast India. It is placed in the Psilorhynchus balitora species group and can be easily distinguished from all other members of this group by a combination of the following characters: dense and prominent tuberculation on the head region; thick and long pre- and post-epiphyseal fontanelles on the neurocranium; 9 + 8 caudal-fin rays; and 34 (24 + 10) vertebrae. Genetic divergence between P. nahlongthai and members of the P. balitora species group from the Brahmaputra and neighbouring drainages, with K2P distances ranging 3.7%-14.7% and 7.4%-20.7% in the mitochondrial COI and cyt b gene datasets respectively, support its report as a new species.

Confinement-Free Wide-Field Ratiometric Tracking of Single Fluorescent Molecules.

Single-molecule fluorescence has been highly instrumental in elucidating interactions and dynamics of biological molecules in the past two decades. Single-molecule fluorescence experiments usually rely on one of two detection geometries, either confocal point-detection or wide-field area detection, typically in a total internal reflection fluorescence (TIRF) format. However, each of these techniques suffers from fundamental drawbacks that limit their application. In this work, we present a new technique, solution wide-field imaging (SWiFi) of diffusing molecules, as an alternative to the existing methods. SWiFi is a simple extension to existing objective-type TIRF microscopes that allows wide-field observations of fast-diffusing molecules down to single fluorophores without the need of tethering the molecules to the surface. We demonstrate that SWiFi enables high-throughput ratiometric measurements with several thousands of individual data points per minute on double-stranded DNA standard (dsDNA) samples containing Förster resonance energy transfer pairs. We further display the capabilities of SWiFi by reporting on mobility and ratiometric characterization of fluorescent nanodiamonds, DNA Holliday junctions, and protein-DNA interactions. The ability of SWiFi for high-throughput, ratiometric measurements of fast-diffusing species renders it a valuable tool for the single-molecule research community by bridging between confocal and TIRF detection geometries in a simple and efficient way.

Recent Advances in Understanding σ70-Dependent Transcription Initiation Mechanisms.

Prokaryotic transcription is one of the most studied biological systems, with relevance to many fields including the development and use of antibiotics, the construction of synthetic gene networks, and the development of many cutting-edge methodologies. Here, we discuss recent structural, biochemical, and single-molecule biophysical studies targeting the mechanisms of transcription initiation in bacteria, including the formation of the open complex, the reaction of initial transcription, and the promoter escape step that leads to elongation. We specifically focus on the mechanisms employed by the RNA polymerase holoenzyme with the housekeeping sigma factor σ70. The recent progress provides answers to long-held questions, identifies intriguing new behaviours, and opens up fresh questions for the field of transcription.

The RNA polymerase clamp interconverts dynamically among three states and is stabilized in a partly closed state by ppGpp.

RNA polymerase (RNAP) contains a mobile structural module, the 'clamp,' that forms one wall of the RNAP active-center cleft and that has been linked to crucial aspects of the transcription cycle, including promoter melting, transcription elongation complex stability, transcription pausing, and transcription termination. Using single-molecule FRET on surface-immobilized RNAP molecules, we show that the clamp in RNAP holoenzyme populates three distinct conformational states and interconvert between these states on the 0.1-1 s time-scale. Similar studies confirm that the RNAP clamp is closed in open complex (RPO) and in initial transcribing complexes (RPITC), including paused initial transcribing complexes, and show that, in these complexes, the clamp does not exhibit dynamic behaviour. We also show that, the stringent-response alarmone ppGpp, which reprograms transcription during amino acid starvation stress, selectively stabilizes the partly-closed-clamp state and prevents clamp opening; these results raise the possibility that ppGpp controls promoter opening by modulating clamp dynamics.

Structural Basis of Transcription Inhibition by Fidaxomicin (Lipiarmycin A3).

Fidaxomicin is an antibacterial drug in clinical use for treatment of Clostridium difficile diarrhea. The active ingredient of fidaxomicin, lipiarmycin A3 (Lpm), functions by inhibiting bacterial RNA polymerase (RNAP). Here we report a cryo-EM structure of Mycobacterium tuberculosis RNAP holoenzyme in complex with Lpm at 3.5-Å resolution. The structure shows that Lpm binds at the base of the RNAP "clamp." The structure exhibits an open conformation of the RNAP clamp, suggesting that Lpm traps an open-clamp state. Single-molecule fluorescence resonance energy transfer experiments confirm that Lpm traps an open-clamp state and define effects of Lpm on clamp dynamics. We suggest that Lpm inhibits transcription by trapping an open-clamp state, preventing simultaneous interaction with promoter -10 and -35 elements. The results account for the absence of cross-resistance between Lpm and other RNAP inhibitors, account for structure-activity relationships of Lpm derivatives, and enable structure-based design of improved Lpm derivatives.

Characterization of OglDREB2A gene from African rice (Oryza glaberrima), comparative analysis and its transcriptional regulation under salinity stress.

In this study, AP2 DNA-binding domain-containing transcription factor, OglDREB2A, was cloned from the African rice (Oryza glaberrima) and compared with 3000 rice genotypes. Further, the phylogenetic and various structural analysis was performed using in silico approaches. Further, to understand its allelic variation in rice, SNPs and indels were detected among the 3000 rice genotypes which indicated that while coding region is highly conserved, yet noncoding regions such as UTR and intron contained most of the variation. Phylogenetic analysis of the OglDREB2A sequence in different Oryza as well as in diverse eudicot species revealed that DREB from various Oryza species were diversed much earlier than other genes. Further, structural features and in silico analyses provided insights into different properties of OglDREB2A protein. The neutrality test on the coding region of OglDREB2A from different genotypes of O. glaberrima showed the lack of selection in this gene. Among the different developmental stages, it was upregulated at tillering and flag leaf under salinity treatment indicating its positive role in seedling and reproductive stage tolerance. Real-time PCR analysis also indicated the conserve expression pattern of this gene under salinity stress across the three different Oryza species having different degree of salinity tolerance.

A peptide-based synthetic transcription factor selectively activates transcription in a mammalian cell.

A peptide-based cell permeable synthetic transcription factor is reported, which binds to its target site with high affinity and specificity. When linked to a HAT-binding peptide, it causes significant upregulation of gene expression in a mammalian cell. Such molecules may be developed for selectively activating repressed genes in mammalian cells.

Specific DNA sequences allosterically enhance protein-protein interaction in a transcription factor through modulation of protein dynamics: implications for specificity of gene regulation.

Most genes are regulated by multiple transcription factors, often assembling into multi-protein complexes in the gene regulatory region. Understanding of the molecular origin of specificity of gene regulatory complex formation in the context of the whole genome is currently inadequate. A phage transcription factor λ-CI forms repressive multi-protein complexes by binding to multiple binding sites in the genome to regulate the lifecycle of the phage. The protein-protein interaction between two DNA-bound λ-CI molecules is stronger when they are bound to the correct pair of binding sites, suggesting allosteric transmission of recognition of correct DNA sequences to the protein-protein interaction interface. Exploration of conformation and dynamics by time-resolved fluorescence anisotropy decay and molecular dynamics suggests a change in protein dynamics to be a crucial factor in mediating allostery. A lattice-based model suggests that DNA-sequence induced allosteric effects could be crucial underlying factors in differentially stabilizing the correct site-specific gene regulatory complexes. We conclude that transcription factors have evolved multiple mechanisms to augment the specificity of DNA-protein interactions in order to achieve an extraordinarily high degree of spatial and temporal specificities of gene regulatory complexes, and DNA-sequence induced allostery plays an important role in the formation of sequence-specific gene regulatory complexes.

Site-specific incorporation of probes into RNA polymerase by unnatural-amino-acid mutagenesis and Staudinger-Bertozzi ligation.

A three-step procedure comprising (1) unnatural-amino-acid mutagenesis with 4-azido-phenylalanine, (2) Staudinger-Bertozzi ligation with a probe-phosphine derivative, and (3) in vitro reconstitution of RNA polymerase (RNAP) enables the efficient site-specific incorporation of a fluorescent probe, a spin label, a cross-linking agent, a cleaving agent, an affinity tag, or any other biochemical or biophysical probe, at any site of interest in RNAP. Straightforward extensions of the procedure enable the efficient site-specific incorporation of two or more different probes in two or more different subunits of RNAP. We present protocols for synthesis of probe-phosphine derivatives, preparation of RNAP subunits and the transcription initiation factor σ, unnatural amino acid mutagenesis of RNAP subunits and σ, Staudinger ligation with unnatural-amino-acid-containing RNAP subunits and σ, quantitation of labelling efficiency and labelling specificity, and reconstitution of RNAP.