Search

CRISPR (clustered regularly interspaced short palindromic repeats) utility relies on a stable Cas effector complex binding to its target site. However, a Cas complex bound to DNA may be removed by motor proteins carrying out host processes and the mechanism governing this removal remains unclear. Intriguingly, during CRISPR interference, RNA polymerase (RNAP) progression is only fully blocked by a bound endonuclease-deficient Cas (dCas) from the protospacer adjacent motif (PAM)-proximal side. By mapping dCas-DNA interactions at high resolution, we discovered that the collapse of the dCas R-loop allows Escherichia coli RNAP read-through from the PAM-distal side for both Sp–dCas9 and As–dCas12a. This finding is not unique to RNAP and holds for the Mfd translocase. This mechanistic understanding allowed us to modulate the dCas R-loop stability by modifying the guide RNAs. This work highlights the importance of the R-loop in dCas-binding stability and provides valuable mechanistic insights for broad applications of CRISPR technology.

During transcription, RNA polymerase (RNAP) supercoils DNA as it translocates. The resulting torsional stress in DNA can accumulate and, in the absence of regulatory mechanisms, becomes a barrier to RNAP elongation, causing RNAP stalling, backtracking, and transcriptional arrest. Here we investigate whether and how a transcription factor may regulate both torque-induced Escherichia coli RNAP stalling and the torque generation capacity of RNAP. Using a unique real-time angular optical trapping assay, we found that RNAP working against a resisting torque was highly prone to extensive backtracking. We then investigated transcription in the presence of GreB, a transcription factor known to rescue RNAP from the backtracked state. We found that GreB greatly suppressed RNAP backtracking and remarkably increased the torque that RNAP was able to generate by 65%, from 11.2 pN⋅nm to 18.5 pN·nm. Variance analysis of the real-time positional trajectories of RNAP after a stall revealed the kinetic parameters of backtracking and GreB rescue. These results demonstrate that backtracking is the primary mechanism by which torsional stress limits transcription and that the transcription factor GreB effectively enhances the torsional capacity of RNAP. These findings suggest a broader role for transcription factors in regulating RNAP functionality and elongation.

During transcription, RNA polymerase (RNAP) supercoils DNA as it forsward-translocates. Accumulation of this torsional stress in DNA can become a roadblock for an elongating RNAP and thus should be subject to regulation during transcription. Here, we investigate whether, and how, a transcription factor may regulate the torque generation capacity of RNAP and torque-induced RNAP stalling. Using a real-time assay based on an angular optical trap, we found that under a resisting torque, RNAP was highly prone to extensive backtracking. However, the presence of GreB, a transcription factor that facilitates the cleavage of the 3’ end of the extruded RNA transcript, greatly suppressed backtracking and remarkably increased the torque that RNAP was able to generate by 65%, from 11.2 to 18.5 pN·nm. Analysis of the real-time trajectories of RNAP position at a stall revealed the kinetic parameters of backtracking and GreB rescue. These results demonstrate that backtracking is the primary mechanism that limits transcription against DNA supercoiling and the transcription factor GreB effectively enhances the torsional capacity of RNAP. These findings broaden the potential impact of transcription factors on RNAP functionality.

Optical trapping is a powerful manipulation and measurement technique widely used in the biological and materials sciences. Miniaturizing optical trap instruments onto optofluidic platforms holds promise for high-throughput lab-on-a-chip applications. However, a persistent challenge with existing optofluidic devices has been achieving controlled and precise manipulation of trapped particles. Here, we report a new class of on-chip optical trapping devices. Using photonic interference functionalities, an array of stable, three-dimensional on-chip optical traps is formed at the antinodes of a standing-wave evanescent field on a nanophotonic waveguide. By employing the thermo-optic effect via integrated electric microheaters, the traps can be repositioned at high speed (∼30 kHz) with nanometre precision. We demonstrate sorting and manipulation of individual DNA molecules. In conjunction with laminar flows and fluorescence, we also show precise control of the chemical environment of a sample with simultaneous monitoring. Such a controllable trapping device has the potential to achieve high-throughput precision measurements on chip.

During transcription, RNA polymerase (RNAP) supercoils DNA as it translocates. The resulting torsional stress in DNA can accumulate and, in the absence of regulatory mechanisms, becomes a barrier to RNAP elongation, causing RNAP stalling, backtracking, and transcriptional arrest. Here we investigate whether and how a transcription factor may regulate both torque-induced Escherichia coli RNAP stalling and the torque generation capacity of RNAP. Using a unique real-time angular optical trapping assay, we found that RNAP working against a resisting torque was highly prone to extensive backtracking. We then investigated transcription in the presence of GreB, a transcription factor known to rescue RNAP from the backtracked state. We found that GreB greatly suppressed RNAP backtracking and remarkably increased the torque that RNAP was able to generate by 65%, from 11.2 pN⋅nm to 18.5 pN·nm. Variance analysis of the real-time positional trajectories of RNAP after a stall revealed the kinetic parameters of backtracking and GreB rescue. These results demonstrate that backtracking is the primary mechanism by which torsional stress limits transcription and that the transcription factor GreB effectively enhances the torsional capacity of RNAP. These findings suggest a broader role for transcription factors in regulating RNAP functionality and elongation.

The nature of the nucleosomal barrier that regulates access to the underlying DNA during many cellular processes is not fully understood. Here we present a detailed map of histone-DNA interactions along the DNA sequence to near base pair accuracy by mechanically unzipping single molecules of DNA, each containing a single nucleosome. This interaction map revealed a distinct approximately 5-bp periodicity that was enveloped by three broad regions of strong interactions, with the strongest occurring at the dyad and the other two about +/-40-bp from the dyad. Unzipping up to the dyad allowed recovery of a canonical nucleosome upon relaxation of the DNA, but unzipping beyond the dyad resulted in removal of the histone octamer from its initial DNA sequence. These findings have important implications for how RNA polymerase and other DNA-based enzymes may gain access to DNA associated with a nucleosome.

The distinct contributions of histone tails and their acetylation to nucleosomal stability were examined by mechanical disruption of individual nucleosomes in a single chromatin fiber using an optical trap. Enzymatic removal of H2A/H2B tails primarily decreased the strength of histone-DNA interactions located approximately +/-36bp from the dyad axis of symmetry (off-dyad strong interactions), whereas removal of the H3/H4 tails played a greater role in regulating the total amount of DNA bound. Similarly, nucleosomes composed of histones acetylated to different degrees by the histone acetyltransferase p300 exhibited significant decreases in the off-dyad strong interactions and the total amount of DNA bound. Acetylation of H2A/H2B appears to play a particularly critical role in weakening the off-dyad strong interactions. Collectively, our results suggest that the destabilizing effects of tail acetylation may be due to elimination of specific key interactions in the nucleosome.

Summary Cancer develops as a result of somatic mutation and clonal selection, but quantitative measures of selection in cancer evolution are lacking. We adapted methods from molecular evolution and applied them to 7,664 tumors across 29 cancer types. Unlike species evolution, positive selection outweighs negative selection during cancer development. On average, 10/tumor in endometrial and colorectal cancers. Half of driver substitutions occur in yet-to-be-discovered cancer genes. With increasing mutation burden, numbers of driver mutations increase, but not linearly. We systematically catalog cancer genes and show that genes vary extensively in what proportion of mutations are drivers versus passengers., Graphical Abstract, Highlights • Unlike the germline, somatic cells evolve predominantly by positive selection • Nearly all (∼99%) coding mutations are tolerated and escape negative selection • Exome-wide estimates of the total number of driver coding mutations per tumor • Half of the coding driver mutations occur outside of known cancer genes, Adapting an evolutionary genomics approach to cancer highlights a limited impact of negative selection on cancer genomes and significant variations in the proportion of coding driver mutations per tumor among different tumor types.

Optical trapping is a powerful manipulation and measurement technique widely employed in biological and materials sciences. Miniaturizing these instruments onto optofluidic platforms holds promise for high throughput lab-on-chip applications. However, a persistent challenge with existing optofluidic devices has been controlled and precise manipulation of trapped particles. Here we report a new class of on-chip optical trapping devices. Using on-chip optical interference functionalities, an array of ultra-stable, three-dimensional optical traps is formed by the evanescent field at the anti-nodes of a standing-wave in a nanophotonic waveguide. By employing the thermo-optic effect via integrated electric microheaters, the traps can be repositioned at high speed (∼ 30 kHz) with nanometer precision. Using this device, we demonstrate trapping, sorting, and transport of particles, as well as manipulation of individual DNA molecules. Such a controllable trapping device has the potential for high-throughput precision measurements on chip.

During transcription, both RNA polymerase (RNAP) and the nascent RNA chain need to rotate relative to DNA at a speed of approximately a few hundred rounds per minute. The resulting accumulation of torsional stress cannot be dissipated immediately, and is therefore able to impact the motion of the RNAP. Despite the fact that torque is an essential aspect of transcription, the extent of its impact remains unknown. By performing single molecule measurements using an angular optical trap, we directly measure the torque that RNAP can generate as well as the transcription rate under torque. This approach provides a framework for investigating the influence of torque on various DNA-based translocases.

Molecules may arrive at targets (receptors, enzymes, etc.) localized on a membrane surface by first adsorbing onto the surface and then surface diffusing to the targets. The flux rate of molecules arriving at targets via this mechanism depends on the surface diffusion coefficient of the molecules and, in some circumstances, on the adsorption/desorption kinetics. The technique of total internal reflection with fluorescence recovery after photobleaching (TIR-FRAP) was used here to study these rate parameters of fluorescein-labeled insulin (f-insulin) interacting with erythrocyte ghosts. Ghosts were adhered to polylysine coated slides for TIR illumination. Some ghosts became flattened and unsealed on the polylysine so that both extracellular and cytoplasmic sides of the membrane were openly exposed to the solution. An aluminum thin film between the polylysine and the fused silica of a slide quenched 'background' fluorescence from f-insulin adsorbed directly onto the polylysine. An interference fringe pattern from two intersecting and totally internally reflecting laser beams provided surface-selective excitation with a spatial variation of illumination intensity across a ghost for surface diffusion measurements. Measured characteristic values of desorption rate constants ranged from 0.043 to 270 s(-1). According to a preexisting theoretical model, the largest desorption rate constant in this range would result in some increase in the total flux rate to a perfect sink target due to capture from the surface, provided that the surface diffusion coefficient was ≥ about 10(-8) cm(2)/s. However, based on TIR-FRAP measurements on our system, we estimate that the surface diffusion coefficient is less than about 5×10(-10) cm(2)/s. The combination of novel techniques presented here may prove valuable to other workers seeking to make diffusive and chemical kinetic rate parameter measurements of biomolecules at biological cell membranes.

During gene expression, RNA polymerase (RNAP) encounters a major barrier at a nucleosome and yet it must access the nucleosomal DNA. Various lines of in vivo evidence suggest that multiple RNAPs might increase transcription efficiency through nucleosomal DNA. Here we have quantitatively investigated this hypothesis by using E. coli RNAP as a model system and directly monitoring its location on the DNA via a single molecule DNA unzipping technique. When a single RNAP encountered a nucleosome, it paused with a distinctive 10-bp periodicity and was backtracked by an average distance of ∼10-15 bp. When two RNAPs were elongating in close proximity, the trailing RNAP exerted an assisting force on the leading RNAP, reducing its backtracking and enhancing its transcription through a nucleosome ∼5-fold. Taken together, our data indicate that histone-DNA interactions within a nucleosome dictate RNAP pausing behavior, and that alleviation of nucleosome-induced backtracking by multiple polymerases is a likely mechanism for overcoming the nucleosomal barrier in vivo.

During gene expression, RNA polymerase (RNAP) encounters a major barrier at a nucleosome and yet must access the nucleosomal DNA. Previous in vivo evidence has suggested that multiple RNAPs might increase transcription efficiency through nucleosomes. Here we have quantitatively investigated this hypothesis using Escherichia coli RNAP as a model system by directly monitoring its location on the DNA via a single-molecule DNA-unzipping technique. When an RNAP encountered a nucleosome, it paused with a distinctive 10-base pair periodicity and backtracked by approximately 10-15 base pairs. When two RNAPs elongate in close proximity, the trailing RNAP apparently assists in the leading RNAP's elongation, reducing its backtracking and enhancing its transcription through a nucleosome by a factor of 5. Taken together, our data indicate that histone-DNA interactions dictate RNAP pausing behavior, and alleviation of nucleosome-induced backtracking by multiple polymerases may prove to be a mechanism for overcoming the nucleosomal barrier in vivo.

The mechanochemical kinetics of transcription elongation was examined with a combination of theoretical and experimental approaches. The predictive power of a sequence-dependent thermal ratchet model for transcription elongation was tested by establishing model parameters based solely on measurements under chemical perturbations and then directly predicting responses under mechanical perturbations without additional model parameters. Agreement between predicted and measured force-velocity curves provides strong support for a simple mechanochemical coupling mechanism.

The fluorescent probe diI was used to study the lateral mobility of lipids in in vitro strains of living adult human keratinocytes grown in four different media. One medium was essential fatty acid deficient (EFAD) and low in calcium ion, a medium known to yield cells that proliferate rapidly and contain lipid with extremely low levels of essential fatty acids. Two other media were supplemented with essential fatty acids (FAS), media that are known to result in cells that grow more slowly and have normalized fatty acid proportions. A fourth medium consisted of 1 microM all-trans-retinoic acid added to the fatty acid-supplemented medium (FAS-RA), a medium known to produce cells that are highly proliferative, with a growth rate greater than that of the FAS strains and similar to that of the EFAD strains. The keratinocytes grown in these four media were studied using the fluorescence recovery after photobleaching (FRAP) technique to determine the lateral diffusion rate of diI in the plasma membranes. Our results showed a positive correlation between growth rate and diffusion coefficient (D): the diffusion coefficient of diI was higher in the EFAD or FAS-RA cells than in the FAS cells. The measurement of D among the FAS cells fell into two groups. One group was similar to the single group seen in the EFAD cells, but the other group was composed of much lower D values. The other FRAP parameters (mobile fraction and bleach depth) were larger in the "slow" group than in the "fast" group. This trend of negative correlation between these parameters and D was also found within the fast group. These results are interpreted in terms of possible changes in membrane structure or morphology that might be indirectly associated with the fatty acid alterations, including the possible presence of areas in senescing keratinocytes where plasma membranes collapse to form an interacting system of lipid bilayers.

Laser/microscope fluorescence methods for studying adsorption/desorption kinetics and surface diffusion of molecules reversibly associated with biological membranes are discussed. These methods, which include fluorescence excitation by evanescent waves generated by total internal reflection, and fluorescence quenching by thin metal films, may also find application in the design of in vivo biosensors and in studies of transmembrane transport and chemical kinetics.