Elastic coupling of nascent apCAM adhesions to flowing actin networks.

Adhesions are multi-molecular complexes that transmit forces generated by a cell's acto-myosin networks to external substrates. While the physical properties of some of the individual components of adhesions have been carefully characterized, the mechanics of the coupling between the cytoskeleton and the adhesion site as a whole are just beginning to be revealed. We characterized the mechanics of nascent adhesions mediated by the immunoglobulin-family cell adhesion molecule apCAM, which is known to interact with actin filaments. Using simultaneous visualization of actin flow and quantification of forces transmitted to apCAM-coated beads restrained with an optical trap, we found that adhesions are dynamic structures capable of transmitting a wide range of forces. For forces in the picoNewton scale, the nascent adhesions' mechanical properties are dominated by an elastic structure which can be reversibly deformed by up to 1 µm. Large reversible deformations rule out an interface between substrate and cytoskeleton that is dominated by a number of stiff molecular springs in parallel, and favor a compliant cross-linked network. Such a compliant structure may increase the lifetime of a nascent adhesion, facilitating signaling and reinforcement.

High-resolution dynamic mapping of histone-DNA interactions in a nucleosome.

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.

Probing SWI/SNF remodeling of the nucleosome by unzipping single DNA molecules.

Chromatin-remodeling enzymes can overcome strong histone-DNA interactions within the nucleosome to regulate access of DNA-binding factors to the genetic code. By unzipping individual DNA duplexes, each containing a uniquely positioned nucleosome flanked by long segments of DNA, we directly probed histone-DNA interactions. The resulting disruption-force signatures were characteristic of the types and locations of interactions and allowed measurement of the positions of nucleosomes with 2.6-base-pair (bp) precision. Nucleosomes remodeled by yeast SWI/SNF were moved bidirectionally along the DNA, resulting in a continuous position distribution. The characteristic distance of motion was approximately 28 bp per remodeling event, and each event occurred with a catalytic efficiency of 0.4 min(-1) per nM SWI/SNF. Remodeled nucleosomes had essentially identical disruption signatures to those of unremodeled nucleosomes, indicating that their overall structure remained canonical. These results impose substantial constraints on the mechanism of SWI/SNF remodeling.

Sequence-dependent kinetic model for transcription elongation by RNA polymerase.

We present a kinetic model for the sequence-dependent motion of RNA polymerase (RNAP) during transcription elongation. For each NTP incorporation, RNAP has a net forward translocation of one base-pair along the DNA template. However, this process may involve the exploration of back-tracked and forward-tracked translocation modes. In our model, the kinetic rates for the reaction pathway, calculated based on the stabilities of the transcription elongation complex (TEC), necessarily lead to sequence-dependent NTP incorporation rates. Simulated RNAP elongation kinetics is in good agreement with data from transcription gels and single-molecule studies. The model provides a kinetic explanation for well-known back-tracked pauses at transcript positions with unstable TECs. It also predicts a new type of pause caused by an energetically unfavorable transition from pre to post-translocation modes.

A single-molecule technique to study sequence-dependent transcription pausing.

We present a technique that allows sequence-dependent analysis of transcription elongation using single-molecule optical trapping techniques. Observation of individual molecules of RNA polymerase (RNAP) allows determination of elongation kinetics that are difficult or impossible to accurately obtain from bulk studies, and provides high temporal resolution of the RNAP motion under a calibrated mechanical load. One limitation of previous single molecule studies was the difficulty in correlating the observed motion of RNAP with its actual position on the DNA template to better than approximately 100 bp. In this work, we improved the spatial precision of optical trapping studies of transcription to approximately 5 bp by using runoff transcription as an unambiguous marker of RNAP template position. This runoff method was sufficient to unequivocally locate and study a single known pause sequence (DeltatR2). By applying various loads to assist RNAP forward translocation, we specifically investigated elongation kinetics within this pause region and found that the dwell time at the pause sequence decreased with increasing assisting load. This observation is consistent with bulk biochemical studies that suggest RNAP reverse translocates, or "backtracks," at the DeltatR2 pause sequence.

Probing protein-DNA interactions by unzipping a single DNA double helix.

We present unzipping force analysis of protein association (UFAPA) as a novel and versatile method for detection of the position and dynamic nature of protein-DNA interactions. A single DNA double helix was unzipped in the presence of DNA-binding proteins using a feedback-enhanced optical trap. When the unzipping fork in a DNA reached a bound protein molecule we observed a dramatic increase in the tension in the DNA, followed by a sudden tension reduction. Analysis of the unzipping force throughout an unbinding "event" revealed information about the spatial location and dynamic nature of the protein-DNA complex. The capacity of UFAPA to spatially locate protein-DNA interactions is demonstrated by noncatalytic restriction mapping on a 4-kb DNA with three restriction enzymes (BsoBI, XhoI, and EcoRI). A restriction map for a given restriction enzyme was generated with an accuracy of approximately 25 bp. UFAPA also allows direct determination of the site-specific equilibrium association constant (K(A)) for a DNA-binding protein. This capability is demonstrated by measuring the cation concentration dependence of K(A) for EcoRI binding. The measured values are in good agreement with previous measurements of K(A) over an intermediate range of cation concentration. These results demonstrate the potential utility of UFAPA for future studies of site-specific protein-DNA interactions.