Fabrication of birefringent nanocylinders for single-molecule force and torque measurement.

Optically anisotropic subwavelength scale dielectric particles have been shown to enable studies of the mechanical properties of bio-molecules via optical trapping and manipulation. However, techniques emphasized to date for fabrication of such particles generally suffer from limited uniformity and control over particle dimensions, or low throughput and high cost. Here, an approach for rapid, low-cost, fabrication of large quantities of birefringent quartz nanocylinders with dimensions optimized for optical torque wrench experiments is described.

Single-molecule measurements of the CCR5 mRNA unfolding pathways.

Secondary or tertiary structure in an mRNA, such as a pseudoknot, can create a physical barrier that requires the ribosome to generate additional force to translocate. The presence of such a barrier can dramatically increase the probability that the ribosome will shift into an alternate reading frame, in which a different set of codons is recognized. The detailed biophysical mechanism by which frameshifting is induced remains unknown.

Effect of handle length and microsphere size on transition kinetics in single-molecule experiments.

When subject to constant tension, a DNA or RNA hairpin will typically make abrupt transitions between the open and closed state. Although the transition kinetics are an intrinsic property of the molecule, the transition rates measured in single-molecule experiments can be influenced by the configuration of the measurement system. We investigate the transition kinetics for a DNA hairpin held under constant force by an optical trap as a function of microsphere size and double-stranded DNA handle length.

Single-molecule study of G-quadruplex disruption using dynamic force spectroscopy.

Guanine-rich sequences in nucleic acids can fold into G quadruplexes, in which four guanines on a single strand combine to form G-tetrad planes stabilized by metallic ions. Sequence motifs which are predicted to form a G quadruplex are found throughout the genome and are believed to regulate a variety of biological processes. Detailed knowledge of the kinetics of G-quadruplex folding and unfolding would provide critical insight into these processes.

Interferometric detection of action potentials.

Individual action potentials in axons can be detected in vitro using intrinsic optical signals. The principles of this detection technique are described here. The technique has been demonstrated using neuronal axons dissected from lobster.

Applied force provides insight into transcriptional pausing and its modulation by transcription factor NusA.

Transcriptional pausing by RNA polymerase (RNAP) plays an essential role in gene regulation. Pausing is modified by various elongation factors, including prokaryotic NusA, but the mechanisms underlying pausing and NusA function remain unclear. Alternative models for pausing invoke blockade events that precede translocation (on-pathway), enzyme backtracking (off-pathway), or isomerization to a nonbacktracked, elemental pause state (off-pathway).

Noise associated with nonconservative forces in optical traps.

It is known that for a particle held in an optical trap the interaction of thermal fluctuations with a nonconservative scattering force can cause a persistent nonequilibrium probability flux in the particle position. We investigate position fluctuations associated with this nonequilibrium flux analytically and through simulation. We introduce a model which reproduces the nonequilibrium effects, and in which the magnitude of additional position fluctuations can be calculated in closed form.

Measuring the folding landscape of a harmonically constrained biopolymer.

Pioneering studies have shown that the probability distribution of opening length for a DNA hairpin, recorded under constant force using an optical trap, can be used to reconstruct the energy landscape of the transition. However, measurements made under constant force are subject to some limitations. Under constant force a system with a sufficiently high energy barrier spends most of its time in the closed or open conformation, with relatively few statistics collected in the transition state region.

E. coli NusG inhibits backtracking and accelerates pause-free transcription by promoting forward translocation of RNA polymerase.

NusG is an essential transcription factor in Escherichia coli that is capable of increasing the overall rate of transcription. Transcript elongation by RNA polymerase (RNAP) is frequently interrupted by pauses of varying durations, and NusG is known to decrease the occupancy of at least some paused states. However, it has not been established whether NusG enhances transcription chiefly by (1) increasing the rate of elongation between pauses, (2) reducing the lifetimes of pauses, or (3) reducing the rate of entry into paused states.

Sequence-resolved detection of pausing by single RNA polymerase molecules.

Transcriptional pausing by RNA polymerase (RNAP) plays an important role in the regulation of gene expression. Defined, sequence-specific pause sites have been identified biochemically. Single-molecule studies have also shown that bacterial RNAP pauses frequently during transcriptional elongation, but the relationship of these "ubiquitous" pauses to the underlying DNA sequence has been uncertain.

Molecular mechanism of transcription inhibition by peptide antibiotic Microcin J25.

21 amino acid peptide Microcin J25 (MccJ25) inhibits transcription by bacterial RNA polymerase (RNAP). MccJ25-resistance mutations cluster in the RNAP secondary channel through which incoming NTP substrates are thought to reach the catalytic center and the 3' end of the nascent RNA is likely to thread in backtracked transcription complexes. The secondary channel also accepts transcript cleavage factors GreA and GreB.

Optical torque wrench: angular trapping, rotation, and torque detection of quartz microparticles.

We describe an apparatus that can measure the instantaneous angular displacement and torque applied to a quartz particle which is angularly trapped. Torque is measured by detecting the change in angular momentum of the transmitted trap beam. The rotational Brownian motion of the trapped particle and its power spectral density are used to determine the angular trap stiffness.

Single molecule analysis of RNA polymerase elongation reveals uniform kinetic behavior.

By using single-molecule measurements, we demonstrate that the elongation kinetics of individual Escherichia coli RNA polymerase molecules are remarkably homogeneous. We find no evidence of distinct elongation states among RNA polymerases. Instead, the observed heterogeneity in transcription rates results from statistical variation in the frequency and duration of pausing.