Ariel Kaplan

Substrate interactions and promiscuity in a viral DNA packaging motor

The ASCE (additional strand, conserved E) superfamily of proteins consists of structurally similar ATPases associated with diverse cellular activities involving metabolism and transport of proteins and nucleic acids in all forms of life. A subset of these enzymes consists of multimeric ringed pumps responsible for DNA transport in processes including genome packaging in adenoviruses, herpesviruses, poxviruses and tailed bacteriophages. Although their mechanism of mechanochemical conversion is beginning to be understood, little is known about how these motors engage their nucleic acid substrates. Questions remain as to whether the motors contact a single DNA element, such as a phosphate or a base, or whether contacts are distributed over several parts of the DNA. Furthermore, the role of these contacts in the mechanochemical cycle is unknown. Here we use the genome packaging motor of the Bacillus subtilis bacteriophage ϕ29 (ref. 4) to address these questions. The full mechanochemical cycle of the motor, in which the ATPase is a pentameric-ring of gene product 16 (gp16), involves two phases—an ATP-loading dwell followed by a translocation burst of four 2.5-base-pair (bp) steps triggered by hydrolysis product release. By challenging the motor with a variety of modified DNA substrates, we show that during the dwell phase important contacts are made with adjacent phosphates every 10-bp on the 5′–3′ strand in the direction of packaging. As well as providing stable, long-lived contacts, these phosphate interactions also regulate the chemical cycle. In contrast, during the burst phase, we find that DNA translocation is driven against large forces by extensive contacts, some of which are not specific to the chemical moieties of DNA. Such promiscuous, nonspecific contacts may reflect common translocase–substrate interactions for both the nucleic acid and protein translocases of the ASCE superfamily.

Acousto-optic lens with very fast focus scanning

We propose and experimentally demonstrate an acousto-optic cylindrical lens with a very fast (400-kHz) focal scanning. The lens is realized by use of two adjacent acousto-optic scanners with counterpropagating acoustic waves that have the same frequency modulation but a pi phase difference. This scheme completely suppresses the lateral scan but adds the linear chirp of the two waves and thus functions as a fast focal-scan lens. We also demonstrate the use of this scanning lens in a very fast confocal profilometer.

Optimized single-beam dark optical trap

We propose a new scheme for constructing a single-beam dark optical trap that minimizes light-induced perturbations of the trapped atoms. The proposed scheme optimizes the trap depth for given trapping laser power and detuning by creating a light envelope with (a) an almost minimal surface area for a given volume and (b) the minimal wall thickness that is allowed by diffraction. The stiffness of the trap’s walls, combined with the large detuning allowed by the efficient distribution of light intensity, yields a low spontaneous photon scattering rate for the trapped atoms. Our trap also optimizes the loading efficiency by maximizing the geometrical overlap between a magneto-optical trap and the dipole trap. We demonstrate this new scheme by generating the proposed light distribution of a single-beam dark trap with a trap depth that is ∼33 times larger than that of existing blue-detuned traps and ∼13 times larger than that of a red-detuned trap with the same diameter, detuning, and laser power. Trapped atoms are predicted to have a decoherence rate that is >200 times smaller than in existing single-beam dark traps and ∼1800 times smaller than in a red-detuned trap with the same diameter, depth, and laser power.

Acousto-optic scanning system with very fast nonlinear scans

A very fast (>100 kHz) acousto-optic scanning system, which relies on two counterpropagating acoustic waves with the same frequency modulation, is proposed and experimentally demonstrated. This scheme completely suppresses linear frequency chirp and thus permits very fast nonlinear scans and nonconstant linear scans. By changing the phase between the modulating signals, this scheme also provides very fast longitudinal scans of the focal point.

Direct measurement of the mechanical work during translocation by the ribosome

A detailed understanding of tRNA/mRNA translocation requires measurement of the forces generated by the ribosome during this movement. Such measurements have so far remained elusive and, thus, little is known about the relation between force and translocation and how this reflects on its mechanism and regulation. Here, we address these questions using optical tweezers to follow translation by individual ribosomes along single mRNA molecules, against an applied force. We find that translocation rates depend exponentially on the force, with a characteristic distance close to the one-codon step, ruling out the existence of sub-steps and showing that the ribosome likely functions as a Brownian ratchet. We show that the ribosome generates ∼13 pN of force, barely sufficient to unwind the most stable structures in mRNAs, thus providing a basis for their regulatory role. Our assay opens the way to characterizing the ribosomes full mechano–chemical cycle. DOI: http://dx.doi.org/10.7554/eLife.03406.001

H2A.Z controls the stability and mobility of nucleosomes to regulate expression of the LH genes

The structure and dynamics of promoter chromatin have a profound effect on the expression levels of genes. Yet, the contribution of DNA sequence, histone post-translational modifications, histone variant usage and other factors in shaping the architecture of chromatin, and the mechanisms by which this architecture modulates expression of specific genes are not yet completely understood. Here we use optical tweezers to study the roles that DNA sequence and the histone variant H2A.Z have in shaping the chromatin landscape at the promoters of two model genes, Cga and Lhb. Guided by MNase mapping of the promoters of these genes, we reconstitute nucleosomes that mimic those located near the transcriptional start site and immediately downstream (+1), and measure the forces required to disrupt these nucleosomes, and their mobility along the DNA sequence. Our results indicate that these genes are basally regulated by two distinct strategies, making use of H2A.Z to modulate separate phases of transcription, and highlight how DNA sequence, alternative histone variants and remodelling machinery act synergistically to modulate gene expression.

Stable islands in chaotic atom-optics billiards, caused by curved trajectories

We investigate the effects of curving trajectories by applying external force fields on a particle in a billiard. We investigate two special cases: a constant force field and a parabolic potential. These perturbations change the stability conditions and can lead to formation of elliptical orbits in otherwise hyperbolic billiards. We demonstrate these effects experimentally with ultra-cold atoms in atom-optic billiards.

Hyperfine spectroscopy of optically trapped atoms

We perform spectroscopy on the ground-state hyperfine splitting of 85Rb atoms trapped in far-off-resonance optical traps. The existence of a spatially dependent shift in the energy levels is shown to induce an inherent dephasing effect, which causes a broadening of the spectroscopic line and hence an inhomogeneous loss of atomic coherence at a much faster rate than the homogeneous one caused by spontaneous photon scattering. We present here a number of approaches for reducing this inhomogeneous broadening, based on trap geometry, additional laser fields, and novel microwave pulse sequences. We then show how hyperfine spectroscopy can be used to study the quantum dynamics of optically trapped atoms.

Nucleosome mobility and the regulation of gene expression: Insights from single‐molecule studies

Nucleosomes at the promoters of genes regulate the accessibility of the transcription machinery to DNA, and function as a basic layer in the complex regulation of gene expression. Our understanding of the role of the nucleosomes spontaneous, thermally driven position changes in modulating expression is lacking. This is the result of the paucity of experimental data on these dynamics, at high‐resolution, and for DNA sequences that belong to real, transcribed genes. We have developed an assay that uses partial, reversible unzipping of nucleosomes with optical tweezers to repeatedly probe a nucleosomes position over time. Using the nucleosomes at the promoters of two model genes, Cga and Lhb, we show that the mobility of nucleosomes is modulated by the sequence of DNA and by the use of alternative histone variants, and describe how the mobility can affect transcription, at the initiation and elongation phases.

Pausing kinetics dominates strand-displacement polymerization by reverse transcriptase

Abstract Reverse transcriptase (RT) catalyzes the conversion of the viral RNA into an integration-competent double-stranded DNA, with a variety of enzymatic activities that include the ability to displace a non-template strand concomitantly with polymerization. Here, using high-resolution optical tweezers to follow the activity of the murine leukemia Virus RT, we show that strand-displacement polymerization is frequently interrupted. Abundant pauses are modulated by the strength of the DNA duplex ∼8 bp ahead, indicating the existence of uncharacterized RT/DNA interactions, and correspond to backtracking of the enzyme, whose recovery is also modulated by the duplex strength. Dissociation and reinitiation events, which induce long periods of inactivity and are likely the rate-limiting step in the synthesis of the genome in vivo, are modulated by the template structure and the viral nucleocapsid protein. Our results emphasize the potential regulatory role of conserved structural motifs, and may provide useful information for the development of potent and specific inhibitors.