Nancy R. Forde

Using mechanical force to probe the mechanism of pausing and arrest during continuous elongation by Escherichia coli RNA polymerase

Escherichia coli RNA polymerase translocates along the DNA discontinuously during the elongation phase of transcription, spending proportionally more time at some template positions, known as pause and arrest sites, than at others. Current models of elongation suggest that the enzyme backtracks at these locations, but the dynamics are unresolved. Here, we study the role of lateral displacement in pausing and arrest by applying force to individually transcribing molecules. We find that an assisting mechanical force does not alter the translocation rate of the enzyme, but does reduce the efficiency of both pausing and arrest. Moreover, arrested molecules cannot be rescued by force, suggesting that arrest occurs by a bipartite mechanism: the enzyme backtracks along the DNA followed by a conformational change of the ternary complex (RNA polymerase, DNA and transcript), which cannot be reversed mechanically.

Calibration of dynamic holographic optical tweezers for force measurements on biomaterials

Holographic optical tweezers (HOTs) enable the manipulation of multiple traps independently in three dimensions in real time. Application of this technique to force measurements requires calibration of trap stiffness and its position dependence. Here, we determine the trap stiffness of HOTs as they are steered in two dimensions. To do this, we trap a single particle in a multiple-trap configuration and analyze the power spectrum of the laser deflection on a position-sensitive photodiode. With this method, the relative trap strengths can be determined independent of exact particle size, and high stiffnesses can be probed because of the high bandwidth of the photodiode. We find a trap stiffness for each of three HOT traps of kappa approximately 26 pN/microm per 100 mW of laser power. Importantly, we find that this stiffness remains constant within +/- 4% over 20 microm displacements of a trap. We also investigate the minimum step size achievable when steering a trap with HOTs, and find that traps can be stepped and detected within approximately 2 nm in our instrument, although there is an underlying position modulation of the traps of comparable scale that arises from SLM addressing. The independence of trap stiffness on steering angle over wide ranges and the nanometer positioning accuracy of HOTs demonstrate the applicability of this technique to quantitative study of force response of extended biomaterials such as cells or elastomeric protein networks.

Microrheological Characterization of Collagen Systems: From Molecular Solutions to Fibrillar Gels

Collagen is the most abundant protein in the extracellular matrix (ECM), where its structural organization conveys mechanical information to cells. Using optical-tweezers-based microrheology, we investigated mechanical properties both of collagen molecules at a range of concentrations in acidic solution where fibrils cannot form and of gels of collagen fibrils formed at neutral pH, as well as the development of microscale mechanical heterogeneity during the self-assembly process. The frequency scaling of the complex shear modulus even at frequencies of ∼10 kHz was not able to resolve the flexibility of collagen molecules in acidic solution. In these solutions, molecular interactions cause significant transient elasticity, as we observed for 5 mg/ml solutions at frequencies above ∼200 Hz. We found the viscoelasticity of solutions of collagen molecules to be spatially homogeneous, in sharp contrast to the heterogeneity of self-assembled fibrillar collagen systems, whose elasticity varied by more than an order of magnitude and in power-law behavior at different locations within the sample. By probing changes in the complex shear modulus over 100-minute timescales as collagen self-assembled into fibrils, we conclude that microscale heterogeneity appears during early phases of fibrillar growth and continues to develop further during this growth phase. Experiments in which growing fibrils dislodge microspheres from an optical trap suggest that fibril growth is a force-generating process. These data contribute to understanding how heterogeneities develop during self-assembly, which in turn can help synthesis of new materials for cellular engineering.

THERMAL PROBING OF E. COLI RNA POLYMERASE OFF-PATHWAY MECHANISMS

RNA polymerase (RNAP) is an essential enzyme for cellular gene expression. In an effort to further understand the enzymes importance in the cells response to temperature, we have probed the kinetic mechanism of Escherichia coli RNAP by studying the force-velocity behavior of individual RNAP complexes at temperatures between 7 and 45 degrees C using optical tweezers. Within this temperature range and at saturating nucleotide concentrations, the pause-free transcription velocity of RNAP was independent of force and increased monotonically with temperature with an elongation activation energy of 9.7+/-0.7 kcal/mol. Interestingly, the pause density at cold temperatures (7 to 21 degrees C) was five times higher than that measured above room temperature. A simple kinetic model revealed a value of 1.29+/-0.05 kcal/mol for the activation energy of pause entry, suggesting that pause entry is indeed a thermally accessible process. The dwell time distribution of all observable pauses was independent of temperature, directly confirming a prediction of the model recently proposed for Pol II in which pauses are diffusive backtracks along the DNA. Additionally, we find that the force at which the polymerase arrests (the arrest force) presents a maximum at 21 degrees C, an unexpected result as this is not the optimum temperature for bacterial growth. This observation suggests that arrest could play a regulatory role in vivo, possibly through interactions with specific elongation factors.

Power spectral analysis for optical trap stiffness calibration from high-speed camera position detection with limited bandwidth

The use of camera imaging enables trap calibration for multiple particles simultaneously. For stiff traps, however, blur from image integration time affects the detected particle positions significantly. In this paper we use power spectral analysis to calibrate stiff optical traps, taking the effects of blur, aliasing and position detection error into account, as put forward by Wong and Halvorsen [Opt. Express 14, 12517 (2006)]. We find agreement with simultaneously obtained photodiode data and the expected relation of corner frequency fc with laser power, up to fc = 3.6 kHz for a Nyquist frequency of 1.25 kHz. Spectral analysis enables easy identification of the contribution of noise. We demonstrate the utility of our approach with simultaneous calibration of multiple holographic optical traps.

The Tumbleweed: Towards a synthetic protein motor

Biomolecular motors have inspired the design and construction of artificial nanoscale motors and machines based on nucleic acids, small molecules, and inorganic nanostructures. However, the high degree of sophistication and efficiency of biomolecular motors, as well as their specific biological function, derives from the complexity afforded by protein building blocks. Here, we discuss a novel bottom‐up approach to understanding biological motors by considering the construction of synthetic protein motors. Specifically, we present a design for a synthetic protein motor that moves along a linear track, dubbed the “Tumbleweed.” This concept uses three discrete ligand‐dependent DNA‐binding domains to perform cyclically ligand‐gated, rectified diffusion along a synthesized DNA molecule. Here we describe how de novo peptide design and molecular biology could be used to produce the Tumbleweed, and we explore the fundamental motor operation of such a design using numerical simulations. The construction of this and more sophisticated protein motors is an exciting challenge that is likely to enhance our understanding of the structure‐function relationship in biological motors.

The influence of local electronic character and nonadiabaticity in the photodissociation of nitric acid at 193 nm

The dissociation of nitric acid upon πnb,O→πNO2* excitation at 193 nm has been studied in a crossed laser-molecular beam apparatus. The primary reaction channels are OH+NO2 and O+HONO. We measure the branching ratio between these two competing processes and determine (OH+NO2)/(O+HONO)=0.50±0.05. Our experiments provide evidence of a minor O+HONO pathway, which we assign to O(3P) and HONO in its lowest triplet state. The dominant pathway correlates to O(1D)+HONO(X 1A′). The translational energy distributions reveal two distinct pathways for the OH+NO2 channel. One pathway produces stable NO2 fragments in the 1 2B2 electronic state. The second pathway produces unstable NO2 fragments which undergo secondary dissociation to NO+O. We examine the influence of nonadiabaticity along the OH+NO2 reaction coordinate in order to explain the significant branching to this other channel. Finally, we introduce a new method for generating correlation diagrams for systems with electronic transitions localized on one moiety...

Stretching single DNA molecules to demonstrate high-force capabilities of holographic optical tweezers.

The well calibrated force-extension behaviour of single double-stranded DNA molecules was used as a standard to investigate the performance of phase-only holographic optical tweezers at high forces. Specifically, the characteristic overstretch transition at 65 pN was found to appear where expected, demonstrating (1) that holographic optical trap calibration using thermal fluctuation methods is valid to high forces; (2) that the holographic optical traps are harmonic out to >250 nm of 2.1 mum particle displacement; and (3) that temporal modulations in traps induced by the spatial light modulator (SLM) do not affect the ability of optical traps to hold and steer particles against high forces. These studies demonstrate a new high-force capability for holographic optical traps achievable by SLM technologies.

Electronic accessibility of dissociation channels in an amide: N,N-dimethylformamide photodissociation at 193 nm

Measurement of the photofragment velocity and angular distributions from the photodissociation of N,N-dimethylformamide at 193 nm in its πnbπ* absorption evidences three competing dissociation channels: HCON(CH3)2→HCO(X 2A′)+N(CH3)2(X 2B1); HCO(X 2A′)+N(CH3)2(A 2A1); and HCONCH3+CH3. (H atom eliminations are not probed.) These products are formed in a ratio of 0.15±0.04:0.49±0.09:0.36±0.07, determined by use of trimethylamine as a calibrant molecule. Nitrogen–carbonyl bond fission occurs on a rapid time scale with an angular distribution of the dissociation products given by β=1.2±0.2. Excited state N(CH3)2 products are formed quasidiabatically from the initial planar geometry, whereas symmetry-breaking vibrations allow one-electron matrix elements to couple the initial electronic configuration to the ground state N(CH3)2+HCO channel. Competition of nitrogen–methyl bond fission is evidence of the strong coupling between the πnbπ* excitation and the nitrogen–methyl reaction coordinate; ab initio calcula...

Using optical tweezers to study mechanical properties of collagen

The mechanical response of biological molecules at the microscopic level contributes significantly to their function. Optical tweezers are instruments that enable scientists to study mechanical properties at microscopic levels. They are based on a highly focused laser beam that creates a trap for microscopic objects such as dielectric spheres, viruses, bacteria, living cells and organelles, and then manipulates them by applying forces in the picoNewton range (a range that is biologically relevant). In this work, mechanical properties of single collagen molecules are studied using optical tweezers. We discuss the challenges of stretching single collagen proteins, whose length is much less than the size of the microspheres used as manipulation handles, and show how instrumental design and biochemistry can be used to overcome these challenges.