TEFM Enhances Transcription Elongation by Modifying mtRNAP Pausing Dynamics.

Regulation of transcription elongation is one of the key mechanisms employed to control gene expression. The single-subunit mitochondrial RNA polymerase (mtRNAP) transcribes mitochondrial genes, such as those related to ATP synthesis. We investigated how mitochondrial transcription elongation factor (TEFM) enhances mtRNAP transcription elongation using a single-molecule optical-tweezers transcription assay, which follows transcription dynamics in real time and allows the separation of pause-free elongation from transcriptional pauses. We found that TEFM enhances the stall force of mtRNAP. Although TEFM does not change the pause-free elongation rate, it enhances mtRNAP transcription elongation by reducing the frequency of long-lived pauses and shortening their durations. Furthermore, we demonstrate how mtRNAP passes through the conserved sequence block II, which is the key sequence for the switch between DNA replication and transcription in mitochondria. Our findings elucidate how both TEFM and mitochondrial genomic DNA sequences directly control the transcription elongation dynamics of mtRNAP.

Portable low-cost instrumentation for monitoring Rayleigh scattering from chemical sensors based on metallic nanoparticles.

Using a Hg(II) sensor based on the aggregation of gold nanoparticles as a model system, we evaluated the performance of two portable low-cost devices that monitor the wavelength-ratiometric resonance Rayleigh scattering signal of the chemical sensor upon white-LED illumination. The first device uses two optical filter-photodiode combinations to detect scattered light while the second employs a novel ultra-compact (grating-free) spectral sensor. Results show that the response of the Hg(II) sensor monitored with these devices is comparable to that measured using a high-end benchtop scanning spectrofluorometer. The great potential of this new LED-spectral sensor was demonstrated with the quantification of Hg(II) in tap and spring water. Due to the promising results obtained, many reported chemical sensors based on Rayleigh scattering from metallic nanoparticles could take advantage of this compact portable instrumentation for cost-effective field-deployable applications.

Full molecular trajectories of RNA polymerase at single base-pair resolution.

In recent years, highly stable optical tweezers systems have enabled the characterization of the dynamics of molecular motors at very high resolution. However, the motion of many motors with angstrom-scale dynamics cannot be consistently resolved due to poor signal-to-noise ratio. Using an acousto-optic deflector to generate a "time-shared" dual-optical trap, we decreased low-frequency noise by more than one order of magnitude compared with conventional dual-trap optical tweezers. Using this instrument, we implemented a protocol that synthesizes single base-pair trajectories, which are used to test a Large State Space Hidden Markov Model algorithm to recover their individual steps. We then used this algorithm on real transcription data obtained in the same instrument to fully uncover the molecular trajectories of Escherichia coli RNA polymerase. We applied this procedure to reveal the effect of pyrophosphate on the distribution of dwell times between consecutive polymerase steps.

Highly sensitive ratiometric quantification of cyanide in water with gold nanoparticles via Resonance Rayleigh Scattering.

A highly sensitive and selective ratiometric sensor for the quantification of cyanide (CN-) in aqueous samples has been developed using spherical gold nanoparticles (AuNPs) stabilized by polysorbate 40 (PS-40). Three different AuNP sizes (14, 40 and 80nm mean diameters) were used to evaluate the response of the sensor using both colorimetric and Resonance Rayleigh Scattering (RRS) detection schemes. The best results were obtained for the sensor using 40nm AuNPs, for which the limits of detection (LODs) were found to be 100nmolL-1 in a benchtop instrument and 500nmolL-1 by the naked eye, values well below the maximum acceptable level for drinking water (1.9µmolL-1) set by the World Health Organization (WHO). The practical use of the 40nm-AuNPs RRS sensor was demonstrated with the determination of CN- in drinking and fresh waters. Finally, the sensor was successfully implemented in a compact portable device consisting of two light-emitting diodes (LEDs) and a miniature spectrometer, turning this sensor into a very potent tool for its application as a quick routine field-deployable analytical method.

Transcription factors IIS and IIF enhance transcription efficiency by differentially modifying RNA polymerase pausing dynamics.

Transcription factors IIS (TFIIS) and IIF (TFIIF) are known to stimulate transcription elongation. Here, we use a single-molecule transcription elongation assay to study the effects of both factors. We find that these transcription factors enhance overall transcription elongation by reducing the lifetime of transcriptional pauses and that TFIIF also decreases the probability of pause entry. Furthermore, we observe that both factors enhance the processivity of RNA polymerase II through the nucleosomal barrier. The effects of TFIIS and TFIIF are quantitatively described using the linear Brownian ratchet kinetic model for transcription elongation and the backtracking model for transcriptional pauses, modified to account for the effects of the transcription factors. Our findings help elucidate the molecular mechanisms by which transcription factors modulate gene expression.

Two-photon fluorescence excitation spectroscopy by pulse shaping ultrabroad-bandwidth femtosecond laser pulses.

A fast and automated approach to measuring two-photon fluorescence excitation (TPE) spectra of fluorophores with high resolution (~2 nm) by pulse shaping ultrabroad-bandwidth femtosecond laser pulses is demonstrated. Selective excitation in the range of 675-990 nm was achieved by imposing a series of specially designed phase and amplitude masks on the excitation pulses using a pulse shaper. The method eliminates the need for laser tuning and is, thus, suitable for non-laser-expert use. The TPE spectrum of Fluorescein was compared with independent measurements and the spectra of the pH-sensitive dye 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) in acidic and basic environments were measured for the first time using this approach.

Atmospheric pressure femtosecond laser imaging mass spectrometry.

A novel atmospheric pressure imaging mass spectrometry approach that offers improved lateral resolution (10 microm) using near-infrared femtosecond laser pulses for nonresonant desorption and ionization of sample constituents without the need of a laser-absorbing matrix is demonstrated. As a proof of concept the method was used to image a two-chemical pattern in paper. To demonstrate the ability of the approach to analyze biological tissue, a monolayer of onion epidermis was imaged allowing the chemical visualization of individual cells using mass spectrometry at ambient conditions for the first time. As the spatial resolution is currently limited by the limit of detection of the setup (approximately 500 fmol limit of detection for citric acid), improvements in sensitivity will increase the achievable spatial resolution.

Asynchronous encrypted information transmission with sub-6 fs laser system at 2.12 GHz repetition rate.

The asynchronous transmission (encoding and decoding) of 64-bit information using binary spectral phase shaping is demonstrated. The accurate introduction and retrieval of the binary information is possible by using multiphoton intrapulse interference phase scan (MIIPS) to measure and correct the spectral phase distortions of the laser and the transmission media. Experimental demonstration is achieved using a sub-6 fs Ti:Sapphire laser with 2.12-GHz repetition rate and an adaptive phase control system.

Broadband 2.12 GHz Ti:sapphire laser compressed to 5.9 femtoseconds using MIIPS.

We report a self-starting prismless femtosecond Ti:sapphire ring laser whose repetition rate has been gradually increased from 1 to 2.12 GHz. A broadband spectrum extending from 650 to 1040 nm, in which 17% of the intracavity power is generated in a single-pass through the crystal, is preserved in spite of the reduction in peak power. An average power of 0.95 W was obtained for 7.5 W of pump power, with very stable operation verified over 22 hours. Pulses from this laser have been fully characterized in spectral phase, and then compressed to 5.9 femtoseconds using multiphoton intrapulse interference phase scan (MIIPS).

Direct measurement of spectral phase for ultrashort laser pulses.

We present an intuitive pulse characterization method that provides an accurate and direct measurement of the spectral phase of ultrashort laser pulses. The method requires the successive imposition of a set of quadratic spectral phase functions on the pulses while recording the corresponding nonlinear spectra. The second-derivative of the unknown spectral phase can be directly visualized and extracted from the experimental 2D contour plot, without any inversion algorithm or mathematical manipulation.

Group-velocity dispersion measurements of water, seawater, and ocular components using multiphoton intrapulse interference phase scan.

The use of femtosecond lasers requires accurate measurements of the dispersive properties of media. Here we measure the second- and third-order dispersion of water, seawater, and ocular components in the range of 660-930 nm using a new method known as multiphoton intrapulse interference phase scan. Our direct dispersion measurements of water have the highest precision and accuracy to date. We found that the dispersion for seawater increases proportionally to the concentration of salt. The dispersion of the vitreous humor was found to be close to that of water. The chromatic dispersion of the cornea-lens complex was measured to obtain the full dispersive properties of the eye.

Pulse shaping of octave spanning femtosecond laser pulses.

Characterization and pulse shaping of octave spanning femtosecond lasers poses a significant challenge. We have constructed a grating-based pulse shaper for an ultra-broad-bandwidth (620-1020 nm)femtosecond laser, and used it to compensate the phase distortions of the laser, spatial-light modulator and optics within 0.1 rad accuracy accross the entire bandwidth using Multiphoton Intrapulse Interference Phase Scan (MIIPS) without a precompressor. The compensated transform limited pulses generated a second harmonic spectrum with a 12,260 cm(-1) spectral width. Binary phase modulation was introduced by this pulse shaping system to demonstrate high resolution control of the second harmonic generation spectrum.