Ping Xie

Escherichia coli RecQ Is a Rapid, Efficient, and Monomeric Helicase

RecQ family helicases play a key role in chromosome maintenance. Despite extensive biochemical, biophysical, and structural studies, the mechanism by which helicase unwinds double-stranded DNA remains to be elucidated. Using a wide array of biochemical and biophysical approaches, we have previously shown that the Escherichia coli RecQ helicase functions as a monomer. In this study, we have further characterized the kinetic mechanism of the RecQ-catalyzed unwinding of duplex DNA using the fluorometric stopped-flow method based on fluorescence resonance energy transfer. Our results show that RecQ helicase binds preferentially to 3′-flanking duplex DNA. Under the pre-steady-state conditions, the burst amplitude reveals a 1:1 ratio between RecQ and DNA substrate, suggesting that an active monomeric form of RecQ helicase is involved in the catalysis. Under the single-turnover conditions, the RecQ-catalyzed unwinding is independent of the 3′-tail length, indicating that functional interactions between RecQ molecules are not implicated in the DNA unwinding. It was further determined that RecQ unwinds DNA rapidly with a step size of 4 bp and a rate of ∼21 steps/s. These kinetic results not only further support our previous conclusion that E. coli RecQ functions as a monomer but also suggest that some of the Superfamily 2 helicases may function through an “inchworm” mechanism.

Ionic effect on combing of single DNA molecules and observation of their force-induced melting by fluorescence microscopy.

Molecular combing is a powerful and simple method for aligning DNA molecules onto a surface. Using this technique combined with fluorescence microscopy, we observed that the length of lambda-DNA molecules was extended to about 1.6 times their contour length (unextended length, 16.2 microm) by the combing method on hydrophobic polymethylmetacrylate coated surfaces. The effects of sodium and magnesium ions and pH of the DNA solution were investigated. Interestingly, we observed force-induced melting of single DNA molecules.

Dynamics of tRNA occupancy and dissociation during translation by the ribosome

Repetitive cycles of protein elongation by the ribosome involve dynamic changes in tRNA occupancy and the deacylated-tRNA dissociation plays an important role in the process. Here we present a detailed analysis of the dynamics for the complicated process. We study analytically the dynamic tRNA occupancy and, specifically, the mean time for occupancy of one tRNA molecule, two tRNA molecules and three tRNA molecules during one cycle of protein elongation. The deacylated-tRNA dissociation probability at each state of the elongation cycle is analytically studied. The analyses give quantitative explanations of the available experimental data and provide interesting results: for example, the deacylated tRNA in hybrid P/E state can also be dissociated from the ribosome, as in post-translocation E/E state, but with a dissociation rate in the former being only about 1/20-fold of that in the latter; at low EF-G.GTP concentration, large fractions of the deacylated tRNA molecules are dissociated before the codon recognition in the A site and the ribosome is most of the time occupied by only one tRNA molecule, while at high and in vivo near EF-G.GTP concentration the ribosome is most of the time occupied by two tRNA molecules and the release of a third tRNA occurs mainly after the decoding step; even at high concentrations of EF-G.GTP and aminoacyl-tRNA.EF-Tu.GTP complex, the mean time for occupancy of three tRNA molecules is in the order of milliseconds in one translation cycle, which is much shorter than the in vitro mean translation time of about 1s.

Efficient second harmonic generation from large band gap II-VI semiconductor photonic crystal

Dramatic enhancement of second harmonic generation (SHG) near the photonic band edge was observed in a one-dimensional ZnSe∕ZnMgS semiconductor photonic crystal (PC) structure. Over two orders of magnitude increase in SHG intensity was observed at the photonic band edge at ∼1400nm compared to the nonphase matching region. The maximum SHG conversion efficiency of 0.8% is observed in only seven micrometers length of crystal. This enhancement came from a combination of large ZnSe second order susceptibility coefficient (χ(2)), high density of optical modes and phase matching of the fundamental and second harmonic waves near the photonic band edge due to modification of the dispersion curve by the PC structure.

THEORETICAL AND EXPERIMENTAL STUDIES OF FANNING EFFECTS IN PHOTOREFRACTIVE CRYSTALS

A model to describe fanning effects in photorefractive crystals is presented. The intensity distribution of the fanning beam for one incident beam and two incident beams in 45°‐cut BaTiO3 crystals has been studied theoretically and experimentally, with good agreement. The theoretical results suggest that, when there is a fanning effect, only at intermediate effective interaction lengths does the signal beam have a high gain in two‐wave mixing. The transient behavior of the fanning beam in a 45°‐cut BaTiO3 crystal has also been studied theoretically and experimentally.

A two‐dimensional theory and propagation of beam fanning in photorefractive crystals

A general two‐dimensional model to describe beam fanning, which takes into consideration the finite width of the input beam and the interaction between different directions of the fanning beam, is presented. By numerical calculation of the coupled equations, the well‐known curved path of a propagating beam inside photorefractive crystals is explained, and the propagation properties of the fanning beam inside the crystals are studied and discussed.

Simultaneous perfect phase matching for second and third harmonic generations in ZnS/YF 3 photonic crystal for visible emissions

Theoretically designed and experimentally realized simultaneous perfect phase matching of second and third harmonic generations were demonstrated in a one-dimensional ZnS/YF(3) photonic crystal (PC) structure. Dramatic enhancement of second harmonic generation (SHG) and third harmonic generation (THG) in forward and backward directions near the photonic band edge were observed. This enhancement came from a combination of large ZnS nonlinear susceptibility coefficients, high density of optical modes and perfect phase matching of the fundamental and the harmonic waves near the photonic band edge due to modification of the dispersion curve by the PC structure. Total SHG and THG conversion efficiency over 4% is measured in only six micrometers length of photonic crystal. Theoretical calculations show good agreement with experimental measurements.

A dynamical model of programmed −1 ribosomal frameshifting

Programmed -1 ribosomal frameshifting is the most widely used translational recoding mechanism of RNA viruses. How the frameshifting occurs at the slippery sequence on the presence of a downstream mRNA pseudoknot has not been fully understood. Here, we present systematical analysis of the -1 frameshifting that can occur during every transition step in elongation phase of protein synthesis by Escherichia coli ribosomes, showing that the -1 frameshifting can occur mainly during three periods. One is during translocation step, another period is after the posttranslocation and before the binding of the ternary complex aminoacyl-tRNA.EF-Tu.GTP, and the third period is after codon recognition and before peptidyl transfer. Of the three periods, the translocation step makes the most contribution to the -1 frameshifting. During the translocation step when the mRNA channel is open, due to the presence of energy barrier to the forward translocation of the ribosome along the mRNA template, which results from unwinding of the mRNA pseudoknot, the reverse ribosomal rotation from ratcheted to non-ratcheted conformation induces the frameshifting and futile translocation besides the effective forward translocation. During the other two slow periods when the mRNA channel is tight, the annealing of the unwound base pair in the mRNA pseudoknot can also promote the -1 frameshifting. Our theoretical results provide a consistent explanation of a lot of independent experimental data and also provide predicted results.

A unified model of nucleic acid unwinding by the ribosome and the hexameric and monomeric DNA helicases.

DNA helicases are enzymes that use the chemical energy to separate DNA duplex into their single-stranded forms. The ribosome, which catalyzes the translation of messenger RNAs (mRNAs) into proteins, can also unwind mRNA duplex. According to their structures, the DNA helicases can fall broadly into hexameric and monomeric forms. A puzzling issue for the monomeric helicases is that although they have similar structures, in vitro biochemical data showed convincingly that in the monomeric forms some have very weak DNA unwinding activities, some have relatively high unwinding activities while others have high unwinding activities. However, in the dimeric or oligomeric forms all of them have high unwinding activities. In addition, in the monomeric forms all of them can translocate efficiently along the single-stranded DNA (ssDNA). Here, we propose a model of the translocation along the ssDNA and DNA unwinding by the monomeric helicases, providing a consistent explanation of these in vitro experimental data. Moreover, by comparing the present model for the monomeric helicases with the model for the hexameric helicases and that for the ribosome which were proposed before, a unified model of nucleic acid unwinding by the three enzymes is proposed.

Simultaneous enhancement of the second- and third-harmonic generations in one-dimensional semiconductor photonic crystals

We report on observation of simultaneous strong enhancements of second-harmonic generation (SHG) and third-harmonic generation (THG) in visible spectra region in a one-dimensional ZnSe-ZnMgS semiconductor photonic crystal. These enhancements come from phase matching and a high density of modes near the photonic band edge. Our result shows that the THG is due to direct /spl chi//sup (3)/ process instead of the cascaded two-step /spl chi//sup (2)/ process. Measured forward SH conversion efficiency close to 1% for only a few microns thick film demonstrates potential device applications. Theoretical calculations are in good agreement with the experimental measurements.