No abstract

Optical tweezers have proven a useful tool for exploring the structure and function of individual molecules, such as proteins, DNA, and RNA. The ability to unfold and refold biological molecules has provided novel insights that complement and go beyond traditional biochemical and structural approaches. With sophisticated optical tweezers instrumentation coming to the market, single-molecule stretching studies now have become feasible and available to a wide range of users. Therefore, a step-by-step protocol for stretching individual biomolecules utilizing a simple experimental geometry is timely and presented here. While we have taken the unfolding of an RNA structure held between two RNA/DNA hybrid handles as an example, the technical protocol should be readily applicable to other biomolecules and may serve as a starting point for more sophisticated experiments.

recombination and repair. In the present work we are investigating mechanical properties of torsionally-denatured DNA at the single-molecule level using an angular optical trap. While applying a constant tension to a DNA molecule, we simultaneously measure the extension change and torque as the DNA is wound up and denatured. We will present measurements on both tensile and torsional properties of denatured DNA. We will also discuss the implications of our findings with respect to previous theoretical work.

Two photon laser scanning microscopy and true N&B analysis allowed determination of the absolute concentration of GFP molecules inside the bacterial cells. We collected data on hundreds of B. subtilis cells expressing GFP under control of the promoters of interest and grown under glycolytic or gluconeogenic conditions. Results showed no regulation of the promoter expressing the gluconeogenic repressor, strong repression of the gluconeogenic enzyme promoters and weak auto-repression of the glycolytic promoter, with a highly asymmetric distribution when repressed. All promoters showed strong evidence for transcriptional bursting. Analysis of the data using stochastic models of gene expression is currently underway. The figure shows number maps of bacterial cells grown on glucose(G) or Malate(M). Each change in color represents 10 molecules up to 180. The flexibilities of single-stranded RNA has not been as well studied as doublestranded RNA, despite their significant biological importance. Their values for persistence lengths have often been inferred from that of single-stranded DNA. In this poster, we compare the persistence lengths of poly rU and poly-dT as measured by single molecule FRET experiments. We show differences in flexibilities when the nucleic acids are in the presence of monovalent and divalent salts. 969-PlatMechanical Unfolding of the Beet Western Yellow Virus À1 Frameshift Signal Using unfolding by optical tweezers and steered molecular dynamics (SMD) simulations we have demonstrated the critical importance of Mg 2þ ions for the mechanical stability of the BWYV RNA pseudoknot. The optical tweezers experiments pointed to a critical role of stem 1 of the pseudoknot, a finding that was confirmed using the SMD simulations. These simulations supported the notion that the stability of stem 1 is critical for À1 frameshifting, a translational recoding event essential for replication of the BWYV. Furthermore, they clarified the precise role of two Mg 2þ ions, Mg45 and Mg52, in À1 frameshifting. The ions were shown to play a critical role in stabilizing stem 1 by two possible mechanisms depending upon the hydration of the Mg 2þ ions. Mg 2þ ions were either directly forming a salt bridge between the strands of stem 1, or they stabilized parallel orientation of the strands in stem 1. Interestingly, these findings explain the drop in frameshifting efficiency, down to null levels, of the C8U mutant. The large effect of this mutant upon the frameshift efficiency seemed surprising as only a single hydrogen bond appeared to have been sacrificed. However, the SMD simulations clarify how the C8U mutation affects the Mg 2þ coordination and destabilizes stem 1 of the pseudoknot. 970-PlatTwo Distinct Overstretched DNA States Double-stranded DNA can undergo an ''overstretching'' transition within a narrow force range when the external force reaches around 60 pN. The basic question of the overstretched DNA is whether the strands are separated or not after transition. Despite numbers of studies, this question still remain...