Single-molecule observation of the induction of k-turn RNA structure on binding L7Ae protein

AbstractThe k-turn is a commonly occurring structural motif that introduces a tight kink into duplex RNA. In free solution, it can exist in an extended form, or by folding into the kinked structure. Binding of proteins including the L7Ae family can induce the formation of the kinked geometry, raising the question of whether this occurs by passive selection of the kinked structure, or a more active process in which the protein manipulates the RNA structure. We have devised a single-molecule experiment whereby immobilized L7Ae protein binds Cy3-Cy5-labeled RNA from free solution. We find that all bound RNA is in the kinked geometry, with no evidence for transitions to an extended form at the millisecond timescale of the camera. Furthermore, real-time binding experiments provide no evidence for a more extended intermediate even at the earliest times, at a time resolution of 16 ms. The data support a passive conformational selection model by which the protein selects a fraction of RNA that is already in the kinked conformation, thereby drawing the equilibrium into this form

Single-molecule fluorescence detection in molecular biology

SMFD techniques offer genuine detection possibilities which are often inaccessible using ensemble methods. This was demonstrated in three projects investigating translocation activity of CHD4 protein, analysis of MS2 phage capsid assembly and in-cell characterization of DNA structure. In other projects, binding interactions between two fluorescent probes and a short oligonucleotide were characterized and all optical depth of focus extended microscope configuration for imaging of individual molecules inside bacterial cells was developed and tested

Dynamic coupling of fast channel gating with slow ATP-turnover underpins protein transport through the Sec-translocon

The Sec-translocon is a highly conserved membrane complex for polypeptide transport across, or into, lipid bilayers. In bacteria, the core protein-channel complex SecYEG resides in the inner-membrane, through which secretion is powered by the cytosolic ATPase SecA. Here, we present a single-molecule FRET dataset which shows that the SecYEG-channel fluctuates between open and closed states much faster than ATP turnover, while the nucleotide status of SecA modulates the rates of opening and closure