Biology subjects

Lerner, E.

Publications and source records attributed to Lerner, E..

4 recordsLinked to original sources

bioRxiv2018

Monte-Carlo Diffusion-Enhanced Photon Inference: Distance Distributions and Conformational Dynamics in single-molecule FRET

Single-molecule Forster Resonance Energy Transfer (smFRET) is utilized to study the structure and dynamics of many bio-molecules, such as proteins, DNA and their various complexes. The structural assessment is based on the well-known Forster relationship between the measured efficiency of energy transfer between a donor (D) and an acceptor (A) dye and the distance between them. Classical smFRET analysis methods called photon distribution analysis (PDA) take into account photon shot-noise, D-A distance distribution and, more recently, interconversion between states in order to extract accurate distance information. It is known that rapid D-A distance fluctuations on the order of the D lifetime (or shorter) can increase the measured mean FRET efficiency and thus decrease the estimated D-A distance. Nonetheless, this effect has been so far neglected in smFRET experiments, potentially leading to biases in estimated distances.\n\nHere we introduce a PDA approach dubbed Monte-Carlo-diffusion-enhanced photon inference (MC-DEPI). MC-DEPI recolor detected photons of smFRET experiments taking into account dynamics of D-A distance fluctuations, multiple interconverting states and photo-blinking. Using this approach, we show how different underlying conditions may yield identical FRET histograms and how the additional information from fluorescence decays helps distinguishing between the different conditions. We also introduce a machine learning fitting approach for retrieving the D-A distance distribution, decoupled from the above-mentioned effects. We show that distance interpretation of smFRET experiments of even the simplest dsDNA is nontrivial and requires decoupling the effects of rapid D-A distance fluctuations on FRET in order to avoid systematic biases in the estimation of the D-A distance distribution.

biophysics↗

bioRxiv2017

The effect of macromolecular crowding on single-round transcription by E. coli RNA polymerase

Biological reactions in the cellular environment differ physicochemically from those performed in dilute buffer solutions due to, in part, slower diffusion of various components in the cellular milieu, increase in their chemical activities, and modulation of their binding affinities and conformational stabilities. In vivo transcription is therefore expected to be strongly influenced by the crowdedness of the cell. Previous studies of transcription under macromolecular crowding conditions have focused mainly on multiple cycles of RNAP-Promoter associations, assuming that the association is the rate-determining step of the entire transcription process. However, recent reports demonstrated that late initiation and promoter escape could be the rate-determining steps for some promoter DNA sequences. The investigation of crowding effects on these steps under single-round conditions is therefore crucial for better understanding of transcription initiation in vivo. Here, we have implemented an in vitro transcription quenched-kinetics single-molecule assay to investigate the dependence of transcription reaction rates on the sizes and concentrations of crowders. Our results demonstrate an expected slowdown of transcription kinetics due to increased viscosity, and an unexpected enhancement in transcription kinetics by large crowding agents (at a given viscosity). More importantly, the enhancements dependence on crowder size significantly deviates from hard-sphere model (scaled-particle theory) predictions, commonly used for description of crowding effects. Our findings shed new light on how enzymatic reactions are affected by crowding conditions in the cellular milieu.

biochemistry↗

bioRxiv2017

Characterizing highly dynamic conformational states: the transcription bubble in RNAP-promoter open complex as an example

Bio-macromolecules carry out complicated functions through structural changes. To understand their mechanism of action, the structure of each step has to be characterized. While classical structural biology techniques allow the characterization of a few structural snapshots along the enzymatic cycle (usually of stable conformations), they do not cover all (and often fast interconverting) structures in the ensemble, where each may play an important functional role. Recently, several groups have demonstrated that structures of different conformations in solution could be solved by measuring multiple distances between different pairs of residues using single-molecule Forster resonance energy transfer (smFRET) and using them as constrains for hybrid/integrative structural modeling. However, this approach is limited in cases where the conformational dynamics is faster than the techniques temporal resolution. In this study, we combine existing tools that elucidate sub-millisecond conformational dynamics together with hybrid/integrative structural modeling to study the conformational states of the transcription bubble in the bacterial RNA polymerase (RNAP)-promoter open complex (RPo). We measured microsecond alternating laser excitation sALEX)-smFRET of differently labeled lacCONS promoter dsDNA constructs. We used a combination of burst variance analysis (BVA), photon-by-photon hidden Markov modelling (H2MM) and the FRET-restrained positioning and screening (FPS) approach to identify two conformational states for RPo. The experimentally-derived distances of one conformational state match the known crystal structure of bacterial RPo. The experimentally-derived distances of the other conformational state have characteristics of a scrunched RPo. These findings support the hypothesis that sub-millisecond dynamics in the transcription bubble are responsible for transcription start site selection.

biophysics↗

bioRxiv2016

Multispot single-molecule FRET: high-throughput analysis of freely diffusing molecules

We describe an 8-spot confocal setup for high-throughput smFRET assays and illustrate its performance with two characteristic experiments. First, measurements on a series of freely diffusing doubly-labeled dsDNA samples allow us to demonstrate that data acquired in multiple spots in parallel can be properly corrected and result in measured sample characteristics identical to those obtained with a standard single-spot setup. We then take advantage of the higher throughput provided by parallel acquisition to address an outstanding question about the kinetics of the initial steps of bacterial RNA transcription. Our real-time kinetic analysis of promoter escape by bacterial RNA polymerase confirms results obtained by a more indirect route, shedding additional light on the initial steps of transcription.\n\nFinally, we discuss the advantages of our multispot setup, while pointing potential limitations of the current single laser excitation design, as well as analysis challenges and their solutions.

biophysics↗