Biology subjects

Elf, J.

Publications and source records attributed to Elf, J..

26 records · Page 2Linked to original sources

bioRxiv2021

Sequence specificity in DNA binding is determined by association rather than dissociation

Sequence-specific binding of proteins to DNA is essential for accessing genetic information. Here, we derive a simple equation for target-site recognition, which uncovers a previously unrecognized coupling between the macroscopic association and dissociation rates of the searching protein. Importantly, this relationship makes it possible to recover the relevant microscopic rates from experimentally determined macroscopic ones. We directly test the equation by observing the binding and unbinding of individual lac repressor (LacI) molecules during target search. We find that LacI dissociates from different target sequences with essentially identical microscopic dissociation rates. Instead, sequence specificity is determined by the efficiency with which the protein recognizes different targets, effectively reducing its risk of being retained on a non-target sequence. Our theoretical framework also accounts for the coupling between off-target binding and unbinding of the catalytically inactive Cas9 (dCas9), showing that the binding pathway can be obtained from macroscopic data. One Sentence SummaryAssociation and dissociation rates are anti-correlated for reactions that include a nonspecific probing step.

biophysics↗

bioRxiv2020

Direct measurements of mRNA translation kinetics in living cells

Ribosome mediated mRNA translation is central to life as we know it. The cycle of translation has, however, not been characterized in a living cell. Here we have developed a live-cell ribosome-labeling method, which allows us to characterize the whole processes of finding an mRNA and translating it, using single-molecule tracking techniques. We find that more than 90% of both bacterial ribosomal subunits are engaged in elongation at any particular time, and that neither of the subunits, in general, continues translation from one open reading frame to the next on a poly-cistronic mRNA. Furthermore, we find that a variety of previously published orthogonal ribosomes, with altered anti-Shine-Dalgarno sequences, show significant binding to endogenous mRNAs, with the rate of translation initiation only modestly affected. Hence, our results suggest that other mRNA elements than the SD sequence play major roles in directing the ribosome to the correct translation start sites.

molecular biology↗

bioRxiv2020

RecA mediated homology search finds segregated sister locus in minutes after a double stranded break

Homologous recombination (HR) is essential for the accurate repair of double-stranded DNA breaks (DSBs); it begins when the RecBCD2 complex resects the ends of the DSB into 3' single-stranded DNA (ssDNA) on which a RecA filament assembles. HR depends on the ability of this RecA-ssDNA filament to locate the homologous repair template on the sister chromosome. The mechanism by which the homology is located among vast amounts of heterologous DNA is not yet understood, despite a long history of research. Here, we directly visualize the repair of DSBs in hundreds of individual cells, using high-throughput microfluidics and fluorescence microscopy. We find that in E. coli, DSB repair is completed in 15 minutes without fitness loss. We further show that the search takes less than 10 minutes and is mediated by a thin, highly dynamic RecA filament that stretches throughout the cell. We propose a model in which the architecture of the RecA filament effectively reduces search dimensionality to two dimensions. The model is corroborated by the observation that the search time does not depend on the length of the cell or the amount of DNA, and also predicts a search time that is consistent with our measurement. Since the RecA family proteins are conserved in all organisms, our results also translate to other systems that rely on homologous recombination.

microbiology↗

bioRxiv2020

Mechanism of DNA surface exploration and operator bypassing during target search

Many proteins that bind specific DNA sequences search the genome by combining three dimensional (3D) diffusion in the cytoplasm with one dimensional (1D) sliding on non-specific DNA1-5. Here we combine resonance energy transfer and fluorescence correlation measurements to characterize how individual lac repressor (LacI) molecules explore DNA during the 1D phase of target search. To track the rotation of sliding LacI molecules on the microsecond time scale during DNA surface search, we use real-time single-molecule confocal laser tracking combined with fluorescence correlation spectroscopy (SMCT-FCS). The fluorescence signal fluctuations are accurately described by rotation-coupled sliding, where LacI traverses ~40 base pairs (bp) per revolution. This distance substantially exceeds the 10.5-bp helical pitch of DNA, suggesting that the sliding protein frequently hops out of the DNA groove, which would result in frequent bypassing of target sequences. Indeed, we directly observe such bypassing by single-molecule fluorescence resonance energy transfer (smFRET). A combined analysis of the smFRET and SMCT-FCS data shows that LacI at most hops one to two grooves (10-20 bp) every 250 s. Overall, our data suggest a speed-accuracy trade-off during sliding; the weak nature of non-specific protein-DNA interactions underlies operator bypassing but also facilitates rapid sliding. We anticipate that our SMCT-FCS method to monitor rotational diffusion on the microsecond time scale while tracking individual molecules with millisecond time resolution will be applicable to the real-time investigation of many other biological interactions and effectively extends the accessible time regime by two orders of magnitude.

biophysics↗

bioRxiv2019

A time-resolved imaging-based CRISPRi screening method

Our ability to connect genotypic variation to biologically important phenotypes has been seriously limited by the gap between live cell microscopy and library-scale genomic engineering. Specifically, this has restricted studies of intracellular dynamics to one strain at a time and thus, generally, to the impact of genes with known function. Here we show how in situ genotyping of a library of E. coli strains after time-lapse imaging in a microfluidic device overcomes this problem. We determine how 235 different CRISPR interference (CRISPRi) knockdowns impact the coordination of the replication and division cycles of E. coli by monitoring the location of replication forks throughout on average >500 cell cycles per knockdown. The single-cell time-resolved assay allows us to determine the distribution of single-cell growth rates, cell division sizes, and replication initiation volumes. Subsequent in situ genotyping allows us to map each phenotype distribution to a specific genetic perturbation in order to determine which genes are important for cell cycle control. The technology presented in this study enables genome-scale screens of virtually all live-cell microscopy assays and, therefore, constitutes a qualitatively new approach to cellular biophysics.

biophysics↗

bioRxiv2018

Direct observation of rotation-coupled protein diffusion along DNA on the microsecond timescale

Many proteins that bind specific DNA sequences search the genome by combining three dimensional (3D) diffusion in the cytoplasm with one dimensional (1D) sliding on non-specific regions of the DNA1-5. It is however not known how sliding proteins are oriented with respect to DNA in order to recognize specific sequences. Here we measure the polarization of fluorescence emission from single fluorescently labeled lac repressor (LacI) molecules sliding on stretched DNA. Real-time feedback-coupled confocal single-particle tracking allows us to measure fluorescence correlation of the sliding molecules. We find that the fluctuations in the fluorescence signal on the s timescale are accurately described by rotation-coupled sliding on DNA. On average, LacI moves [~]50 base pairs per revolution, which is significantly longer than the 10.5 bp helical periodicity of DNA. Our data support a facilitated diffusion model1 where the transcription factor (TF) scans the DNA grooves for hydrogen bonding opportunities in a pre-aligned orientation with occasional slippage out of the groove.

biophysics↗

bioRxiv2018

Variational algorithms for analyzing noisy multi-state diffusion trajectories

Single particle tracking offers a non-invasive high-resolution probe of biomolecular reactions inside living cells. However, efficient data analysis methods that correctly account for various noise soures are needed to realize the full quantitative potential of the method. We report new algorithms for hidden Markov based analysis of single particle tracking data, which incorporate most sources of experimental noise, including heterogeneuous localization errors and missing positions. Compared to previous implementations, the algorithms offer significant speed-ups, support for a wider range of inference methods, and a simple user interface. This will enable more advanced and exploratory quantitative analysis of single particle tracking data.

biophysics↗

bioRxiv2017

In Situ Genotyping Of A Pooled Strain Library After Characterizing Complex Phenotypes

So far, it has not been possible to perform advanced microscopy on pool generated strain libraries and at the same time know each strains genotype. We have overcome this barrier by identifying the genotypes for individual cells in situ after a detailed characterization of the phenotype. The principle is demonstrated by single molecule fluorescence imaging of E. coli strains harboring barcoded plasmids that express a sgRNA which suppress different genes through dCas9.

systems biology↗