The lac operon is a well-known example of gene expression regulation, based on the specific interaction of Lac repressor protein (LacI) with its target DNA sequence (operator). We recently developed an ultrafast force-clamp laser trap technique capable of probing molecular interactions with sub-ms temporal resolution, under controlled pN-range forces. With this technique, we tested the interaction of LacI with different DNA constructs. Based on position along the DNA sequence, the observed interactions can be interpreted as specific binding to operator sequences and transient interactions with nonspecific sequences.
The lac operon is a well known example of gene expression regulation, based on the specific interaction of Lac repressor protein (LacI) with its target DNA sequence (operator). LacI and other DNA-binding proteins bind their specific target sequences with rates higher than allowed by 3D diffusion alone. Generally accepted models predict a combination of free 3D diffusion and 1D sliding along non-specific DNA. We recently developed an ultrafast force-clamp laser trap technique capable of probing molecular interactions with sub-ms temporal resolution, under controlled pN-range forces. With this technique, we tested the interaction of LacI with two different DNA constructs: a construct with two copies of the O1 operator separated by 300 bp and a construct containing the native E.coli operator sequences. Our measurements show at least two classes of LacI-DNA interactions: long (in the tens of s range) and short (tens of ms). Based on position along the DNA sequence, the observed interactions can be interpreted as specific binding to operator sequences (long events) and transient interactions with nonspecific sequences (short events). Moreover, we observe continuous sliding of the protein along DNA, passively driven by the force applied with the optical tweezers.
The maintenance of intact genetic information, as well as the deployment of transcription for specific sets of genes,
critically rely on a family of proteins interacting with DNA and recognizing specific sequences or features. The
mechanisms by which these proteins search for target DNA are the subject of intense investigations employing a variety
of methods in biology. A large interest in these processes stems from the faster-than-diffusion association rates,
explained in current models by a combination of 3D and 1D diffusion. Here, we describe the combination of optical
tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the
opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in
other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence.
Forces play a fundamental role in a wide array of biological processes, regulating enzymatic activity, kinetics of
molecular bonds, and molecular motors mechanics. Single molecule force spectroscopy techniques have enabled the
investigation of such processes, but they are inadequate to probe short-lived (millisecond and sub-millisecond) molecular
complexes. We developed an ultrafast force-clamp spectroscopy technique that uses a dual trap configuration to apply
constant loads to a single intermittently interacting biological polymer and a binding protein. Our system displays a delay
of only ∼10 μs between formation of the molecular bond and application of the force and is capable of detecting
interactions as short as 100 μs. The force-clamp configuration in which our assay operates allows direct measurements of
load-dependence of lifetimes of single molecular bonds. Moreover, conformational changes of single proteins and
molecular motors can be recorded with sub-nanometer accuracy and few tens of microseconds of temporal resolution.
We demonstrate our technique on molecular motors, using myosin II from fast skeletal muscle and on protein-DNA
interaction, specifically on Lactose repressor (LacI). The apparatus is stabilized to less than 1 nm with both passive and
active stabilization, allowing resolving specific binding regions along the actin filament and DNA molecule. Our
technique extends single-molecule force-clamp spectroscopy to molecular complexes that have been inaccessible up to
now, opening new perspectives for the investigation of the effects of forces on biological processes.
We recently developed an ultrafast force-clamp laser trap capable to probe, under controlled force, bimolecular
interactions with unprecedented temporal resolution. Here we present the technique in the framework of protein-DNA
interactions, specifically on Lactose repressor protein (LacI). The high temporal resolution of the method reveals the
kinetics of both short- and long-lived interactions of LacI along the DNA template (from ∼100 μs to tens of seconds), as
well the dependence on force of such interaction kinetics. The two kinetically well-distinct populations of interactions
observed clearly represent specific interactions with the operator sequences and a fast scanning of LacI along non-cognate
DNA. These results demonstrate the effectiveness of the method to study the sequence-dependent affinity of
DNA-binding proteins along the DNA and the effects of force on a wide range of interaction durations, including μs time
scales not accessible to other single-molecule methods. This improvement in time resolution provides also important
means of investigation on the long-puzzled mechanism of target search on DNA and possible protein conformational
changes occurring upon target recognition.
Here we report the effect of DNA tension on lac repressor 1D-diffusion through a combination of single-molecule
localization and optical trapping. The diffusion coefficient shows a parabolic dependence on DNA tension.
Forces play a fundamental role in a wide array of biological processes, regulating enzymatic activity, kinetics of
molecular bonds, and molecular motors mechanics. Single molecule force spectroscopy techniques have enabled the
investigation of such processes, but they are inadequate to probe short-lived (millisecond and sub-millisecond) molecular
complexes. We developed an ultrafast force-clamp spectroscopy technique that uses a dual trap configuration to apply
constant loads to a single intermittently interacting biological polymer and a binding protein. Our system displays a delay
of only ∼10 μs between formation of the molecular bond and application of the force and is capable of detecting
interactions as short as 100 μs. The force-clamp configuration in which our assay operates allows direct measurements of
load-dependence of lifetimes of single molecular bonds. Moreover, conformational changes of single proteins and
molecular motors can be recorded with sub-nanometer accuracy and few tens of microseconds of temporal resolution.
We demonstrate our technique on molecular motors, using myosin II from fast skeletal muscle and on protein-DNA
interaction, specifically on Lactose repressor (LacI). The apparatus is stabilized to less than 1 nm with both passive and
active stabilization, allowing resolving specific binding regions along the actin filament and DNA molecule. Our
technique extends single-molecule force-clamp spectroscopy to molecular complexes that have been inaccessible up to
now, opening new perspectives for the investigation of the effects of forces on biological processes.
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