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Interconvertible lac repressor-DNA loops revealed by single-molecule experiments.

Wong OK, Guthold M, Erie DA, Gelles J - PLoS Biol. (2008)

Bottom Line: Unexpectedly, repeated spontaneous transitions between two distinct loop structures were observed in individual protein-DNA complexes.The results imply a dynamic equilibrium between a novel loop structure with the repressor in its crystallographic "V" conformation and a second structure with a more extended linear repressor conformation that substantially lessens the DNA bending strain.The ability to switch between different loop structures may help to explain how robust transcription regulation is maintained even though the mechanical work required to form a loop may change substantially with metabolic conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Brandeis University, Waltham, Massachusetts, USA.

ABSTRACT
At many promoters, transcription is regulated by simultaneous binding of a protein to multiple sites on DNA, but the structures and dynamics of such transcription factor-mediated DNA loops are poorly understood. We directly examined in vitro loop formation mediated by Escherichia coli lactose repressor using single-molecule structural and kinetics methods. Small ( approximately 150 bp) loops form quickly and stably, even with out-of-phase operator spacings. Unexpectedly, repeated spontaneous transitions between two distinct loop structures were observed in individual protein-DNA complexes. The results imply a dynamic equilibrium between a novel loop structure with the repressor in its crystallographic "V" conformation and a second structure with a more extended linear repressor conformation that substantially lessens the DNA bending strain. The ability to switch between different loop structures may help to explain how robust transcription regulation is maintained even though the mechanical work required to form a loop may change substantially with metabolic conditions.

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Related in: MedlinePlus

State Lifetime Distributions (A–E) and the Mechanisms and Rate Constants Determined from the TPM Data (F and G)For each state, N measured lifetimes were used to construct a histogram (circles) of bin width W, which is plotted here as a scaled probability density (see Materials and Methods); also shown (lines) are the theoretical distributions calculated from the mechanisms and rate constants.(A) O-153-O unlooped state (N = 110, W = 15 s); (B) O-153-O looped state (N = 108, W = 18 s); (C) O-158-O unlooped state (N = 168, W = 15 s); (D) O-158-O long-tether loop (N = 245, W = 40 s); (E) O-158-O short-tether loop (N = 80, W = 30 s). (F and G) Proposed mechanisms for repressor-mediated looping of O-153-O (F) and O-158-O (G). O2 (lines), di-operator DNA molecule; R (squares), repressor tetramer. For simplicity, only one of the two identical reaction steps linking each of the two equivalent O2R linear species with the other states is shown; the rate constants are the microscopic rate constants for each of the individual reaction steps. Similarly, unlooping rate constants k-loop, k2, and k6 are for the dissociation of repressor–single operator interactions; there are two such interactions in a loop so that the overall unlooping rate constant is twice the value given. ka* is the pseudo first-order rate constant for repressor association with operator. Numbers in parentheses are the standard error (S.E.) of the final digit of the corresponding rate constant. Rate constants were determined from observed state lifetimes, equilibrium constants, and kinetic partition ratios as described in Materials and Methods. The O-158-O rate constant in brackets was not well determined by the experimental data and was instead assumed to be equal to the corresponding O-153-O rate constant (see Materials and Methods).
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pbio-0060232-g003: State Lifetime Distributions (A–E) and the Mechanisms and Rate Constants Determined from the TPM Data (F and G)For each state, N measured lifetimes were used to construct a histogram (circles) of bin width W, which is plotted here as a scaled probability density (see Materials and Methods); also shown (lines) are the theoretical distributions calculated from the mechanisms and rate constants.(A) O-153-O unlooped state (N = 110, W = 15 s); (B) O-153-O looped state (N = 108, W = 18 s); (C) O-158-O unlooped state (N = 168, W = 15 s); (D) O-158-O long-tether loop (N = 245, W = 40 s); (E) O-158-O short-tether loop (N = 80, W = 30 s). (F and G) Proposed mechanisms for repressor-mediated looping of O-153-O (F) and O-158-O (G). O2 (lines), di-operator DNA molecule; R (squares), repressor tetramer. For simplicity, only one of the two identical reaction steps linking each of the two equivalent O2R linear species with the other states is shown; the rate constants are the microscopic rate constants for each of the individual reaction steps. Similarly, unlooping rate constants k-loop, k2, and k6 are for the dissociation of repressor–single operator interactions; there are two such interactions in a loop so that the overall unlooping rate constant is twice the value given. ka* is the pseudo first-order rate constant for repressor association with operator. Numbers in parentheses are the standard error (S.E.) of the final digit of the corresponding rate constant. Rate constants were determined from observed state lifetimes, equilibrium constants, and kinetic partition ratios as described in Materials and Methods. The O-158-O rate constant in brackets was not well determined by the experimental data and was instead assumed to be equal to the corresponding O-153-O rate constant (see Materials and Methods).

Mentions: To more fully characterize the mechanism by which repressor interacts with O-153-O, the complete set of O-153-O TPM records (e.g., Figure 2C) was analyzed to determine the lifetime distributions of the unlooped (Figure 3A) and looped (Figure 3B) states. The loop lifetime histogram is well fit by a simple exponential function, consistent with the interpretation that the O-153-O looped state is a single chemical species, not an unresolved mixture of two states. In contrast, the unlooped state lifetime histogram requires a distribution function that is the sum of at least two exponential terms to produce an acceptable fit. A multiexponential distribution is expected [23] because we know a priori that an unlooped di-operator DNA can exist in a minimum of four different states, an “O2R2” state that has two bound repressor molecules, two equivalent “O2R linear” states in which a single repressor molecule interacts with one operator, and an “O2” state with no bound repressor. These four unlooped species, together with the single looped species, comprise the minimal kinetic scheme (Figure 3F) for the interaction of repressor with O-153-O DNA. The scheme has only four independent rate constants; because the TPM experiments provide the shapes of the lifetime distributions (for both the looped and aggregate unlooped states) and the equilibrium constant (between the looped and aggregate unlooped states), they allow determination of well-constrained values for all four (Figure 3F; see Materials and Methods). The shapes of the lifetime distributions predicted by this scheme reproduce the empirical data within experimental uncertainty (Figure 3A and 3B); similarly, the value of the equilibrium constant predicted by this scheme and that determined by experiment are also in good agreement (0.48 and 0.53 ± 0.11, respectively).


Interconvertible lac repressor-DNA loops revealed by single-molecule experiments.

Wong OK, Guthold M, Erie DA, Gelles J - PLoS Biol. (2008)

State Lifetime Distributions (A–E) and the Mechanisms and Rate Constants Determined from the TPM Data (F and G)For each state, N measured lifetimes were used to construct a histogram (circles) of bin width W, which is plotted here as a scaled probability density (see Materials and Methods); also shown (lines) are the theoretical distributions calculated from the mechanisms and rate constants.(A) O-153-O unlooped state (N = 110, W = 15 s); (B) O-153-O looped state (N = 108, W = 18 s); (C) O-158-O unlooped state (N = 168, W = 15 s); (D) O-158-O long-tether loop (N = 245, W = 40 s); (E) O-158-O short-tether loop (N = 80, W = 30 s). (F and G) Proposed mechanisms for repressor-mediated looping of O-153-O (F) and O-158-O (G). O2 (lines), di-operator DNA molecule; R (squares), repressor tetramer. For simplicity, only one of the two identical reaction steps linking each of the two equivalent O2R linear species with the other states is shown; the rate constants are the microscopic rate constants for each of the individual reaction steps. Similarly, unlooping rate constants k-loop, k2, and k6 are for the dissociation of repressor–single operator interactions; there are two such interactions in a loop so that the overall unlooping rate constant is twice the value given. ka* is the pseudo first-order rate constant for repressor association with operator. Numbers in parentheses are the standard error (S.E.) of the final digit of the corresponding rate constant. Rate constants were determined from observed state lifetimes, equilibrium constants, and kinetic partition ratios as described in Materials and Methods. The O-158-O rate constant in brackets was not well determined by the experimental data and was instead assumed to be equal to the corresponding O-153-O rate constant (see Materials and Methods).
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2553838&req=5

pbio-0060232-g003: State Lifetime Distributions (A–E) and the Mechanisms and Rate Constants Determined from the TPM Data (F and G)For each state, N measured lifetimes were used to construct a histogram (circles) of bin width W, which is plotted here as a scaled probability density (see Materials and Methods); also shown (lines) are the theoretical distributions calculated from the mechanisms and rate constants.(A) O-153-O unlooped state (N = 110, W = 15 s); (B) O-153-O looped state (N = 108, W = 18 s); (C) O-158-O unlooped state (N = 168, W = 15 s); (D) O-158-O long-tether loop (N = 245, W = 40 s); (E) O-158-O short-tether loop (N = 80, W = 30 s). (F and G) Proposed mechanisms for repressor-mediated looping of O-153-O (F) and O-158-O (G). O2 (lines), di-operator DNA molecule; R (squares), repressor tetramer. For simplicity, only one of the two identical reaction steps linking each of the two equivalent O2R linear species with the other states is shown; the rate constants are the microscopic rate constants for each of the individual reaction steps. Similarly, unlooping rate constants k-loop, k2, and k6 are for the dissociation of repressor–single operator interactions; there are two such interactions in a loop so that the overall unlooping rate constant is twice the value given. ka* is the pseudo first-order rate constant for repressor association with operator. Numbers in parentheses are the standard error (S.E.) of the final digit of the corresponding rate constant. Rate constants were determined from observed state lifetimes, equilibrium constants, and kinetic partition ratios as described in Materials and Methods. The O-158-O rate constant in brackets was not well determined by the experimental data and was instead assumed to be equal to the corresponding O-153-O rate constant (see Materials and Methods).
Mentions: To more fully characterize the mechanism by which repressor interacts with O-153-O, the complete set of O-153-O TPM records (e.g., Figure 2C) was analyzed to determine the lifetime distributions of the unlooped (Figure 3A) and looped (Figure 3B) states. The loop lifetime histogram is well fit by a simple exponential function, consistent with the interpretation that the O-153-O looped state is a single chemical species, not an unresolved mixture of two states. In contrast, the unlooped state lifetime histogram requires a distribution function that is the sum of at least two exponential terms to produce an acceptable fit. A multiexponential distribution is expected [23] because we know a priori that an unlooped di-operator DNA can exist in a minimum of four different states, an “O2R2” state that has two bound repressor molecules, two equivalent “O2R linear” states in which a single repressor molecule interacts with one operator, and an “O2” state with no bound repressor. These four unlooped species, together with the single looped species, comprise the minimal kinetic scheme (Figure 3F) for the interaction of repressor with O-153-O DNA. The scheme has only four independent rate constants; because the TPM experiments provide the shapes of the lifetime distributions (for both the looped and aggregate unlooped states) and the equilibrium constant (between the looped and aggregate unlooped states), they allow determination of well-constrained values for all four (Figure 3F; see Materials and Methods). The shapes of the lifetime distributions predicted by this scheme reproduce the empirical data within experimental uncertainty (Figure 3A and 3B); similarly, the value of the equilibrium constant predicted by this scheme and that determined by experiment are also in good agreement (0.48 and 0.53 ± 0.11, respectively).

Bottom Line: Unexpectedly, repeated spontaneous transitions between two distinct loop structures were observed in individual protein-DNA complexes.The results imply a dynamic equilibrium between a novel loop structure with the repressor in its crystallographic "V" conformation and a second structure with a more extended linear repressor conformation that substantially lessens the DNA bending strain.The ability to switch between different loop structures may help to explain how robust transcription regulation is maintained even though the mechanical work required to form a loop may change substantially with metabolic conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Brandeis University, Waltham, Massachusetts, USA.

ABSTRACT
At many promoters, transcription is regulated by simultaneous binding of a protein to multiple sites on DNA, but the structures and dynamics of such transcription factor-mediated DNA loops are poorly understood. We directly examined in vitro loop formation mediated by Escherichia coli lactose repressor using single-molecule structural and kinetics methods. Small ( approximately 150 bp) loops form quickly and stably, even with out-of-phase operator spacings. Unexpectedly, repeated spontaneous transitions between two distinct loop structures were observed in individual protein-DNA complexes. The results imply a dynamic equilibrium between a novel loop structure with the repressor in its crystallographic "V" conformation and a second structure with a more extended linear repressor conformation that substantially lessens the DNA bending strain. The ability to switch between different loop structures may help to explain how robust transcription regulation is maintained even though the mechanical work required to form a loop may change substantially with metabolic conditions.

Show MeSH
Related in: MedlinePlus