Interconvertible Lac Repressor–DNA Loops Revealed by Single-Molecule Experiments

Wong, Oi Kwan; Guthold, Martin; Erie, Dorothy A.; Gelles, Jeff

doi:10.1371/journal.pbio.0060232

Cited by 76 publications

(139 citation statements)

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“…For example, AFM does not have enough resolution to decipher the details of repressor binding to DNA. However, it permits visualization of the loop shape and protein position within the loop (15). Similarly, LacI was deduced to mediate A1 loops on a negatively supercoiled plasmid with 197 bp interoperator distance (16).…”

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confidence: 99%

“…Therefore, a further refinement of the role of various loops can be accomplished only by using an ensemble averaging free single-molecule approach, which enables dynamic observation of loop formation/breakdown and, therefore, offers the potential to identify diverse and temporally separate species, as demonstrated in the work by Finzi and Gelles using tethered particle motion (TPM) (21). A subsequent experiment on a DNA construct with shorter (158 bp and 153 bp) inter-operator spacings led to the observation of two interconverting looped states, attributed to a V-shape and an open tetramer conformer (15).…”

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confidence: 99%

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Tetramer opening in LacI-mediated DNA looping

Rutkauskas

Zhan²,

Matthews

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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Lactose repressor protein (LacI) controls transcription of the genes involved in lactose metabolism in bacteria. Essential to optimal LacI-mediated regulation is its ability to bind simultaneously to two operators, forming a loop on the intervening DNA. Recently, several lines of evidence (both theoretical and experimental) have suggested various possible loop structures associated with different DNA binding topologies and LacI tetramer structural conformations (adopted by flexing about the C-terminal tetramerization domain). We address, specifically, the role of protein opening in loop formation by employing the single-molecule tethered particle motion method on LacI protein mutants chemically cross-linked at different positions along the cleft between the two dimers. Measurements on the wild-type and uncross-linked LacI mutants led to the observation of two distinct levels of short tether length, associated with two different DNA looping structures. Restricting conformational flexibility of the protein by chemical cross-linking induces pronounced effects. Crosslinking the dimers at the level of the N-terminal DNA binding head (E36C) completely suppresses looping, whereas cross-linking near the C-terminal tetramerization domain (Q231C) results in changes of looping geometry detected by the measured tether length distributions. These observations lead to the conclusion that tetramer opening plays a definite role in at least a subset of LacI/DNA loop conformations. (1). This effect is due to the fact that LacI is a homotetrameric protein assembled as a dimer of dimers, with each dimer presenting a DNA-binding domain, enabling the tetramer to bind simultaneously to two operators (2) and form a loop in the intervening DNA ( Fig. 1 A and B). Consequently, LacI binding to one of the auxiliary operators increases the proximity of the remaining free DNA-binding domain to the primary operator, thus enhancing the probability of transcription blocking. Moreover, upon dissociation from the primary operator, the repressor is likely to remain bound to one of the auxiliary operators, avoiding diffusion into the cytosol and retaining proximity to the primary operator to facilitate rebinding. Thus, LacI-mediated DNA looping is fundamental for the efficiency of repression, as demonstrated by the drastic effects of deletion of auxiliary operators (3).In general, DNA looping is a recurring motif among several other DNA-binding proteins with different functions (4). The looping probability is quantified by the J m factor (5), defined as the local concentration of the LacI bound to one of the operators with respect to the remaining free operator. This factor is governed by the DNA-protein mechanics, as manifest in the profound dependence of in vivo repression (6) or in vitro cyclization efficiency (7) on interoperator spacing. Modulation of this property with the period of helical repeat is due to a large DNA-twisting-associated energetic penalty and, therefore, certain phase requirements for joining the loose DNA ends for cycliz...

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confidence: 99%

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confidence: 99%

Tetramer opening in LacI-mediated DNA looping

Rutkauskas

Zhan²,

Matthews

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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“…The LacI particles could be catalogued as either in the closed or open conformations22, 23 that were especially clear in DNA molecules with LacI‐mediated loops [Fig. 1(B)].…”

Section: Resultsmentioning

confidence: 99%

Proteins mediating DNA loops effectively block transcription

et al. 2017

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Loops are ubiquitous topological elements formed when proteins simultaneously bind to two noncontiguous DNA sites. While a loop‐mediating protein may regulate initiation at a promoter, the presence of the protein at the other site may be an obstacle for RNA polymerases (RNAP) transcribing a different gene. To test whether a DNA loop alters the extent to which a protein blocks transcription, the lac repressor (LacI) was used. The outcome of in vitro transcription along templates containing two LacI operators separated by 400 bp in the presence of LacI concentrations that produced both looped and unlooped molecules was visualized with scanning force microscopy (SFM). An analysis of transcription elongation complexes, moving for 60 s at an average of 10 nt/s on unlooped DNA templates, revealed that they more often surpassed LacI bound to the lower affinity O2 operator than to the highest affinity Os operator. However, this difference was abrogated in looped DNA molecules where LacI became a strong roadblock independently of the affinity of the operator. Recordings of transcription elongation complexes, using magnetic tweezers, confirmed that they halted for several minutes upon encountering a LacI bound to a single operator. The average pause lifetime is compatible with RNAP waiting for LacI dissociation, however, the LacI open conformation visualized in the SFM images also suggests that LacI could straddle RNAP to let it pass. Independently of the mechanism by which RNAP bypasses the LacI roadblock, the data indicate that an obstacle with looped topology more effectively interferes with transcription.

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“…This early study also indicated that the primary mechanism for loop breakdown was not tetramer dissociation into two bound dimers, but tetramer dissociation from operator DNA (7). Further TPM experimentation (11,29) accompanied by AFM imaging (30) showed that the loop is preferentially antiparallel and that LacI may adopt two structures in dynamic equilibrium: the "V" conformation observed in crystallographic studies and a more extended conformation that considerably relieves the strain due to DNA bending. Protein flexure may facilitate the ability to switch between different structures in simple short loops.…”

Section: Specific Dna Looping and Flexibility Of The Closure Proteinmentioning

confidence: 99%

Single-molecule Approaches to Probe the Structure, Kinetics, and Thermodynamics of Nucleoprotein Complexes That Regulate Transcription

Finzi¹,

Dunlap

2010

Journal of Biological Chemistry

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Single-molecule experimentation has contributed significantly to our understanding of the mechanics of nucleoprotein complexes that regulate epigenetic switches. In this minireview, we will discuss the application of the tethered-particle motion technique, magnetic tweezers, and atomic force microscopy to (i) directly visualize and thermodynamically characterize DNA loops induced by the lac, gal, and repressors and (ii) understand the mechanistic role of DNA-supercoiling and DNAbending cofactors in both prokaryotic and eukaryotic systems.Transcriptional regulation involving protein-mediated DNA bending, wrapping, and looping occurs ubiquitously in all organisms. By enabling long-distance interaction between transcription factors, limiting the accessibility to, and/or mechanically deforming promoters, nucleoprotein complexes can tune transcription effectively. In other words, protein-mediated bends, wraps, or loops in DNA constitute elements for transcriptional regulation. Some of the best known examples of DNA bends are induced by the IHF 2 protein in prokaryotes (1) and by the HMG1 protein in eukaryotes (2). The wrapping of DNA around histone octamers is a fundamental structure in eukaryotic chromatin (3), but most, if not all, DNA-binding proteins are hypothesized to be able to wrap and thereby organize flanking regions of DNA (4). Examples of DNA looping include the large chromatin loops proposed to explain insulator domains in eukaryotes (5) and the loops induced by prokaryotic repressors, such as the lac and gal repressors (see below). Although these examples pertain to transcriptional regulation, the same conformational changes are common in the regulation of most DNA transactions, such as replication, recombination, etc. Therefore, characterizing the kinetics and thermodynamics of the formation of nucleoprotein complexes involving DNA bending, wrapping, or looping, as well as their structure and stoichiometry, is paramount to understand their functions. Enhanced understanding of epigenetic regulation also might enable the design of mechanistic mutations for biomedical applications. Single-molecule experiments are incisive means with which to explore protein-induced conformational changes in DNA because they avoid ensemble averaging and reveal heterogeneities that might go undetected in bulk measurements. The following is a review of recent TPM, MT, and AFM experiments on nucleoprotein complexes that regulate transcription. TPM, MT, AFM, and Advantages of Single-molecule ExperimentsSingle-molecule assays yield distributions of individual measurements instead of averages for large ensembles of molecules. This provides a wealth of information and often reveals heterogeneous behaviors obscured in bulk experiments. Such discriminatory power has produced new insight even into "well understood" paradigms, such as prokaryotic transcriptional repressors (see below). Single-molecule microscopy and spectroscopy can be used to distinguish protein-induced DNA bending, looping, and wrapping, and they permit facile cont...

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Interconvertible Lac Repressor–DNA Loops Revealed by Single-Molecule Experiments

Cited by 76 publications

References 79 publications

Tetramer opening in LacI-mediated DNA looping

Tetramer opening in LacI-mediated DNA looping

Proteins mediating DNA loops effectively block transcription

Single-molecule Approaches to Probe the Structure, Kinetics, and Thermodynamics of Nucleoprotein Complexes That Regulate Transcription

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