Global DNA Compaction in Stationary-Phase Bacteria Does Not Affect Transcription.

In stationary-phase Escherichia coli, Dps (DNA-binding protein from starved cells) is the most abundant protein component of the nucleoid. Dps compacts DNA into a dense complex and protects it from damage. Dps has also been proposed to act as a global regulator of transcription. Here, we directly examine the impact of Dps-induced compaction of DNA on the activity of RNA polymerase (RNAP). Strikingly, deleting the dps gene decompacted the nucleoid but did not significantly alter the transcriptome and only mildly altered the proteome during stationary phase. Complementary in vitro assays demonstrated that Dps blocks restriction endonucleases but not RNAP from binding DNA. Single-molecule assays demonstrated that Dps dynamically condenses DNA around elongating RNAP without impeding its progress. We conclude that Dps forms a dynamic structure that excludes some DNA-binding proteins yet allows RNAP free access to the buried genes, a behavior characteristic of phase-separated organelles.

Bridging DNA contacts allow Dps from E. coli to condense DNA.

The DNA-binding protein from starved cells (Dps) plays a crucial role in maintaining bacterial cell viability during periods of stress. Dps is a nucleoid-associated protein that interacts with DNA to create biomolecular condensates in live bacteria. Purified Dps protein can also rapidly form large complexes when combined with DNA in vitro. However, the mechanism that allows these complexes to nucleate on DNA remains unclear. Here, we examine how DNA topology influences the formation of Dps-DNA complexes. We find that DNA supercoils offer the most preferred template for the nucleation of condensed Dps structures. More generally, bridging contacts between different regions of DNA can facilitate the nucleation of condensed Dps structures. In contrast, Dps shows little affinity for stretched linear DNA before it is relaxed. Once DNA is condensed, Dps forms a stable complex that can form inter-strand contacts with nearby DNA, even without free Dps present in solution. Taken together, our results establish the important role played by bridging contacts between DNA strands in nucleating and stabilizing Dps complexes.

Live to fight another day: The bacterial nucleoid under stress.

The bacterial chromosome is both highly supercoiled and bound by an ensemble of proteins and RNA, causing the DNA to form a compact structure termed the nucleoid. The nucleoid serves to condense, protect, and control access to the bacterial chromosome through a variety of mechanisms that remain incompletely understood. The nucleoid is also a dynamic structure, able to change both in size and composition. The dynamic nature of the bacterial nucleoid is particularly apparent when studying the effects of various stresses on bacteria, which require cells to protect their DNA and alter patterns of transcription. Stresses can lead to large changes in the organization and composition of the nucleoid on timescales as short as a few minutes. Here, we summarize some of the recent advances in our understanding of how stress can alter the organization of bacterial chromosomes.

Water Leakage Pathway Leads to Internal Hydration of the p53 Core Domain.

The gene encoding the p53 tumor suppressor protein is the most frequently mutated oncogene in cancer patients; yet, generalized strategies for rescuing the function of different p53 mutants remain elusive. This work investigates factors that may contribute to the low inherent stability of the wild-type p53 core domain (p53C) and structurally compromised Y220C mutant. Pressure-induced unfolding of p53C was compared to p63C, the p53 family member with the highest stability, the engineered superstable p53C hexamutant (p53C HM), and lower stability p53C Y220C cancer-associated mutant. The following pressure unfolding values (P50% bar) were obtained: p53C 3346, p53C Y220C 2217, p53C HM 3943, and p63C 4326. Molecular dynamics (MD) simulations revealed that p53C Y220C was most prone to water infiltration, followed by p53C, whereas the interiors of p53C HM and p63C remained comparably dry. A strong correlation (r2 = 0.92) between P50% and extent of interior hydration was observed. The pathways of individual water molecule entry and exit were mapped and analyzed, revealing a common route preserved across the p53 family involving a previously reported pocket, along with a novel surface cleft, both of which appear to be targetable by small molecules. Potential determinants of propensity to water incursion were assessed, including backbone hydrogen bond protection and combined sequence and structure similarity. Collectively, our results indicate that p53C has an intrinsic susceptibility to water leakage, which is exacerbated in a structural class mutant, suggesting that there may be a common avenue for rescuing p53 function.

Curvature-based sorting of eight lipid types in asymmetric buckled plasma membrane models.

Curvature is a fundamental property of biological membranes and has essential roles in cellular function. Bending of membranes can be induced by their lipid and protein compositions, as well as peripheral proteins, such as those that make up the cytoskeleton. An important aspect of membrane function is the grouping of lipid species into microdomains, or rafts, which serve as platforms for specific biochemical processes. The fluid mosaic model of membranes has evolved to recognize the importance of curvature and leaflet asymmetry, and there are efforts toward evaluating their functional roles. This work investigates the effect of curvature on the sorting of lipids in buckled asymmetric bilayers containing eight lipid types, approximating an average mammalian plasma membrane, through coarse-grained (CG) molecular dynamics (MD) simulations with the Martini force field. The simulations reveal that 1) leaflet compositional asymmetry can induce curvature asymmetry, 2) lipids are sorted by curvature to different extents, and 3) curvature-based partitioning trends show moderate to strong correlations with lipid molecular volumes and head to tail bead ratios, respectively. The findings provide unique insights into the role of curvature in membrane organization, and the curvature-based sorting trends should be useful references for later investigations and potentially interpreting the functional roles of specific lipids.

Do Go Chasing Waterfalls: Enoyl Reductase (FabI) in Complex with Inhibitors Stabilizes the Tetrameric Structure and Opens Water Channels.

The enzyme enoyl-ACP reductase (FabI) is the limiting step of the membrane's fatty acid biosynthesis in bacteria and a druggable target for novel antibacterial agents. The FabI active form is a homotetramer, which displays the highest affinity to inhibitors. Herein, molecular dynamics studies were carried out using the structure of FabI in complex with known inhibitors to investigate their effects on tetramerization. Our results suggest that multimerization is essential for the integrity of the catalytic site and that inhibitor binding enables the multimerization by stabilizing the substrate binding loop (SBL, L:195-200) coupled with changes in the H4/5 (QR interface). We also observed that AFN-1252 (naphtpyridinone derivative) promotes unique conformational changes affecting monomer-monomer interfaces. These changes are induced by AFN-1252 interaction with key residues in the binding sites (Ala95, Tyr146, and Tyr156). In addition, the analysis of water trajectories indicated that AFN-1252 complexes allow more water molecules to enter the binding site than triclosan and MUT056399 complexes. FabI-AFN-1252 simulations show accumulation of water molecules near the Tyr146/147 pocket, which can become a hotspot to the design of novel FabI inhibitors.

Lipid distributions and transleaflet cholesterol migration near heterogeneous surfaces in asymmetric bilayers.

Specific and nonspecific protein-lipid interactions in cell membranes have important roles in an abundance of biological functions. We have used coarse-grained (CG) molecular dynamics (MD) simulations to assess lipid distributions and cholesterol flipping dynamics around surfaces in a model asymmetric plasma membrane containing one of six structurally distinct entities: aquaporin-1 (AQP1), the bacterial ß-barrel outer membrane proteins OmpF and OmpX, the KcsA potassium channel, the WALP23 peptide and a carbon nanotube (CNT). Our findings revealed varied lipid partitioning and cholesterol flipping times around the different solutes and putative cholesterol binding sites in AQP1 and KcsA. The results suggest that protein-lipid interactions can be highly variable, and that surface-dependent lipid profiles are effectively manifested in CG simulations with the Martini force field.

Biophysical characterization of p53 core domain aggregates.

Aggregation is the cause of numerous protein conformation diseases. A common facet of these maladies is the transition of a protein from its functional native state into higher order forms, such as oligomers and amyloid fibrils. p53 is an essential tumor suppressor that is prone to such conformational transitions, resulting in its compromised ability to avert cancer. This work explores the biophysical properties of early-, mid-, and late-stage p53 core domain (p53C) aggregates. Atomistic and coarse-grained molecular dynamics (MD) simulations suggest that early- and mid-stage p53C aggregates have a polymorphic topology of antiparallel and parallel ß-sheets that localize to the core amyloidogenic sequence. Both topologies involve similar extents of interstrand mainchain hydrogen bonding, while sidechain interactions could play a role in regulating strand orientation. The free energy difference between the antiparallel and parallel states was within statistical uncertainty. Negative stain electron microscopy of mature fibrils shows a wide distribution of fiber widths, indicating that polymorphism may extend to the quaternary structure level. Circular dichroism of the fibrils was indicative of ß-sheet rich structures in atypical conformations. The Raman spectrum of aggregated p53C was consistent with a mixture of arranged ß-sheets and heterogeneous structural elements, which is compatible with the MD findings of an ordered ß-sheet nucleus flanked by disordered structure. Structural polymorphism is a common property of amyloids; however, because certain polymorphs of the same protein can be more harmful than others, going forward it will be pertinent to establish correlations between p53C aggregate structure and pathology.

Aggregation-primed molten globule conformers of the p53 core domain provide potential tools for studying p53C aggregation in cancer.

The functionality of the tumor suppressor p53 is altered in more than 50% of human cancers, and many individuals with cancer exhibit amyloid-like buildups of aggregated p53. An understanding of what triggers the pathogenic amyloid conversion of p53 is required for the further development of cancer therapies. Here, perturbation of the p53 core domain (p53C) with subdenaturing concentrations of guanidine hydrochloride and high hydrostatic pressure revealed native-like molten globule (MG) states, a subset of which were highly prone to amyloidogenic aggregation. We found that MG conformers of p53C, probably representing population-weighted averages of multiple states, have different volumetric properties, as determined by pressure perturbation and size-exclusion chromatography. We also found that they bind the fluorescent dye 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) and have a native-like tertiary structure that occludes the single Trp residue in p53. Fluorescence experiments revealed conformational changes of the single Trp and Tyr residues before p53 unfolding and the presence of MG conformers, some of which were highly prone to aggregation. p53C exhibited marginal unfolding cooperativity, which could be modulated from unfolding to aggregation pathways with chemical or physical forces. We conclude that trapping amyloid precursor states in solution is a promising approach for understanding p53 aggregation in cancer. Our findings support the use of single-Trp fluorescence as a probe for evaluating p53 stability, effects of mutations, and the efficacy of therapeutics designed to stabilize p53.

More than just a phase: the search for membraneless organelles in the bacterial cytoplasm.

The bacterial cytoplasm, once thought to be a relatively undifferentiated reaction medium, has now been recognized to have extensive microstructure. This microstructure includes bacterial microcompartments, inclusion bodies, granules, and even some membrane-bound vesicles. Several recent papers suggest that bacteria may also organize their cytoplasm using an additional mechanism: phase-separated membraneless organelles, a strategy commonly used by eukaryotes. Phase-separated membraneless organelles such as Cajal bodies, the nucleolus, and stress granules allow proteins to become concentrated in sub-compartments of eukaryotic cells without being surrounded by a barrier to diffusion. In this review, we summarize the known structural organization of the bacterial cytoplasm and discuss the recent evidence that phase-separated membraneless organelles might also play a role in bacterial systems. We specifically focus on bacterial ribonucleoprotein complexes and two different protein components of the bacterial nucleoid that may have the ability to form subcellular partitions within bacteria cells.

Targeting the Prion-like Aggregation of Mutant p53 to Combat Cancer.

Prion-like behavior of several amyloidogenic proteins has been demonstrated in recent years. Despite having functional roles in some cases, irregular aggregation can have devastating consequences. The most commonly known amyloid diseases are Alzheimer's, Parkinson's, and Transmissible Spongiform Encephalopathies (TSEs). The pathophysiology of prion-like diseases involves the structural transformation of wild-type (wt) proteins to transmissible forms that can convert healthy proteins, generating aggregates. The mutant form of tumor suppressor protein, p53, has recently been shown to exhibit prion-like properties. Within the context of p53 aggregation and the search for ways to avert it, this review emphasizes discoveries, approaches, and research from our laboratory and others. Although its standard functions are strongly connected to tumor suppression, p53 mutants and aggregates are involved in cancer progression. p53 aggregates are heterogeneous assemblies composed of amorphous aggregates, oligomers, and amyloid-like fibrils. Evidence of these structures in tumor tissues, the in vitro capability for p53 mutants to coaggregate with wt protein, and the detection of cell-to-cell transmission indicate that cancer has the basic characteristics of prion and prion-like diseases. Various approaches aim to restore p53 functions in cancer. Methods include the use of small-molecule and peptide stabilizers of mutant p53, zinc administration, gene therapy, alkylating and DNA intercalators, and blockage of p53-MDM2 interaction. A primary challenge in developing small-molecule inhibitors of p53 aggregation is the large number of p53 mutations. Another issue is the inability to recover p53 function by dissociating mature fibrils. Consequently, efforts have emerged to target the intermediate species of the aggregation reaction. Φ-value analysis has been used to characterize the kinetics of the early phases of p53 aggregation. Our experiments using high hydrostatic pressure (HHP) and chemical denaturants have helped to clarify excited conformers of p53 that are prone to aggregation. Molecular dynamics (MD) and phasor analysis of single Trp fluorescence signals point toward the presence of preamyloidogenic conformations of p53, which are not observed for p63 or p73. Exploring the features of competent preamyloidogenic states of wt and different p53 mutants may provide a framework for designing personalized drugs for the restoration of p53 function. Protection of backbone hydrogen bonds (BHBs) has been shown to be an important factor for the stability of amyloidogenic proteins and was employed to identify and stabilize the structural defect resulting from the p53 Y220C mutation. Using MD simulations, we compared BHB protection factors between p53 family members to determine the donor-acceptor pairs in p53 that exhibit lower protection. The identification of structurally vulnerable sites in p53 should provide new insights into rational designs that can rapidly be screened using our experimental methodology. Through continued and combined efforts, the outlook is positive for the development of strategies for regulating p53 amyloid transformation.

Neuroprotective Effect of Ilex Paraguariensis Intake on Brain Myelin of Lung Adenocarcinoma-Bearing Male Balb/c Mice.

Ilex paraguariensis (IP) is widely consumed as tea with high nutritional value. This plant contains several bioactive phenolic compounds, which are antioxidant and anti-inflammatory. On the other hand, lung adenocarcinoma (LAC) deleteriously involves neoplastic progression, inflammatory paraneoplastic syndromes, and death. Given that brain is a frequent target of this illness, our objective was to determine the neuroprotective effect of IP consumption in LAC-bearing mice. They were orally treated with 50 mg of IP extract/kg/day (IP50) for 3 weeks. Results (phenolic compounds, lipid peroxides, interleukin 6-IL-6-, tumor necrosis factor alpha -TNFα-, and luxol-stained myelination) were compared with respect to untreated controls (C) by the T test. IP50 significantly lowed brain IL-6 (2858.12 ± 57.81 pg g-1 vs. 3801.30 ± 27.34 pg g-1), whereas other variables differed in a less extent. C brains showed demyelination (low luxol-staining contrast between gray and white matters), with IP50 increasing myelination (P < 0.05). In conclusion, LAC deleterious effects on murine brain were prevented by dietary IP, which is an original discovery to develop a nutritional approach against cancer neurological compromise.

Heterogeneity in Adipose Stem Cells.

Adipose stem cells (ASCs) are the basis of procedures intended for tissue regeneration. These cells are heterogeneous, owing to various factors, including the donor age, sex, body mass index, and clinical condition; the isolation procedure (liposuction or fat excision); the place from where the cells were sampled (body site and depth of each adipose depot); culture surface; type of medium (whether supplemented with fetal bovine serum or xeno-free), that affect the principal phenotypic features of ASCs. The features related to ASCs heterogeneity are relevant for the success of therapeutic procedures; these features include proliferation capacity, differentiation potential, immunophenotype, and the secretome. These are important characteristics for the success of regenerative tissue engineering, not only because of their effects upon the reconstruction and healing exerted by ASCs themselves, but also because of the paracrine signaling of ASCs and its impact on recipient tissues. Knowledge of sources of heterogeneity will be helpful in the standardization of ASCs-based procedures. New avenues of research could include evaluation of the effects of the use of more homo1geneous ASCs for specific purposes, the study of ASCs-recipient interactions in heterologous cell transplantation, and the characterization of epigenetic changes in ASCs, as well as investigations of the effect of the metabolome upon ASCs behavior in culture.

Hysteresis in DNA compaction by Dps is described by an Ising model.

In all organisms, DNA molecules are tightly compacted into a dynamic 3D nucleoprotein complex. In bacteria, this compaction is governed by the family of nucleoid-associated proteins (NAPs). Under conditions of stress and starvation, an NAP called Dps (DNA-binding protein from starved cells) becomes highly up-regulated and can massively reorganize the bacterial chromosome. Although static structures of Dps-DNA complexes have been documented, little is known about the dynamics of their assembly. Here, we use fluorescence microscopy and magnetic-tweezers measurements to resolve the process of DNA compaction by Dps. Real-time in vitro studies demonstrated a highly cooperative process of Dps binding characterized by an abrupt collapse of the DNA extension, even under applied tension. Surprisingly, we also discovered a reproducible hysteresis in the process of compaction and decompaction of the Dps-DNA complex. This hysteresis is extremely stable over hour-long timescales despite the rapid binding and dissociation rates of Dps. A modified Ising model is successfully applied to fit these kinetic features. We find that long-lived hysteresis arises naturally as a consequence of protein cooperativity in large complexes and provides a useful mechanism for cells to adopt unique epigenetic states.

Effects of bioavailable phenolic compounds from Ilex paraguariensis on the brain of mice with lung adenocarcinoma.

Lung carcinoma is one of the most common cancers and has a high mortality. Recently, we showed that it produces neurological paraneoplastic syndrome, with Ilex paraguariensis (IP) extract exerting palliative effects due to its content of phenolic compounds. It is possible, therefore, that these diet agents can arrive at the brain and exert neuroprotection, after the oral intake of IP. Here, the aim was to investigate the protective role of bioavailable IP compounds on the telencephalon and diencephalon in lung adenocarcinoma-bearing BALB/cJ males. Mice aged 2 months were treated for 3 weeks with 0-100 IP mg·kg-1 ·day-1 . HPLC-UV revealed the presence of chlorogenic acid and quercetin in brain regions, liver, and tumour, in an IP dose-dependent manner. Brain was also evaluated histologically, and interleukin-6 was measured by ELISA. Chlorogenic acid was the major compound found in brain, whereas quercetin was observed at the diencephalon to a lesser extent. Both compounds were involved in IP dose-dependent diencephalic interleukin-6 reduction. Histology suggested cellular protection with less apoptosis in chlorogenic-exposed areas. Taken together, chlorogenic acid and quercetin from dietary IP were bioavailable and bioactive in brain, thereby attenuating lung cancer-related neuroinflammation and damage. These findings support plant-based strategies to improve prognosis.

Testing Wallace's intuition: water type, reproductive isolation and divergence in an Amazonian fish.

Alfred Russel Wallace proposed classifying Amazon rivers based on their colour and clarity: white, black and clear water. Wallace also proposed that black waters could mediate diversification and yield distinct fish species. Here, we bring evidence of speciation mediated by water type in the sailfin tetra (Crenuchus spilurus), a fish whose range encompasses rivers of very distinct hydrochemical conditions. Distribution of the two main lineages concords with Wallace's water types: one restricted to the acidic and nutrient-poor waters of the Negro River (herein Rio Negro lineage) and a second widespread throughout the remaining of the species' distribution (herein Amazonas lineage). These lineages occur over a very broad geographical range, suggesting that despite occurring in regions separated by thousands of kilometres, individuals of the distinct lineages fail to occupy each other's habitats, hundreds of metres apart and not separated by physical barrier. Reproductive isolation was assessed in isolated pairs exposed to black-water conditions. All pairs with at least one individual of the lineage not native to black waters showed significantly lower spawning success, suggesting that the water type affected the fitness and contributed to reproductive isolation. Our results endorse Wallace's intuition and highlight the importance of ecological factors in shaping diversity of the Amazon fish fauna.

Differential Potentiation of Retinoic Acid Effects against Human Breast Cancer Cells by Unsaturated Fatty Acids.

Retinoic acid (RA) and unsaturated fatty acids (UFA) are proposed as nutritional anticancer agents. Nonetheless, the activity of their combination on human breast cancer needs further study. Our aim was to evaluate this activity on the MCF-7 and ZR-75-1 cell lines treated with 1 µM RA and 50 µM of γ-linoleic (GLA, ω-6), eicosapentaenoic (EPA, ω-3), oleic (OA, ω-9), or eicosatrienoic (ETA, ω-9) acids. The following cellular responses were compared by ANOVA and Fisher test (P < 0.05): fatty acids, E-cadherin, actin (differentiation), conjugated dienes, γ-glutamyltranspeptidase activity (stress), and viability, which were correlated by partial least squares regression. Although both cell lines responded differentially, RA modified unsaturated fatty acids, increased differentiation, reduced γ-glutamyltranspeptidase, and viability. RA differentiating activity on ZR-75-1 was morphologically enhanced by UFA. Stress induction with γ-glutamyltranspeptidase decrease and conjugated dienes was promoted by ETA in MCF-7, and EPA and OA in ZR-75-1. RA-related reduced viability was potentiated by EPA and OA in both lines. GLA was less active. Therefore, unsaturated fatty acids (ω-3/ω-9) potentiated the multitarget retinoic acid activity against these human breast cancer cells.

Modulation of Fatty Acids and Interleukin-6 in Glioma Cells by South American Tea Extracts and their Phenolic Compounds.

Dietary phenolic compounds are plant metabolites with beneficial effects on the central nervous system. Thus, our aim was to identify anti-inflammatory compounds from South American plants on glia, which regulates neuro-immune response. The compounds were extracted from Lantana grisebachii (LG), Aspidosperma quebracho-blanco (AQB), and Ilex paraguariensis (IP) teas and identified by HPLC-DAD-MS. Extracts (0-200 µg/ml) were tested on human T98-G and rat C6 glioma lines. Cellular viability (by the resazurin assay), fatty acid profile (by gas chromatography) and pro-inflammatory interleukin-6 release (IL-6 by ELISA) were determined. Data were analyzed by partial least-square regression to discriminate bioactive compounds. Twenty-one compounds were determined in LG, mainly iridoids, which were linked to ω-3 and ω-6 polyunsaturated fatty acids, but not to IL-6 release. Thirty-one compounds were found in AQB, mostly hydroxybenzoic derivatives, which were positively related to IL-6 release. Twenty-three compounds were identified in IP, including caffeoylquinic derivatives and mainly chlorogenic acid. They increased the ω-7 palmitoleic fatty acid, which was related to IL-6 decrease. These results enhances phytochemical knowledge of widely available plants, and suggest the lipid-related anti-inflammatory activity of IP phenolic compounds, which give nutritional relevance to the tea.

Investigation of the binding mode of a novel cruzain inhibitor by docking, molecular dynamics, ab initio and MM/PBSA calculations.

Chagas disease remains a major health problem in South America, and throughout the world. The two drugs clinically available for its treatment have limited efficacy and cause serious adverse effects. Cruzain is an established therapeutic target of Trypanosoma cruzi, the protozoan that causes Chagas disease. Our group recently identified a competitive cruzain inhibitor (compound 1) with an IC50 = 15 µM that is also more synthetically accessible than the previously reported lead, compound 2. Prior studies, however, did not propose a binding mode for compound 1, hindering understanding of the structure-activity relationship and optimization. Here, the cruzain binding mode of compound 1 was investigated using docking, molecular dynamics (MD) simulations with ab initio derived parameters, ab initio calculations, and MM/PBSA. Two ligand protonation states and four binding poses were evaluated. A careful ligand parameterization method was employed to derive more physically meaningful parameters than those obtained by automated tools. The poses of unprotonated 1 were unstable in MD, showing large conformational changes and diffusing away from the binding site, whereas the protonated form showed higher stability and interaction with negatively charged residues Asp161 and Cys25. MM/PBSA also suggested that these two residues contribute favorably to binding of compound 1. By combining results from MD, ab initio calculations, and MM/PBSA, a binding mode of 1 is proposed. The results also provide insights for further optimization of 1, an interesting lead compound for the development of new cruzain inhibitors.

DNA binding proteins explore multiple local configurations during docking via rapid rebinding.

Finding the target site and associating in a specific orientation are essential tasks for DNA-binding proteins. In order to make the target search process as efficient as possible, proteins should not only rapidly diffuse to the target site but also dynamically explore multiple local configurations before diffusing away. Protein flipping is an example of this second process that has been observed previously, but the underlying mechanism of flipping remains unclear. Here, we probed the mechanism of protein flipping at the single molecule level, using HIV-1 reverse transcriptase (RT) as a model system. In order to test the effects of long-range attractive forces on flipping efficiency, we varied the salt concentration and macromolecular crowding conditions. As expected, increased salt concentrations weaken the binding of RT to DNA while increased crowding strengthens the binding. Moreover, when we analyzed the flipping kinetics, i.e. the rate and probability of flipping, at each condition we found that flipping was more efficient when RT bound more strongly. Our data are consistent with a view that DNA bound proteins undergo multiple rapid re-binding events, or short hops, that allow the protein to explore other configurations without completely dissociating from the DNA.