scholarly journals Direct substitution and assisted dissociation pathways for turning off transcription by a MerR-family metalloregulator

2012 ◽  
Vol 109 (38) ◽  
pp. 15121-15126 ◽  
Author(s):  
Chandra P. Joshi ◽  
Debashis Panda ◽  
Danya J. Martell ◽  
Nesha May Andoy ◽  
Tai-Yen Chen ◽  
...  
Keyword(s):  
Dna Binding ◽  
Single Molecule ◽  
Dna Sequences ◽  
Metal Binding ◽  
Metal Resistance ◽  
Binding Modes ◽  
Dynamic Interactions ◽  
Single Molecule Fret ◽  
Waste Energy ◽  
Kinetics Of

Metalloregulators regulate transcription in response to metal ions. Many studies have provided insights into how transcription is activated upon metal binding by MerR-family metalloregulators. In contrast, how transcription is turned off after activation is unclear. Turning off transcription promptly is important, however, as the cells would not want to continue expressing metal resistance genes and thus waste energy after metal stress is relieved. Using single-molecule FRET measurements we studied the dynamic interactions of the copper efflux regulator (CueR), a Cu+-responsive MerR-family metalloregulator, with DNA. Besides quantifying its DNA binding and unbinding kinetics, we discovered that CueR spontaneously flips its binding orientation at the recognition site. CueR also has two different binding modes, corresponding to interactions with specific and nonspecific DNA sequences, which would facilitate recognition localization. Most strikingly, a CueR molecule coming from solution can directly substitute for a DNA-bound CueR or assist the dissociation of the incumbent CueR, both of which are unique examples for any DNA-binding protein. The kinetics of the direct protein substitution and assisted dissociation reactions indicate that these two unique processes can provide efficient pathways to replace a DNA-bound holo-CueR with apo-CueR, thus turning off transcription promptly and facilely.

scholarly journals Single-molecule imaging uncover novel pathways in transcriptional regulation

2014 ◽  
Vol 70 (a1) ◽  
pp. C109-C109
Author(s):  
Peng Chen
Keyword(s):  
Transcriptional Regulation ◽  
Single Molecule ◽  
Metal Binding ◽  
Metal Resistance ◽  
Bacterial Cells ◽  
Dynamic Interactions ◽  
Single Molecule Tracking ◽  
Single Molecule Fret ◽  
Waste Energy ◽  
Kinetics Of

Metalloregulators regulate transcription in response to metal ions. Many studies have provided insights into how transcription is activated upon metal binding by MerR-family metalloregulators. In contrast, how transcription is turned off after activation is unclear. Turning off transcription promptly is important, however, as the cells would not want to continue expressing metal resistance genes and thus waste energy after metal stress is relieved. Using single-molecule FRET measurements, we studied the dynamic interactions of CueR, a Cu+-responsive MerR-family metalloregulator, with DNA. We discovered that a CueR molecule coming from solution can directly substitute for a DNA-bound CueR or assist the dissociation of the incumbent CueR. The kinetics of the direct protein substitution and assisted dissociation reactions indicate that these two novel processes can provide efficient pathways to replace a DNA-bound holo-CueR with apo-CueR, thus turning off transcription promptly and facilely. Using single-molecule tracking experiments, we further show that these novel pathways also operate in living bacterial cells.

scholarly journals Real-time kinetic studies of Mycobacterium tuberculosis LexA-DNA interaction

2021 ◽  
Author(s):  
Chitral Chatterjee ◽  
Soneya Majumdar ◽  
Sachin Deshpande ◽  
Deepak Pant ◽  
Saravanan Matheshwaran
Keyword(s):  
Amino Acids ◽  
Mycobacterium Tuberculosis ◽  
Dna Binding ◽  
Kinetic Parameters ◽  
Dna Sequences ◽  
Kinetic Studies ◽  
Dna Interaction ◽  
Damage Repair ◽  
Inducible Genes ◽  
Kinetics Of

Transcriptional repressor, LexA, regulates the “SOS” response, an indispensable bacterial DNA damage repair machinery.  Compared to its E.coli ortholog, LexA from Mycobacterium tuberculosis (Mtb) possesses a unique N-terminal extension of additional 24 amino acids in its DNA binding domain (DBD) and 18 amino acids insertion at its hinge region that connects the DBD to the C-terminal dimerization/autoproteolysis domain. Despite the importance of LexA in “SOS” regulation, Mtb LexA remains poorly characterized and the functional importance of its additional amino acids remained elusive. In addition, the lack of data on kinetic parameters of Mtb LexA-DNA interaction prompted us to perform kinetic analyses of Mtb LexA and its deletion variants using Bio-layer Interferometry (BLI). Mtb LexA is seen to bind to different “SOS” boxes, DNA sequences present in the operator regions of damage-inducible genes, with comparable nanomolar affinity. Deletion of 18 amino acids from the linker region is found to affect DNA binding unlike the deletion of the N-terminal stretch of extra 24 amino acids. The conserved RKG motif has been found to be critical for DNA binding. Overall, this study provides insights into the kinetics of the interaction between Mtb LexA and its target “SOS” boxes. The kinetic parameters obtained for DNA binding of Mtb LexA would be instrumental to clearly understand the mechanism of “SOS” regulation and activation in Mtb.

scholarly journals p53 dynamically directs TFIID assembly on target gene promoters

2016 ◽  
Author(s):  
R. A. Coleman ◽  
Z. Qiao ◽  
S. K. Singh ◽  
C. S. Peng ◽  
M. Cianfrocco ◽  
...  
Keyword(s):  
Dna Binding ◽  
Single Molecule ◽  
Transcription Initiation ◽  
Gene Networks ◽  
Target Gene ◽  
Core Promoter ◽  
Gene Promoters ◽  
Binding Modes ◽  
Dissociation Kinetics ◽  
Target Promoters

AbstractThe p53 tumor suppressor protein is a central regulator that turns on vast gene networks to maintain cellular integrity upon various stimuli. p53 activates transcription initiation in part by aiding recruitment of TFIID to the promoter. However, the precise means by which p53 dynamically interacts with TFIID to facilitate assembly on target gene promoters remains elusive. To address this key question, we have undertaken an integrated approach involving single molecule fluorescence microscopy, single particle cryo-electron microscopy, and biochemistry. Our real-time single molecule imaging demonstrates that TFIID alone binds poorly to native p53 target promoters. p53 unlocks TFIID’s ability to bind DNA by increasing TFIID contacts with both the core promoter and a region surrounding p53’s response element (RE). Analysis of single molecule dissociation kinetics reveals that TFIID interacts with promoters via transient and prolonged DNA binding modes that are each regulated by p53. Importantly, our structural work reveals that TFIID’s conversion from a canonical form to a rearranged DNA-binding conformation is enhanced in the presence of DNA and p53. Notably, TFIID’s interaction with DNA induces p53 to rapidly dissociate, effectively liberating the RE on the promoter. Collectively, these findings indicate that p53 dynamically escorts and loads the basal transcription machinery onto its target promoters.

scholarly journals The HMGB chromatin protein Nhp6A can bypass obstacles when traveling on DNA

2020 ◽  
Vol 48 (19) ◽  
pp. 10820-10831
Author(s):  
Kiyoto Kamagata ◽  
Kana Ouchi ◽  
Cheng Tan ◽  
Eriko Mano ◽  
Sridhar Mandali ◽  
...  
Keyword(s):  
Dna Binding ◽  
Single Molecule ◽  
Binding Proteins ◽  
Dna Binding Proteins ◽  
Binding Modes ◽  
Dna Structures ◽  
Bacterial Proteins ◽  
Associated Proteins ◽  
Diffusion Mechanisms ◽  
Dynamics Simulations

Abstract DNA binding proteins rapidly locate their specific DNA targets through a combination of 3D and 1D diffusion mechanisms, with the 1D search involving bidirectional sliding along DNA. However, even in nucleosome-free regions, chromosomes are highly decorated with associated proteins that may block sliding. Here we investigate the ability of the abundant chromatin-associated HMGB protein Nhp6A from Saccharomyces cerevisiae to travel along DNA in the presence of other architectural DNA binding proteins using single-molecule fluorescence microscopy. We observed that 1D diffusion by Nhp6A molecules is retarded by increasing densities of the bacterial proteins Fis and HU and by Nhp6A, indicating these structurally diverse proteins impede Nhp6A mobility on DNA. However, the average travel distances were larger than the average distances between neighboring proteins, implying Nhp6A is able to bypass each of these obstacles. Together with molecular dynamics simulations, our analyses suggest two binding modes: mobile molecules that can bypass barriers as they seek out DNA targets, and near stationary molecules that are associated with neighboring proteins or preferred DNA structures. The ability of mobile Nhp6A molecules to bypass different obstacles on DNA suggests they do not block 1D searches by other DNA binding proteins.

scholarly journals A conformational checkpoint between DNA binding and cleavage by CRISPR-Cas9

2017 ◽  
Author(s):  
Yavuz S. Dagdas ◽  
Janice S. Chen ◽  
Samuel H. Sternberg ◽  
Jennifer A. Doudna ◽  
Ahmet Yildiz
Keyword(s):  
Dna Binding ◽  
Single Molecule ◽  
Dna Cleavage ◽  
Genome Engineering ◽  
Divalent Cations ◽  
Guide Rna ◽  
Single Molecule Fret ◽  
Target Sites ◽  
Multiple Conformations ◽  
Dna Binding And Cleavage

AbstractThe Cas9 endonuclease is widely utilized for genome engineering applications by programming its single-guide RNA and ongoing work is aimed at improving the accuracy and efficiency of DNA targeting. DNA cleavage of Cas9 is controlled by the conformational state of the HNH nuclease domain, but the mechanism that governs HNH activation at on-target DNA while reducing cleavage activity at off-target sites remains poorly understood. Using single-molecule FRET, we identified an intermediate state of S. pyogenes Cas9, representing a conformational checkpoint between DNA binding and cleavage. Upon DNA binding, the HNH domain transitions between multiple conformations before docking into its active state. HNH docking requires divalent cations, but not strand scission, and this docked conformation persists following DNA cleavage. Sequence mismatches between the DNA target and guide RNA prevent transitions from the checkpoint intermediate to the active conformation, providing selective avoidance of DNA cleavage at stably bound off-target sites.

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