Dataset Information

Tracking Low-Copy Transcription Factors in Living Bacteria: The Case of the lac Repressor.

ABSTRACT: Transcription factors control the expression of genes by binding to specific sites in DNA and repressing or activating transcription in response to stimuli. The lac repressor (LacI) is a well characterized transcription factor that regulates the ability of bacterial cells to uptake and metabolize lactose. Here, we study the intracellular mobility and spatial distribution of LacI in live bacteria using photoactivated localization microscopy combined with single-particle tracking. Since we track single LacI molecules in live cells by stochastically photoactivating and observing fluorescent proteins individually, there are no limitations on the copy number of the protein under study; as a result, we were able to study the behavior of LacI in bacterial strains containing the natural copy numbers (?40 monomers), as well as in strains with much higher copy numbers due to LacI overexpression. Our results allowed us to determine the relative abundance of specific, near-specific, and non-specific DNA binding modes of LacI in vivo, showing that all these modes are operational inside living cells. Further, we examined the spatial distribution of LacI in live cells, confirming its specific binding to lac operator regions on the chromosome; we also showed that mobile LacI molecules explore the bacterial nucleoid in a way similar to exploration by other DNA-binding proteins. Our work also provides an example of applying tracking photoactivated localization microscopy to studies of low-copy-number proteins in living bacteria.

SUBMITTER: Garza de Leon F

PROVIDER: S-EPMC5390046 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Tracking Low-Copy Transcription Factors in Living Bacteria: The Case of the lac Repressor.

Garza de Leon Federico F Sellars Laura L Stracy Mathew M Busby Stephen J W SJW Kapanidis Achillefs N AN

Biophysical journal 20170401 7

Transcription factors control the expression of genes by binding to specific sites in DNA and repressing or activating transcription in response to stimuli. The lac repressor (LacI) is a well characterized transcription factor that regulates the ability of bacterial cells to uptake and metabolize lactose. Here, we study the intracellular mobility and spatial distribution of LacI in live bacteria using photoactivated localization microscopy combined with single-particle tracking. Since we track s ...[more]

PMID: 28402875

Similar Datasets

Project description:In a process known as facilitated diffusion, DNA-binding proteins find their target sites by combining three-dimensional diffusion and one-dimensional scanning of the DNA. Following the trade-off between speed and stability, agile exploration of DNA requires loose binding, whereas, at the DNA target site, the searching protein needs to establish tight interactions with the DNA. To enable both efficient search and stable binding, DNA-binding proteins and DNA often switch conformations upon recognition. Here, we study the one-dimensional diffusion and DNA binding of the dimeric lac repressor (LacI), which was reported to adopt two different conformations when binding different conformations of DNA. Using coarse-grained molecular dynamic simulations, we studied the diffusion and the sequence-specific binding of these conformations of LacI, as well as their truncated or monomeric variants, with two DNA conformations: straight and bent. The simulations were compared to experimental observables. This study supports that linear diffusion along DNA combines tight rotation-coupled groove tracking and rotation-decoupled hopping, where the protein briefly dissociates and reassociates just a few base pairs away. Tight groove tracking is crucial for target-site recognition, while hopping speeds up the overall search process. We investigated the diffusion of different LacI conformations on DNA and show how the flexibility of LacI's hinge regions ensures agility on DNA as well as faithful groove tracking. If the hinge regions instead form α-helices at the protein-DNA interface, tight groove tracking is not possible. On the contrary, the helical hinge region is essential for tight binding to bent, specific DNA, for the formation of the specific complex. Based on our study of different encounter complexes, we argue that the conformational change in LacI and DNA bending are somewhat coupled. Our findings underline the importance of two distinct protein conformations for facilitated diffusion and specific binding, respectively.

Dataset Information

Tracking Low-Copy Transcription Factors in Living Bacteria: The Case of the lac Repressor.

Publications

Tracking Low-Copy Transcription Factors in Living Bacteria: The Case of the lac Repressor.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets