Related Experiment Videos
Induced bending of plasmid pLS1 DNA by the plasmid-encoded protein RepA
J Pérez-Martín1, G H del Solar, R Lurz
1Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid E, Spain.
Abstract:
The broad host range streptococcal plasmid pLS1 encodes for a 5.1-kDa repressor protein, RepA. This protein has affinity for DNA (linear or supercoiled) and is translated from the same mRNA as the replication initiator protein RepB. By gel retardation assays, we observed that RepA shows specificity for binding to the plasmid HinfID fragment, which includes the target of the protein. The target of RepA within the plasmid DNA molecule has been located around the plasmid single site ApaLI. This site is included in a region that contains the promoter for the repA and repB genes and is contiguous to the plasmid ori(+). A complex sequence-directed DNA curvature is observed in this region of pLS1. Upon addition of RepA to plasmid linear DNA or to circularly permuted restriction fragments, this intrinsic curvature was greatly enhanced. Thus, a strong RepA-induced bending could be located in the vicinity of the ApaLI site. Visualization of the bent DNA was achieved by electron microscopy of complexes between RepA and plasmid DNA fragments containing the RepA target.
Insights
The streptococcal plasmid pLS1 repressor protein, RepA, binds to a specific DNA region. RepA enhances intrinsic DNA curvature at the ApaLI site, influencing gene regulation.
Area of Science:
- Molecular Biology
- Genetics
- Microbiology
Background:
- Streptococcal plasmid pLS1 encodes the RepA repressor protein.
- RepA is co-translated with the replication initiator protein RepB from the same mRNA.
- RepA exhibits DNA-binding affinity for both linear and supercoiled DNA.
Purpose of the Study:
- To investigate the DNA-binding specificity and mechanism of the RepA repressor protein from plasmid pLS1.
- To identify the precise target site of RepA on the plasmid DNA.
- To elucidate the effect of RepA binding on plasmid DNA structure, particularly DNA curvature.
Main Methods:
- Gel retardation assays were employed to determine RepA's DNA-binding specificity.
- Restriction fragment analysis, including circularly permuted fragments, was used to map the RepA target site.
- Electron microscopy was utilized to visualize RepA-DNA complexes and assess DNA bending.
Main Results:
- RepA specifically binds to a HinfID fragment of plasmid pLS1 containing its target site.
- The RepA target site was localized near the ApaLI restriction site, within a region containing the repA/repB promoter and the ori(+).
- RepA binding significantly enhanced the intrinsic DNA curvature in this region, inducing a strong bend near the ApaLI site.
Conclusions:
- RepA acts as a sequence-specific DNA-binding protein that targets a region crucial for plasmid replication and gene expression.
- RepA binding induces significant DNA bending, suggesting a role in modulating the accessibility of the promoter or origin of replication.
- The findings provide insights into the regulatory mechanisms of plasmid pLS1 replication and gene expression through DNA-protein interactions and structural changes.