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Two forms of replication initiator protein: positive and negative controls
1Department of Bacteriology, University of Wisconsin, Madison, WI 53706, USA.
Abstract:
The pir gene of plasmid R6K encodes the protein, pi, a replication and transcription factor. Two translational options for the pir gene give rise to two forms of pi protein: a 35.0-kDa form (pi35.0) and a shortened 30.5-kDa form (pi30.5). Although both proteins bind to a series of 22-bp direct repeats essential for plasmid R6K replication, only pi35.0 can bind to a site in the (A.T)-rich segment of its gamma ori and activate the gamma ori in vivo and in vitro. However, unlike pi35.0, pi30.5can inhibit in vivo and in vitro replication (activated by pi35.0). We propose that the two forms of pi might have distinct functions in replication. We show that although both forms of pi produce dimers, the nature of these dimers is not identical. The N-terminal 37 amino acid residues appear to control the formation of the more stable pi35.0 dimers, whereas another, apparently weaker interface holds together dimers of pi30.5. We speculate that the leucine zipper-like motif, absent in pi30.5, controls very specific functions of pi protein.
Insights
The pir gene produces two pi proteins that regulate plasmid R6K replication. While both bind DNA, pi35.0 activates replication, and pi30.5 inhibits it, suggesting distinct roles.
Area of Science:
- Molecular Biology
- Microbiology
Background:
- The pir gene of plasmid R6K encodes the pi replication and transcription factor.
- Two translational start sites yield pi35.0 and pi30.5 protein isoforms.
Purpose of the Study:
- To investigate the distinct functional roles of pi35.0 and pi30.5 in plasmid R6K replication.
- To elucidate the structural basis for differential protein activity.
Main Methods:
- Analysis of protein-DNA interactions in vitro and in vivo.
- Characterization of protein dimerization using biochemical assays.
- Site-directed mutagenesis to identify functional domains.
Main Results:
- Both pi35.0 and pi30.5 bind to 22-bp direct repeats crucial for R6K replication.
- Only pi35.0 activates replication by binding to the gamma origin.
- pi30.5 inhibits replication, while pi35.0 activates it.
- Both isoforms form dimers, but with different stability and interfaces.
- The N-terminal 37 amino acids influence pi35.0 dimer stability.
Conclusions:
- The two pi protein isoforms, pi35.0 and pi30.5, possess distinct functions in plasmid R6K replication.
- Differential dimerization and the presence of a leucine zipper-like motif in pi35.0 likely mediate their opposing roles.