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.
Highlights
From the ZCentro de Investigaciones Biologicas, ConsejoSuperior de Znvestigaciones Cientificas, 144 Veluzquez Madrid E-28006, Spain and the §Max-Planck Institut furMolekulare Genetik, 73 Ihnestrasse, 0-1000 Berlin 33, Federal Republic of Germany
The target of RepA within the plasmid DNA molecule has been located around the plasmid single site ApaLI
Recent analysis of crystals of the E. coli trp repressor/operator complex supports the growing evidence that curvaturesin DNA play an essential role in the recognition of regulatory proteins for their targets (Otwinowski et al, 1988)
Summary
Location of the RepA-binding Region-We have found previously that the 443-bp HinfID fragment of pLSl shows a temperature-dependent abnormal electrophoretic mobility. The target of RepA was contained in a region of 247 bp which included the plasmid ori(+) and therepAB promoter Within this region, the pLSlsingle site ApaLI is located (coordinate 607, Fig. L4).Digestion of the plasmids with HincII (which generates four fragments) followed by incubation with RepA andfurther digestion with differenrtestriction enzymes showed that thefragments were only insensitive to digestion with ApaLI (results not shown). The pLSlsingle site ApaLI is located (coordinate 607, Fig. L4).Digestion of the plasmids with HincII (which generates four fragments) followed by incubation with RepA andfurther digestion with differenrtestriction enzymes showed that thefragments were only insensitive to digestion with ApaLI (results not shown) These results showed that the region around coordinate607was occupied by the protein,
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