The isolated N-terminal DNA binding domain of thec repressor of bacteriophage16-3 is functional in DNA binding in vivo and in vitro
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The 197 amino acid c repressor of the temperateRhizobium meliloti phage16-3 still regulates theO R operator of the phage after removal of its carboxyl terminal region. When cloned in the low-copy-number plasmid pGA46, a severely truncated variant (R1-77), which retains only the first 77 amino acids of the intact protein, repressed in vivo transcription from the phage promoterP R. When theR1-77 repressor was fused toE. coli β-galactosidase, the hybrid protein boundO R operator DNA in vitro. The behavior of fusion proteins derived from a point mutant is consistent with the assignment of DNA binding specificity to the amino-terminal region. Furthermore two repressor alleles bearingts mutations that mapped in theR1-77 region (near a helix-turn-helix motif) were also temperature sensitive for regulation of theO R site, while an 18 by “in frame” deletion mutant, which mapped in the carboxyl terminal segment, regulated theO R operator in wild-type fashion. The carboxyl terminal region of the repressor is however necessary for the control of lysogenic development of16-3.
Key wordsRepressor Operator binding domain Repressor-βal fusion Temperate phage Rhizobium
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