Structural analysis of the carboxy terminus of bacteriophage lambda repressor determined by antipeptide antibodies.
To analyze lambda repressor function and structure, antibodies were generated with synthetic peptides corresponding to sequences believed to be involved in prophage induction. These site-directed antibodies seemed to recognize preferentially the primary sequence of repressor because they reacted better in competition experiments with the oligopeptide and with the partially denatured forms of repressor than with the native molecules. This information, together with the characteristic ability of the antibodies to immunoprecipitate or react with repressor in immunoblots, allowed us to infer some conformational properties of the specific regions that the antibodies recognized. The antibodies reacted less with some mutant repressors that had a single amino acid substitution within the cognitive sequences. RecA-catalyzed cleavage of repressor was inhibited to different extents in relation to the proportion of repressor that each antipeptide immunoglobulin G (IgG) was able to immunoprecipitate. The antipeptide IgGs did not affect specific binding of repressor to operator DNA, whereas the antirepressor IgG was inhibitory. The three different IgGs competed for binding to repressor in an enzyme-linked immunosorbent assay additivity test, which suggested that the three regions of conserved amino acids are probably located on the same side of the carboxyl domain of repressor and possibly close together in the tertiary structure.
Sussman
R
R
Marine Biological Laboratory, Woods Hole, Massachusetts 02543.
Alexander
H B
HB
D000595:Amino Acid Sequence; D000906:Antibodies; D000936:Antigen-Antibody Complex; D010582:Bacteriophage lambda / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics*; D007074:Immunoglobulin G; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis; D010447:Peptide Hydrolases; D011938:Rec A Recombinases / Q000502:physiology; D012097:Repressor Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics*
D000906:Antibodies; D000936:Antigen-Antibody Complex; D007074:Immunoglobulin G; D010446:Peptide Fragments; D012097:Repressor Proteins; D014157:Transcription Factors; D011938:Rec A Recombinases; D010447:Peptide Hydrolases