Function of nucleoside triphosphate and polynucleotide in Escherichia coli recA protein-directed cleavage of phage lambda repressor.
Escherichia coli recA protein catalyzes a specific proteolytic cleavage of repressors in vitro when it is activated by interaction with a single-stranded polynucleotide and nucleoside triphosphate. The ATP analogue adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S) satisfies the NTP requirement. We show here that despite its activity in repressor cleavage, ATP gamma S is hydrolyzed at a negligible rate by the recA protein DNA-dependent nucleoside triphosphatase activity. In the presence of DNA, ATP gamma S binds tightly to recA protein in a complex that can be detected because it is trapped by a nitrocellulose filter. One ATP gamma S molecule is bound per recA monomer. These results suggest that a ternary complex of recA protein, DNA, and nucleoside triphosphate is the species active in repressor cleavage. The activation of recA protein by small, defined oligonucleotides in place of DNA is described and characterized.
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000345:Affinity Labels / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D010582:Bacteriophage lambda / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000235:genetics*; D007700:Kinetics; D009841:Oligonucleotides / Q000378:metabolism; D011119:Polynucleotides / Q000378:metabolism*; D011938:Rec A Recombinases; D012097:Repressor Proteins / Q000378:metabolism*; D012265:Ribonucleotides / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins
D000345:Affinity Labels; D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009841:Oligonucleotides; D011119:Polynucleotides; D012097:Repressor Proteins; D012265:Ribonucleotides; D014157:Transcription Factors; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D011938:Rec A Recombinases