Purification and structure of the intact Ada regulatory protein of Escherichia coli K12, O6-methylguanine-DNA methyltransferase.
The ada gene of Escherichia coli K12, the regulatory gene for the adaptive response of bacteria to alkylating agents, was cloned and placed under the control of the lac regulatory region on a multicopy runaway plasmid, thereby yielding a hybrid plasmid pYN3059. Ada protein with a molecular weight of about 38,000 was overproduced when cells harboring pYN3059 were incubated at 42 degrees C in the presence of a lac inducer, isopropyl-beta-D-thiogalactoside. Taking advantage of overproduction of Ada protein, we purified the protein to apparent physical homogeneity. The purified 38,000-dalton Ada protein transferred the methyl group from the O6-methylguanine residue of alkylated DNA to the Ada protein, per se. Although the Ada protein was degraded to smaller polypeptides when crude extracts or partially purified preparations were incubated in a high ionic-strength buffer at neutral pH, the purified Ada protein remained stable under the same conditions, indicating that the Ada protein may not undergo autodegradation. An amino-terminal sequence and total amino acid composition of the purified Ada protein were in accord with nucleotide sequence of the ada gene, determined by the dideoxy method using M13 phage. It was deduced that Ada protein comprises 354 amino acids and its molecular weight is 39,385. The promoter for the ada gene was determined by S1 nuclease mapping.
Nakabeppu
Y
Y
Kondo
H
H
Kawabata
S
S
Iwanaga
S
S
Sekiguchi
M
M
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D004262:DNA Restriction Enzymes; D004720:Endonucleases; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D005796:Genes; D005798:Genes, Bacterial; D008780:Methyltransferases / Q000235:genetics / Q000302:isolation & purification*; D008970:Molecular Weight; D019853:O(6)-Methylguanine-DNA Methyltransferase; D010446:Peptide Fragments / Q000032:analysis; D010957:Plasmids; D015719:Single-Strand Specific DNA and RNA Endonucleases; D014157:Transcription Factors
D000596:Amino Acids; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D010446:Peptide Fragments; D014157:Transcription Factors; D008780:Methyltransferases; C045675:Ada protein, E coli; D019853:O(6)-Methylguanine-DNA Methyltransferase; D004720:Endonucleases; D004262:DNA Restriction Enzymes; D015719:Single-Strand Specific DNA and RNA Endonucleases