Sequence analysis of the DNA encoding the Eco RI endonuclease and methylase.
The Eco RI endonuclease and methylase recognize the same hexanucleotide substrate sequence. We have determined the sequence of a fragment of DNA which encodes these enzymes using the chain-termination method of Sanger (Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467). The amino acid sequences of both enzymes were derived from the DNA sequence. The coding regions selected include the only open translational frames of sufficient length to accommodate the enzymes. They coincide with previously established gene boundaries and orientation. The predicted amino acid sequences correlate well with analyses of the purified protein. Comparison of the nucleotide and protein sequences reveals no homology between the endonuclease and methylase which might provide insight into the origin of the restriction-modification system or the mechanism of common substrate recognition. Based on secondary structure predictions, the two enzymes also have grossly different molecular architecture. The base composition of the sequence is 65% A + T, and the codon usage is significantly different from that observed in several Escherichia coli chromosomal genes. In some cases, frequently selected codons are recognized by minor tRNA species. A spontaneous mutation in the endonuclease gene was isolated. Serine replaces arginine at residue 187. In crude extracts, Eco RI specific cleavage is approximately 0.3% wild type.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001483:Base Sequence; D003062:Codon; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics*; D004262:DNA Restriction Enzymes / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics*; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000201:enzymology*; D005815:Genetic Code; D008780:Methyltransferases / Q000235:genetics*; D010957:Plasmids; D014176:Protein Biosynthesis; D011487:Protein Conformation; D014158:Transcription, Genetic
D000596:Amino Acids; D003062:Codon; D004269:DNA, Bacterial; D004274:DNA, Recombinant; D008780:Methyltransferases; D004248:DNA (Cytosine-5-)-Methyltransferases; D004262:DNA Restriction Enzymes