Identification of the site of translational frameshifting required for production of the transposase encoded by insertion sequence IS 1.
Previous genetic analyses indicated that translational frameshifting in the--1 direction occurs within the run of six adenines in the sequence 5'-TTAAAAAACTC-3' at nucleotide positions 305-315 in IS 1, where the two out-of-phase reading frames insA and B'-insB overlap, to produce transposase with a polypeptide segment Leu-Lys-Lys-Leu at residues 84-87. IS 1 mutants with a 1 bp insertion, which encode mutant transposases with an amino acid substitution within the polypeptide segment at residues 84-87, did not efficiently mediate cointegration, except for an IS 1 mutant which encodes a mutant transposase with a Leu-Arg-Lys-Leu segment instead of Leu-Lys-Lys-Leu. An IS 1 mutant with the DNA segment 5'-CTTAAAAACTC-3' at positions 305-315 carrying the termination codon TAA in the B'-insB reading frame could still mediate cointegration, indicating that codon AAA for Lys corresponding to second, third and fourth positions in the run of adenines is the site of frameshifting. The beta-galactosidase activity specified by several IS 1-lacZ fusion plasmids, in which B'-insB is in-frame with lacZ, showed that the region 292-377 is sufficient for frameshifting. The protein produced by frameshifting from the IS 1-lacZ plasmid in fact contained the polypeptide segment Leu-Lys-Lys-Leu encoded by the DNA segment 5'-TTAAAAAACTC-3', indicating that--1 frameshifting does occur within the run of adenines.
Sekine
Y
Y
Institute of Applied Microbiology, University of Tokyo, Japan.
Nagasawa
H
H
Ohtsubo
E
E
D000225:Adenine / Q000032:analysis; D000595:Amino Acid Sequence; D001483:Base Sequence; D003062:Codon; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D016368:Frameshift Mutation; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000235:genetics* / Q000378:metabolism; D016366:Open Reading Frames; D010957:Plasmids; D014176:Protein Biosynthesis; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015183:Restriction Mapping; D019895:Transposases; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D003062:Codon; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; D009713:Nucleotidyltransferases; D019895:Transposases; D001616:beta-Galactosidase; D000225:Adenine