A cryptic ribosome binding site, false signals in reporter systems and avoidance of protein translation chaos.
The expression of reporter gene may be induced by activation of cryptic signalling sequences, as we found while constructing the mutS-lacZ fusion gene. We cloned the Escherichia coli lacZ gene encoding beta-galactosidase into a plasmid vector carrying the Thermus thermophilus mutS gene. The clones expected to produce beta-galactosidase as the C-terminal fusion were selected for the complementation of beta-galactosidase activity in a lacZ deficient E. coli strain. Surprisingly, one of the clones, though displaying beta-galactosidase activity, did not produce the fusion protein. As shown by DNA sequencing a 92 bp fragment in the 3' part of mutS gene was substituted by a 19 bp sequence. As the consequence of the resultant frameshift, a truncated MutS peptide was translated instead of beta-galactosidase fusion. The cloned lacZ gene lacked its ribosome binding site, so lacZ expression could be explained by activation of a cryptic ribosome binding site in the 3' end of mutS gene. This observation shows that fusion domains in reporter systems are possible to produce accidentally misleading signals. This observation also suggests that some triplets like AGG and AGA, present in the canonical ribosome binding sequence, are rarely used codons to prevent chaotic protein translation.
Sachadyn
Paweł
P
Gdańsk University of Technology, Department of Microbiology, ul. Narutowicza 11/12, 80-233 Gdańsk, Poland. psach@altis.chem.pg.gda.pl
Stanisławska-Sachadyn
Anna
A
Kabat
Ewa Maria
EM
Zielińska
Aleksandra
A
Kur
Józef
J
D001665:Binding Sites; D017930:Genes, Reporter; D051719:MutS DNA Mismatch-Binding Protein / Q000235:genetics; D009154:Mutation / Q000235:genetics; D014176:Protein Biosynthesis; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D016963:Thermus thermophilus / Q000235:genetics; D001616:beta-Galactosidase / Q000235:genetics
D011993:Recombinant Fusion Proteins; D001616:beta-Galactosidase; D051719:MutS DNA Mismatch-Binding Protein