Quantitation of insertion sequence IS10 transposase gene expression by a method generally applicable to any rarely expressed gene.
We have found that IS10 transposase is synthesized in tiny amounts, about 0.15 polypeptide chain per cell per generation on average, as judged from the beta-galactosidase activity of a single chromosomal copy of a suitable transposase-lacZ gene fusion. Enzymatic activity from the fusion gene is a factor of 10 lower in a permeabilized whole cell assay than in cell extracts. Probably, most cells contain fewer than four polypeptide chains, and these chains can assemble into active tetramers only after cell disruption. This interpretation permits formulation of two equations relating enzyme activities to transcription and translation rates, solution of which reveals that the fusion gene is expressed at the average rate of only 0.25 transcript per cell per generation, with an average of only 0.58 translation product per transcript. This methodology is generally applicable to analysis of any gene from which fewer than four polypeptide chains are synthesized per cell per generation.
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics*; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005786:Gene Expression Regulation; D005821:Genetic Techniques; D009713:Nucleotidyltransferases / Q000096:biosynthesis / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D013269:Stochastic Processes; D019895:Transposases; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D011994:Recombinant Proteins; D009713:Nucleotidyltransferases; D019895:Transposases; D001616:beta-Galactosidase