MOLECULAR-BASIS OF THE EXCLUSIVE LOW-TEMPERATURE SYNTHESIS OF AN ENZYME IN ESCHERICHIA-COLI - PENICILLIN ACYLASE

The enzyme penicillin acylase is synthesized by Escherichia coli only at growth temperatures below 30-degrees-C. The biochemical basis of th is strict temperature-dependent formation of an enzyme was investigate d. When the gene (pac) was under the control of the lacUV5 promoter it showed the same temperature-dependent expression as the chromosomally encoded gene transcribed from its own promoter. This indicates that t ranslation of the pac mRNA rather than transcription of the gene is te mperature-dependent. This conclusion could be further confirmed by Nor thern hybridisation and by analysis of pac-lacZ transcriptional fusion s. TnphoA insertion mutagenesis and experiments in which the promoter and 5' sequence encoding the signal peptide of the pac gene was exchan ged with those of the cyclodextrin glycosyltransferase gene from Klebs iella oxytoca localised the region of pac mRNA responsible for the tem perature-sensitive translation to the 5'-untranslated region and/or th e signal peptide. Extension of the 5 nucleotide long spacer separating the Shine-Dalgarno motif from the AUG initiation codon by one or thre e nucleotides lead to partial or full synthesis of penicillin acylase precursor at 40-degrees-C, respectively. The precursor of penicillin a cylase formed at 40-degrees-C by the mutant variants or when placed un der the control of a heterologous upstream region was associated with the membrane but could not be translocated. Taken together these data suggest that transport and translation of the penicillin acylase precu rsor are coupled and that the short Shine-Dalgarno-AUG distance interf eres with a competent interaction between the translation initiation c omplex and the export system at high temperature. Moreover, evidence w as also provided which indicates a direct effect of temperature on the conformation of the precursor and it is proposed that the lack of tra nslation at high temperatures has been selected to prevent the accumul ation of transport-incompetent protein locked in the membrane.