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

被引:34
作者
KEILMANN, C
WANNER, G
BOCK, A
机构
[1] UNIV MUNNCHEN,INST GENET & MIKROBIOL,MARIA WARD STR 1A,D-80638 MUNICH,GERMANY
[2] UNIV MUNICH,INST BOT,D-80638 MUNICH,GERMANY
来源
BIOLOGICAL CHEMISTRY HOPPE-SEYLER | 1993年 / 374卷 / 10期
关键词
POLYPROTEINS; PROTEIN EXPORT; TRANSLATIONAL CONTROL; TEMPERATURE-DEPENDENT EXPRESSION;
D O I
10.1515/bchm3.1993.374.7-12.983
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The enzyme penicillin acylase is synthesized by Escherichia coli only at growth temperatures below 30-degrees-C. The biochemical basis of this strict temperature-dependent formation of an enzyme was investigated. 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 translation of the pac mRNA rather than transcription of the gene is temperature-dependent. This conclusion could be further confirmed by Northern hybridisation and by analysis of pac-lacZ transcriptional fusions. TnphoA insertion mutagenesis and experiments in which the promoter and 5' sequence encoding the signal peptide of the pac gene was exchanged with those of the cyclodextrin glycosyltransferase gene from Klebsiella oxytoca localised the region of pac mRNA responsible for the temperature-sensitive translation to the 5'-untranslated region and/or the signal peptide. Extension of the 5 nucleotide long spacer separating the Shine-Dalgarno motif from the AUG initiation codon by one or three nucleotides lead to partial or full synthesis of penicillin acylase precursor at 40-degrees-C, respectively. The precursor of penicillin acylase formed at 40-degrees-C by the mutant variants or when placed under 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 precursor are coupled and that the short Shine-Dalgarno-AUG distance interferes with a competent interaction between the translation initiation complex and the export system at high temperature. Moreover, evidence was also provided which indicates a direct effect of temperature on the conformation of the precursor and it is proposed that the lack of translation at high temperatures has been selected to prevent the accumulation of transport-incompetent protein locked in the membrane.
引用
收藏
页码:983 / 992
页数:10
相关论文
共 35 条
[31]   THE ROLE OF PENICILLIN AMIDASES IN NATURE AND IN INDUSTRY [J].
VALLE, F ;
BALBAS, P ;
MERINO, E ;
BOLIVAR, F .
TRENDS IN BIOCHEMICAL SCIENCES, 1991, 16 (01) :36-40
[32]  
Vandamme E. J., 1980, ECON MICROBIOL, V5, P467
[33]   PREPARATIVE AND ANALYTICAL PURIFICATION OF DNA FROM AGAROSE [J].
VOGELSTEIN, B ;
GILLESPIE, D .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1979, 76 (02) :615-619
[34]   TRANSCRIPTION SIGNALS FOR STABLE RNA GENES IN METHANOCOCCUS [J].
WICH, G ;
HUMMEL, H ;
JARSCH, M ;
BAR, U ;
BOCK, A .
NUCLEIC ACIDS RESEARCH, 1986, 14 (06) :2459-2479
[35]   IMPROVED M13 PHAGE CLONING VECTORS AND HOST STRAINS - NUCLEOTIDE-SEQUENCES OF THE M13MP18 AND PUC19 VECTORS [J].
YANISCHPERRON, C ;
VIEIRA, J ;
MESSING, J .
GENE, 1985, 33 (01) :103-119