Mutational analysis of the Streptomyces scabies esterase signal peptide.
Ten site-directed mutations affecting the predicted 39-amino-acid signal peptide of the Streptomyces scabies esterase were used to examine start-codon usage and esterase secretion in S. lividans. The first of two in-frame AUG codons was preferred for translation initiation. Removal of 2 of the 4 positively charged amino acids at the amino terminus of the signal peptide reduced esterase expression more than 100-fold; however, deletion of all 4 charged residues reduced expression by only 2- to 5-fold. Deletion of 4 or 8 amino acids from the hydrophobic core of the signal peptide reduced esterase production more than 200-fold, and a signal peptide processing site deletion completely disrupted esterase expression. For all constructs in which a mutation in the signal sequence decreased esterase production, esterase mRNA levels were also reduced, suggesting that a defect in secretion or processing affected esterase transcript abundance.
Hale
V A
VA
Department of Biochemistry, University of Minnesota, St Paul 55108, USA.
Schottel
J L
JL
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D018387:Codon, Initiator / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004950:Esterases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010442:Peptide Chain Initiation, Translational / Q000235:genetics; D021382:Protein Sorting Signals / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism
D018387:Codon, Initiator; D004269:DNA, Bacterial; D021382:Protein Sorting Signals; D012333:RNA, Messenger; D004950:Esterases