Nucleotide sequence of the promoter and NH2-terminal signal peptide region of the alpha-amylase gene from Bacillus amyloliquefaciens

Palva, I.; Pettersson, R.F.; Kalkkinen, N.; Lehtovaara, P.; Sarvas, M.; Söderlund, H.; Takkinen, K.; Kääriäinen, L.

Gene 15(1): 43-51


ISSN/ISBN: 0378-1119
PMID: 6170539
DOI: 10.1016/0378-1119(81)90103-7
Accession: 043768327

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We have isolated and partially sequenced the gene coding for alpha-amylase (EC from Bacillus amyloliquefaciens by molecular cloning in the plasmid pUB110 using Bacillus subtilis as a host. The nucleotide sequence of the NH2-terminal region of the cloned gene was determined and found to contain a 31-residue-long stretch of amino acids preceding the NH2-terminal sequence of the extracellular alpha-amylase. Within this sequence there is a 15-residue-long stretch of uncharged amino acids similar to that found at the NH2 terminus of other precursors to exported proteins. This "signal sequence" is probably removed in conjunction with the translocation of alpha-amylase through the cytoplasmic membrane. In vitro labeling of alpha-amylase with radioactive amino acids in a coupled transcription-translation system followed by partial sequencing established the exact location of the NH2 terminus of the alpha-amylase gene. The nucleotide sequence preceding the NH2 terminus has properties resembling the RNA-polymerase- and ribosome-binding sites found at the 5' terminus of many prokaryotic genes.