Nucleotide sequence of the promoter and amino terminal signal peptide region of the alpha amylase ec gene from bacillus amyloliquefaciens

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

Gene 15(1): 43-52


ISSN/ISBN: 0378-1119
Accession: 006005898

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The gene coding for .alpha.-amylase (EC from B. amyloliquefaciens was studied by molecular cloning in plasmid pUB110 using B. subtilis as a host. The nucleotide sequence of the NH2-terminal region of the cloned gene was determined and contained 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.