The amino acid specificity of the N-terminus of pro-o k required for processing by the metalloprotease SpoIVFB in sporulating Bacillus subtilis
Cunliffe, Chelsea L.
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Bacillus subtilis is a commonly studied model for temporal and spatial regulation of gene expression due to its extensive sporulation pathway. The RNA polymerase subunit responsible for the spore coat formation is o k, which is produced in the mother cell as the inactive precursor protein pro-o k. The inactive precursor pro-d' is processed by the zinc metalloprotease SpoIVFB via a mechanism of regulated intramembrane proteolysis (RIP). Using site-directed mutagenesis, in-frame point mutations were made around the cleavage site of pro-o k and along the N terminus of SpoIVFB-processed o k. We found that single point mutations of each amino acid from serine-20 to leucine-31 using an alanine scan had an inhibitory effect on the SpoIVFB dependent processing of pro-o k. The deletion of amino acids 2 through 9 from the pro-sequence was inconclusive. Overall, the single amino acid specificity of the substrate to the enzyme indicates that, unlike many substrates that are processed by other members of the intramembrane cleaving protease family, many of the amino acid residues of pro-o k are individually critical for recognition by the enzyme SpoIVFB.