Initiation of translation at an AUA codon for an archaebacterial protein gene expressed in E.coli.

Overexpression of the Sulfolobus solfataricus L12 ribosomal protein gene in E.coli cells yielded two products of different size. If the E.coli cells carrying the overexpression plasmid were induced in the early stage of bacterial growth, the smaller of the two products was almost exclusively produced. However, induction in a late stage of bacterial growth yielded the larger product in significant excess. The larger protein was identified as the translation product of the entire SsoL12 gene, while the smaller product was a N-terminally shortened version of the L12 protein (sh-SsoL12), starting with a N-terminal methionine at position 22 of the coded protein and continuing with the predicted protein sequence. Position 22 is an isoleucine in the complete SsoL12 protein sequence, coded by an AUA codon. A subclone (SsoL12**) of the SsoL12 gene containing overexpression plasmid, lacking the regular AUG start codon and the putative Shine Dalgarno sequence, was constructed to determine if E.coli ribosomes could initiate at this AUA codon. During overexpression the SsoL12** construct yielded exclusively the sh-SsoL12 product in significant amounts. An AUA start codon has never been found before in a natural message. However, experiments utilizing site directed mutagenesis to generate AUA start codons showed that this codon can be functional for initiation in prokaryotes and eukaryotes. The findings presented in this paper show that AUA acts as an initiation codon in a natural message expressed in a heterologous organism.