Mutant analysis of Prevotella sp. plaA-lacZ fusion protein expression in Escherichia coli: support for an essential role of the stem-loop

This study investigated the involvement of RNA folding in the synthesis of a fusion protein with beta-galactosidase activity. The coding gap region of the Prevotella loescheii adhesin gene plaA was fused in-frame with the Escherichia coli lacZ gene on plasmid pSK105. N-Terminal sequencing of the expressed plaA-lacZ protein indicated that it resulted from translational initiation at a fortuitous ribosomal-binding site within the plaA sequence at nt 570. Specific mutations were introduced in the stem-loop region that precedes the gap sequence. Analysis of stem-loop mutants, together with the introduction of compensatory mutations that restored activity, supports a requirement for stem-loop formation within the plaA sequence preceding the translational initiation site. A mutation reducing the predicted size of the loop, but preserving the stem structure, inactivated fusion protein synthesis. A suppressor mutation predicted to restore the size of the loop restored efficient fusion protein synthesis. In addition, the sequence preceding the translational start site of the plaA-lacZ fusion has several similarities to sequences that function as translational enhancers in prokaryotes. These include a stem-loop structure, an A-U rich region preceding the initiation codon, and a region of homology to 16S rRNA.