Post‐transcriptional regulation by distal S hine‐ D algarno sequences in the grp E ‐ dna K intergenic region of S treptococcus mutans

Summary A unique 373 bp region ( igr66 ) between grp E and dna K of S treptococcus mutans lacks a promoter but is required for optimal production of DnaK . Northern blotting using probes specific to hrc A , igr66 or dna K revealed multiple transcripts produced from the dna K operon and 5′‐ RACE mapped 5′ termini of multiple dna K transcripts within igr66 . One product mapped to a predicted 5′‐ SL (stem‐loop) and two others mapped just 5′ to S hine‐ D algarno ( SD )‐like sequences located immediately upstream to dna K and to a predicted SL 120 bp upstream of the dna K start codon (3′‐ SL ). A collection of cat reporter‐gene strains containing mutant derivatives of igr66 were engineered. Chloramphenicol acetyltransferase ( CAT ) activity varied greatly between strains, but there were no correlative changes in cat m RNA levels. Interestingly, mutations introduced into the SD ‐like sequences 5′ to the 3′‐ SL resulted in an 83–98% decrease in CAT activity. Markerless point mutations introduced upstream of dnaK in the SD ‐like sequences impaired growth at elevated temperatures and resulted in up to a 40% decrease in DnaK protein after heat shock. Collectively, these results indicate processing within igr66 enhances translation in a temperature dependent manner via non‐canonical ribosome binding sites positioned > 120 bp upstream of dna K .