The 5' untranslated region regulates dnaK expression in Streptococcus mutans

Objective:  The dnaK gene encodes a universally conserved molecular chaperone of the Hsp70 family (heat-shock protein) that is upregulated in response to heat and acid stress. Previous work by our group indicates that a unique 373-bp region between grpE and dnaK (IGR66) does not contain a promoter, yet is required for optimal production of DnaK (Lemos, J., et al. 2007). The objective of this research was to dissect the mechanisms by which IGR66 regulates dnaK expression. Method:  Nine reporter-gene fusions containing different mutant derivatives of IGR66, including strains lacking large predicted 5’ and 3’ stem:loops (SL) structures, were engineered. All constructs contained the RBS of dnaK fused to a chloramphenicol acetyltransferase (CAT) gene, with transcription driven by the lactate dehydrogenase promoter (pLDH) of S. mutans UA159. The pLDH promoter fused directly to the putative RBS of DnaK, without IGR66, was used as a control. qRT-PCR was used to determine the level of cat gene mRNA and CAT assays were performed to determine the effect of IGR66 or its derivatives on CAT activity.  Result:  The amount of CAT activity varied profoundly among constructs, ranging from 7±1 units for the control strain to 2,816±256 units for the construct containing only the 3’ SL of IGR66. Strains that contained both the 3’ and 5’ SLs displayed activity ranging from 669±68 to 2103±26 units, depending on the mutation. Surprisingly, qRT-PCR revealed no significant differences in the amount of cat mRNA produced by all strains. Conclusion:  IGR66 enhances the production of gene products by a post-transcriptional mechanism involving the SL structures. Since DnaK is essential to the virulence of S. mutans, and IGR66 and its effects on DnaK production appear unique to S. mutans, further investigation into how IGR66 controls translation are warranted.