IDENTIFICATION OF ELEMENTS ON GENEX-SECA RNA OF ESCHERICHIA-COLI REQUIRED FOR SECA BINDING AND SECA AUTOREGULATION

The protein translocation ATPase of Escherichia coli, SecA protein, au to-regulates its translation by binding to its translation initiation region in geneX-secA mRNA. To analyze this regulation further the seco ndary structure of this portion of geneX-secA RNA was investigated uti lizing structure-specific nucleases and chemical probing approaches. T he results of this analysis were consistent with the existence of two adjacent helices, helix I and the lower portion of helix II, whose fun ction in secA activation and repression, respectively, has been demons trated. Binding of SecA protein to geneX-secA RNA or various mutant de rivatives of this RNA was studied by measurement of affinity constants , RNA footprint analysis, and quantitation of auto-repression in vivo. This analysis showed that the SecA-binding site in geneX-secA RNA was remarkably large spanning a region of 96 nucleotides including a 3' p ortion of helix II, the secA translation initiation region and distal sequences. From the size of the SecA-binding site and the plasticity o f its response to mutational alteration, it is suggested that SecA pro tein contains two distinct RNA-binding sites. Finally, it was shown th at SecA binding was not sufficient to promote auto-regulation and that sequences both upstream (helix I) and within the binding site can con tribute to auto-regulation without affecting SecA-binding affinity. (C ) 1997 Academic Press Limited