In vivo and in vitro structural analysis of the rplJ mRNA leader of Escherichia coli. Protection by bound L10-L7/L12

Abstract

The rplJL-rpoBC operon of Escherichia coli is regulated in part at the level of translation by an autogenous mechanism (feedback regulation) that involves ribosomal protein L10-L7/L12. Feedback regulation occurs as the result of L10-L7/L12 binding to a site on the untranslated leader region of the rplJ mRNA that is located more than 100 nucleotides upstream from the translation start site. Previous studies have indicated that the secondary structure of the rplJ leader region is important for efficient translation and feedback regulation. We have done chemical modification experiments to examine the secondary structure of approximately 200 nucleotides of the rplJ leader region, and we propose a secondary structure that is consistent with the experimental data. RNA structure was probed in vitro by treating samples of total cellular RNA with diethyl pyrocarbonate and in vivo by treating log-phase cultures with dimethyl sulfate. Modified bases were detected by primer extension using three different oligonucleotide primers. The proposed structure includes five double-stranded regions designated I to V, separated by single-stranded segments numbered 1 to 5. We have also identified specific nucleotides in the rplJ mRNA leader that are protected by purified L10-L7/L12 from methylation by dimethyl sulfate in vitro. The protected bases are located within a bulge-loop of region IV, a portion of the mRNA that has been shown genetically to be necessary for feedback regulation.