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

Abstract

The rpWL-rpoBC operon of Escherichia coli is regulated in part at the level of translation by an autogenous mechanism (feedback regulation) that involves ribosomal protein LlO-L7/L12.Feedback regulation occurs as the result of LlO-L7/L12 binding to a site on the untranslated leader region of the rpW mRNA that is located more than 100 nucleotides upstream from the translation start site.Previous studies have indicated that the secondary structure of the rpW 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 rpW 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 rpW mRNA leader that are protected by purified LlO-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.The regulation of ribosomal protein (r-protein)' synthesis in Escherichia coli occurs in part at the level of translation by an autogenous mechanism termed feedback regulation.At least one of the gene products of each r-protein operon can act as a translational repressor to prevent the synthesis of most or all of the r-proteins encoded by its polycistronic mRNA (Dennis and Fiil, 1979; Lindahl and Zengel, 1979; Dean et al., 1981a, 1981b; Yates and Nomura, 1980, 1981; Yates et al., 1981).Both genetic and biochemical studies have