Probing the structure of RNAIII, the Staphylococcus aureus agr regulatory RNA, and identification of the RNA domain involved in repression of proteinA expression

RNBIII, a 514-nt RNA molecule, regulates the expression of many Staphylococ cus aureus genes encoding exoproteins and cell-wail-associated proteins. We have studied the structure of RNAIII in solution, using a combination of c hemical and enzymatic probes. A model of the secondary structure was derive d from experimental data with the help of computer simulation of RNA foldin g. The model contains 14 hairpin structures connected by unpaired nucleotid es. The date also point to three helices formed by distant nucleotides that close off structural domains. This model was generally compatible with the results of in vivo probing experiments with dimethylsulfate in late expone ntial-phase cultures. Toe-printing experiments revealed that the ribosome b inding site of hid, which is encoded by RNAIII, was accessible to the Esche richia coli 30S ribosomal subunit, suggesting that the in vitro structure r epresented a translatable form of RNAIII. We also found that, within the 3' end of RNAIII, the conserved hairpin 13 and the terminator form an intrins ic structural domain that exerts specific regulatory activity on protein A gene expression.