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

Abstract

RNAIII, a 514-nt RNA molecule, regulates the expression of many Staphylococcus aureus genes encoding exoproteins and cell-wall-associated proteins. We have studied the structure of RNAIII in solution, using a combination of chemical and enzymatic probes. A model of the secondary structure was derived from experimental data with the help of computer simulation of RNA folding. The model contains 14 hairpin structures connected by unpaired nucleotides. The data 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 exponential-phase cultures. Toe-printing experiments revealed that the ribosome binding site of hld, which is encoded by RNAIII, was accessible to the Escherichia coli 30S ribosomal subunit, suggesting that the in vitro structure represented a translatable form of RNAIII. We also found that, within the 39 end of RNAIII, the conserved hairpin 13 and the terminator form an intrinsic structural domain that exerts specific regulatory activity on protein A gene expression.

RNAIII of the Staphylococcus aureus agr system activates global regulator MgrA by stabilizing mRNA

“…Importantly, our RNAIII structure prediction is in close agreement with the published RNAIII structure by Benito et al (9), with only minor differences, which validates our SHAPE methodology. Based on the predicted structure, the 14-nt sequence and the adjacent 33-nt sequence of the mgrA UTR are not totally unpaired in the absence of RNAIII.…”

“…We also modeled the RNAIII structure based on the reactivity data (Fig. S6B), which matches well with the structure of RNAIII predicted using chemical and enzymatic probing (9). The high similarity between our predicted RNAIII structure and the previously established structure indirectly validates our RNA SHAPE results.…”

“…A 514-nt RNA, RNAIII, is the effector through which most genes in the agr regulon are regulated (7,8). RNAIII is one of the largest known sRNAs that regulates gene expression via antisense mechanism by base pairing with target mRNAs (5,9). RNAIII is capable of forming a stable structure that is characterized by 14 stem-loop motifs and three long-distance interacting helices (9).…”

“…RNAIII is one of the largest known sRNAs that regulates gene expression via antisense mechanism by base pairing with target mRNAs (5,9). RNAIII is capable of forming a stable structure that is characterized by 14 stem-loop motifs and three long-distance interacting helices (9). Several examples of regulation by direct interaction between RNAIII and its target mRNAs have been reported (10)(11)(12)(13)(14).…”

Quorum sensing in bacterial virulence

“…Transcription from P3 leads to the production of RNAIII, which is the effector molecule of the agr system (Roux et al, 2009). RNAIII is a 514 nt regulatory RNA, which also functions as the mRNA for the d-toxin (Balaban & Novick, 1995;Benito et al, 2000;Kong et al, 2006;Novick et al, 1995). The 59 end is thought to upregulate a-haemolysin, while the 39 end is required for the repression of protein A synthesis (Kong et al, 2006;Morfeldt et al, 1995).…”