Inhibition of rot translation by RNAIII, a key feature of agr function

Abstract

SummaryRNAIII is a 514 nt regulatory RNA that is the effector molecule of the staphylococcal agr quorum-sensing system, regulating a large set of virulence and other accessory genes at the level of transcription. RNAIII was discovered nearly 20 years ago and we long ago hypothesized that it would function by regulating the synthesis or activity of one or more intermediary transcription factors. We have finally confirmed this hypothesis, showing that Staphylococcus aureus RNAIII regulates the synthesis of a major pleiotropic transcription factor, Rot, by blocking its translation. RNAIII has a complex secondary structure with several stable hairpins that have highly C-rich end loops, unusual in an AT-rich organism. We noted that these loops are complementary to two G-rich stem loops of the rot mRNA translation initiation region (TIR). Pairing of the complementary RNAs would be predicted to occlude the rot Shine-Dalgarno (SD) site and to block rot translation. Through a combination of transcriptional and translational fusions and Northern and Western blot hybridization analyses, we show that RNAIII does, indeed, block rot translation. Through alterations in the C-rich loops of RNAIII and the G-rich loops of rot, we show that the sequences of these loops are critical for inhibition of rot translation and suggest that this inhibition is affected by pairing between the complementary stem loops, followed by the cleavage of rot mRNA. We propose that the RNAIII-rot mRNA interaction plays a key role in agr regulation of staphylococcal virulence.

Regulation and characterization of rot transcription in Staphylococcus aureus

“…This result was supported by the observation that the 15.6 kDa Rot protein (corresponding to 133 residues) co-eluted with SarA, MgrA and SarR in an affinity pull-down assay from whole-cell lysates with a spa promoter fragment (Oscarsson et al, 2005). In addition, studies by Geisinger et al (2006) with the C-terminal histidine fusion of the rot gene in a multicopy plasmid and immunoblot analysis with antihistidine antibody indicated the production of a functional Rot of 17 kDa in the various isogenic S. aureus strains. The published S. aureus N315 genome reveals at least ten proteins with significant homology to SarA (Kuroda et al, 2001), of which nine have been characterized.…”

“…A diffused pattern of transcripts appeared particularly in the lateexponential and post-exponential phases, probably due to the presence of the multiple transcripts as evidenced in the primer extension experiment. Alternatively, it may have been due to cleavage of the rot transcripts by the base pairing of the agr RNAIII molecule as suggested by Geisinger et al (2006) or the degradation of the rot transcripts at the postexponential phase of growth. In addition, it is consistent with the primer extension results that the sarA gene product acts as a repressor for rot transcription.…”

“…2) is probably the Rot translational start codon and that Rot is a 133-residue polypeptide. Indeed, recent transcriptional fusion analysis of the upstream promoter region of the rot gene suggested that translation of rot initiated from the ATG codon located at the +92 nt position (Geisinger et al, 2006). Analysis of the downstream sequence of the rot gene disclosed the presence of a potential hairpin structure or rho-independent transcriptional terminator sequence from a position 13-48 bp immediately after the stop codon (TAA).…”

“…Although rot has been shown to be a global regulator for virulence gene expression, the regulation of rot transcription, particularly by other members of the sarA family of genes, is not known and physical mapping of rot transcript has not been performed. However, recently it has been shown that the translation of rot transcript is inhibited by the agr RNAIII molecule (Geisinger et al, 2006).…”

The SarA protein family of Staphylococcus aureus

“…Transcriptional profiling studies indicate that Rot affects the transcription of 168 genes (SaidSalim et al, 2003), many of which reflect an agr minus phenotype. A recent study suggested that the agr RNAIII molecule may interact with rot mRNA to inhibit Rot translation (Geisinger et al, 2006).…”