Stabilization of Hfq-mediated translational repression by the co-repressor Crc in Pseudomonas aeruginosa.

Ewelina M Malecka,Tanino G Albanese,Flavia Bassani,Armin Resch,Ben Luisi,Sarah Woodson,Marlena Rozner,Udo Bläsi,Elisabeth Sonnleitner,Tom Dendooven

doi:10.1093/nar/gkab510

Abstract

In Pseudomonas aeruginosa the RNA chaperone Hfq and the catabolite repression control protein (Crc) govern translation of numerous transcripts during carbon catabolite repression. Here, Crc was shown to enhance Hfq-mediated translational repression of several mRNAs. We have developed a single-molecule fluorescence assay to quantitatively assess the cooperation of Hfq and Crc to form a repressive complex on a RNA, encompassing the translation initiation region and the proximal coding sequence of the P. aeruginosa amiE gene. The presence of Crc did not change the amiE RNA-Hfq interaction lifetimes, whereas it changed the equilibrium towards more stable repressive complexes. This observation is in accord with Cryo-EM analyses, which showed an increased compactness of the repressive Hfq/Crc/RNA assemblies. These biophysical studies revealed how Crc protein kinetically stabilizes Hfq/RNA complexes, and how the two proteins together fold a large segment of the mRNA into a more compact translationally repressive structure. In fact, the presence of Crc resulted in stronger translational repression in vitro and in a significantly reduced half-life of the target amiE mRNA in vivo. Although Hfq is well-known to act with small regulatory RNAs, this study shows how Hfq can collaborate with another protein to down-regulate translation of mRNAs that become targets for the degradative machinery.

Highlights

The opportunistic human pathogen P. aeruginosa (Pae) features two extensive post-transcriptional networks that depend on Hfq [1,2] and the CsrA-like Rsm proteins [3,4]
catabolite repression control protein (Crc) only slightly decreased the fraction of non-specific amiE 3 ARNmut RNA complexes in favor of stable Hfq complexes. These results indicated that Crc can only stabilize Hfq/RNA complexes when Hfq suitably interacts with the AA(R)N motifs located up- and downstream of the amiE start codon, indicating that Crc repression depends on sequence-specific recognition of the mRNA by Hfq
Together with the results shown above, we conclude that Crc compacts and stabilizes Hfq/Crc/RNA assemblies, strengthening translational repression, which leads to rapid degradation of target mRNAs during carbon catabolite repression (CCR) when their encoded functions are dispensable

Summary

Introduction

The opportunistic human pathogen P. aeruginosa (Pae) features two extensive post-transcriptional networks that depend on Hfq [1,2] and the CsrA-like Rsm proteins [3,4]. As in Enterobacteriaceae [11], Hfq was shown to exert these functions in Pae by assisting riboregulation by small RNAs (sRNAs; 10,12–14) as well as by directly acting as a translational repressor on target mRNAs [2,7]. The CsrA/RsmA family of dimeric translational repressors control virulence gene expression in pathogenic Bacteria [3,15] including Pae [16]. Members of this family recognize a GGA motif usually present in the Shine and Dalgarno sequence of target mRNAs that is exposed in the loop of a hairpin [17,18].

Methods

Results

Conclusion