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

In Pseudomonas aeruginosa the RNA chaperone Hfq and the catabolite repression control protein (Crc) govern translation of numerous transcripts during carbon catabolite repression. Crc can be deemed as translational co-repressor as it was shown to enhance Hfq-mediated translational repression of several mRNAs. A single-molecule fluorescence assay was developed to quantitatively assess the impact of Crc on the formation of a Hfq/Crc 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 increased the longevity of the repressive complexes indicating that it changes the equilibrium towards more stable complexes.. This observation is underscored by Cryo-EM studies after 2D classification and 3D reconstruction of such complexes, which revealed an increased compactness of the repressive Hfq/Crc/RNA assemblies when compared with Hfq/RNA complexes alone. Using in vitro translation assays we further show that the presence of Crc results in stronger translational repression, i.e. increased shielding from competing ribosomes. As a consequence, the half-life of the target amiE was found to be significantly reduced in vivo.