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

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.

The Small RNA AmiL Regulates Quorum Sensing-Mediated Virulence in Pseudomonas aeruginosa PAO1

“…In the amiEBCRS operon, amiL gene precedes amiE , a gene that encodes aliphatic amidase AmiE ( 33 , 34 ). AmiE is a well-studied enzyme that involves in carbon-nitrogen metabolic processes ( 35 , 36 ). Recent research found AmiE could exercise additional functions in regulating P. aeruginosa virulence ( 37 ).…”

Polymorphic ribonucleoprotein folding as a basis for translational regulation

“…Thus, the ability of Crc to engage Hfq-mRNA intermediates arises through the cooperative effects of the interactions in the higher order assemblies. In line with this model, single-molecule fluorescence assays and molecular dynamics simulations showed that Crc interacts with transient, pre-organized Hfq/RNA complexes and shifts the equilibrium towards assemblies with increased stability so that the effector complex blocks translation more efficiently (Malecka et al ., 2021; Krepl et al ., 2021). Thus, the cooperation of Hfq with Crc effectively stabilizes the repressive complex, excluding the 30S ribosomal subunit more effectively than Hfq alone.…”

“…One system depends on the CsrA-like Rsm proteins, which act as translational repressors and engage GGA motifs in the Shine-Dalgarno sequence of target mRNAs that are often exposed in loops of stem-loop structures (Dubey et al ., 2005; Schubert et al ., 2007, Goodman et al ., 2016; Holmqvist et al ., 2016; Romero et al ., 2018; Gebhardt et al ., 2020). The other system depends on the RNA chaperone Hfq, a member of the widely occurring Lsm/Sm protein family, which facilitates the actions of small regulatory RNAs (sRNAs; Pusic et al ., 2021), and can act as a translational repressor of target mRNAs (Sonnleitner & Bläsi, 2014; Sonnleitner et al ., 2018; Kambara et al ., 2018; Gebhardt et al ., 2020; Malecka et al ., 2021). Through these activities, Hfq contributes to the coordination of stress responses (Lu et al, 2016), metabolism (Sonnleitner & Bläsi, 2014), quorum sensing (Sonnleitner et al, 2006; Yang et al, 2015), virulence (Sonnleitner et al ., 2003), and affects complex processes such as biofilm formation and the antibiotic susceptibility (Fernàndez et al ., 2016; Pusic et al ., 2016, Zhang et al .…”

Specific and Global RNA Regulators in Pseudomonas aeruginosa

“…Under conditions of carbon catabolite repression (CCR), Hfq and Crc form a repressive complex on target mRNAs, the encoded proteins of which are involved in uptake and/or utilization of carbon and nitrogen sources other than the preferred one [29,147,197]. In the repressive complex, Crc acts as a translational co-repressor that promotes Hfq-mediated translational repression of target mRNAs [29] by binding to Hfq/mRNA complexes [147,148], thereby increasing stable Hfq/Crc/RNA complex formation [198]. A recent ChIP-seq analysis and a combined RNA-seq/proteomics study identified 100 [142] and 244 mRNAs [172], respectively, to be co-regulated by Hfq and Crc.…”