CsrA-Mediated Translational Activation of ymdA Expression in Escherichia coli

Abstract

The sequence-specific RNA-binding protein CsrA is the central component of the conserved global regulatory Csr system. In Escherichia coli, CsrA regulates many cellular processes, including biofilm formation, motility, carbon metabolism, iron homeostasis, and stress responses. Such regulation often involves translational repression by CsrA binding to an mRNA target, thereby inhibiting ribosome binding. While CsrA also extensively activates gene expression, no detailed mechanism for CsrA-mediated translational activation has been demonstrated. An integrated transcriptomic study identified ymdA as having the strongest CsrA-mediated activation across the E. coli transcriptome. Here, we determined that CsrA activates ymdA expression posttranscriptionally. Gel mobility shift, footprint, toeprint, and in vitro coupled transcription-translation assays identified two CsrA binding sites in the leader region of the ymdA transcript that are critical for translational activation. Reporter fusion assays confirmed that CsrA activates ymdA expression at the posttranscriptional level in vivo. Furthermore, loss of binding at either of the two CsrA binding sites abolished CsrA-dependent activation. mRNA half-life studies revealed that CsrA also contributes to stabilization of ymdA mRNA. RNA structure prediction revealed an RNA hairpin upstream of the ymdA start codon that sequesters the Shine-Dalgarno (SD) sequence, which would inhibit ribosome binding. This hairpin also contains one of the two critical CsrA binding sites, with the other site located just upstream. Our results demonstrate that bound CsrA destabilizes the SD-sequestering hairpin such that the ribosome can bind and initiate translation. Since YmdA represses biofilm formation, CsrA-mediated activation of ymdA expression may repress biofilm formation under certain conditions. IMPORTANCE The Csr system of E. coli controls gene expression and physiology on a global scale. CsrA protein, the central component of this system, represses translation initiation of numerous genes by binding to target transcripts, thereby competing with ribosome binding. Variations of this mechanism are so common that CsrA is sometimes called a translational repressor. Although CsrA-mediated activation mechanisms have been elucidated in which bound CsrA inhibits RNA degradation, no translation activation mechanism has been defined. Here, we demonstrate that CsrA binding to two sites in the 5′ untranslated leader of ymdA mRNA activates translation by destabilizing a structure that otherwise prevents ribosome binding. The extensive role of CsrA in activating gene expression suggests the common occurrence of similar activation mechanisms.

CsrA Enhances Cyclic-di-GMP Biosynthesis and Yersinia pestis Biofilm Blockage of the Flea Foregut by Alleviating Hfq-Dependent Repression of the hmsT mRNA

“…Therefore, to determine if Y. pestis rcsB and hfq mRNAs are candidate mRNA targets of CsrA, a position matrix scan ( 44 ) was applied to the 5′ UTR of rcsB and hfq mRNAs to identify putative CsrA binding sites. Up to −250 bases upstream from the translational initiation codon were queried, in keeping with ranges for to-date validated CsrA targeted 5′ UTRs ( 36 , 53 , 54 ). Two potential binding sites were found in the hfq transcript ( Fig.…”

“…CsrA-dependent biofilm regulation can occur through multiple mechanisms in bacteria. For example, in E. coli , CsrA represses translation of mRNAs encoding DGCs ( 25 ), PgaABCD, a homolog to HmsHFRS ( 36 ), and the NhaR transcriptional regulator that activates pgaABCD transcription ( 70 ) but promotes translation of the ymdA mRNA involved in inhibiting biofilm production ( 54 ). Similarly, we predict that CsrA targets other mRNAs involved in biofilm formation in Y. pestis .…”

Diverse Mechanisms and Circuitry for Global Regulation by the RNA-Binding Protein CsrA

“…The ymdA gene was identified as the most strongly activated direct mRNA target of CsrA in the E. coli genome in an integrated transcriptomics study (Potts et al, 2017) and may play a role in biofilm formation (Kim and Kim, 2017). Reporter assays and mRNA half-life studies confirmed that CsrA posttranscriptionally activates ymdA expression and stabilizes the ymdA transcript (Renda et al, 2020). In the absence of CsrA, ymdA mRNA forms a hairpin that sequesters the SD sequence, inhibiting ribosome binding.…”

The World of Stable Ribonucleoproteins and Its Mapping With Grad-Seq and Related Approaches

“…This preference makes CsrA family proteins potent translational repressors whose high-affinity binding can only be overcome by a dedicated protein antagonist, such as FliW or CesT, or a special group of “sponge” RNAs, which will be described in the next section ( Mukherjee et al, 2011 , 2016 ; Altegoer et al, 2016 ; Dugar et al, 2016 ; Katsowich et al, 2017 ; Sowa et al, 2017 ; Ye et al, 2018 ; Oshiro et al, 2019 ). In rare cases, CsrA upregulates the expression of its target through mRNA stabilization or translational activation ( Yakhnin et al, 2013 ; Renda et al, 2020 ).…”