The RNA-binding protein CspA favors the progression of the ribosomes during translation under cold-shock conditions

CspA is a small protein expressed at high levels during cold-shock in Escherichia coli. This protein contains a conserved OB fold domain, found in the three domains of life, which mediates RNA-binding. Furthermore, E. coli CspA is endowed with the capacity to stimulate the translation process at low temperature. To understand how CspA accomplishes this activity, we characterized the interaction between CspA and its own mRNA, a transcript that adopts two alternative structures, one at 37°C and another at temperatures ≤ 20°C. Notably, this protein promotes the in vitro translation only of the 37°C-form, which contains more extended helices and has a structure overall less accessible to the ribosome at low temperature. Furthermore, this stimulation is present only when the reaction is carried out at low temperature. By crosslinking and probing experiments we identified several CspA binding sites along the two cspA mRNA forms, the majority of which are partially localized in apical or internal loops and comprise one or two YYR motifs. We also verified by isothermal calorimetry that this cold-shock protein does not specifically interact with ribosomes. Therefore, CspA binds both forms of cspA mRNA, but favors the translation only of the one that is more structured. However, CspA is neither able to unwind these secondary structures by itself nor capable of stimulating the formation of the translation initiation complex. Indeed, our experiments demonstrate that CspA helps the progression of the ribosome during translation, likely by promoting melting of the secondary structures stabilized by the low temperature as the ribosome moves along the mRNA. Invariably, in all probed mRNAs we have found a strong CspA cross-link positioned 9-25 nucleotides downstream the initiation codon