Escherichia coli CspA stimulates translation in the cold of its own mRNA by promoting ribosome progression

Abstract

Escherichia coli CspA is an RNA binding protein that accumulates during cold-shock and stimulates translation of several mRNAs—including its own. Translation in the cold of cspA mRNA involves a cis-acting thermosensor element, which enhances ribosome binding, and the trans-acting action of CspA. Using reconstituted translation systems and probing experiments we show that, at low temperature, CspA specifically promotes the translation of the cspA mRNA folded in the conformation less accessible to the ribosome, which is formed at 37°C but is retained upon cold shock. CspA interacts with its mRNA without inducing large structural rearrangements, but allowing the progression of the ribosomes during the transition from translation initiation to translation elongation. A similar structure-dependent mechanism may be responsible for the CspA-dependent translation stimulation observed with other probed mRNAs, for which the transition to the elongation phase is progressively facilitated during cold acclimation with the accumulation of CspA.

FAST, a method based on split-GFP for the detection in solution of proteins synthesized in cell-free expression systems

“…Gfp1-10 sequence is followed by the Ribosome Binding Site (RBS) of the cold-shock gene cspA, which is a soluble small β-barrel protein highly abundant in E. coli during cold stress as well as at 37°C during the initial phases of cell growth (Brandi et al, 1999). The expression of CspA is mainly controlled at the translational level (Giuliodori et al, 2010(Giuliodori et al, , 2023, and the 30-base pair of the cspA's RBS cloned in the pMal-c5x plasmid contains both the Shine-Dalgarno (SD) sequence and the AT-rich region preceding it, which is a putative binding site for the ribosomal protein S1 (Giuliodori et al, 2010). This RBS is followed by the cspA coding sequence fused at the C-terminus to a linker encoding the sequence (Gly-Gly-Gly-Ser)2, and then by the gfp11 fragment.…”

“…Cell-free protein synthesis systems (CFPS) exploit the cell's transcriptional/translational apparatus to obtain the desired protein product by providing the corresponding DNA or mRNA template. The aim of this approach is to monitor the quality/quantity of protein synthesis for various purposes, ranging from understanding the genetic code (Nirenberg and Matthaei, 1961), to synthetic biology studies that seek to expand the code (for a review see Gao et al, 2019, Khambhati et al, 2019, from studying the cis-and trans-elements that regulate gene expression at the translational level (Giuliodori et al, 2010, Giuliodori et al, 2023, to the production of otherwise insoluble or toxic proteins or to perform high-throughput screening of therapeutic proteins (for a review see Gregorio et al, 2019, Khambhati et al, 2019 and molecules with antibiotic properties (Brandi et al, 2007).…”