Cold-Responsive Regions of Paradigm Cold-Shock and Non-Cold-Shock mRNAs Responsible for Cold Shock Translational Bias

Abstract

In Escherichia coli, the mRNA transcribed from the main cold-shock gene cspA is a thermosensor, which at low temperature adopts a conformation particularly suitable for translation in the cold. Unlike cspA, its paralogue cspD is expressed only at 37 °C, is toxic so cannot be hyper-expressed in E. coli and is poorly translated in vitro, especially at low temperature. In this work, chimeric mRNAs consisting of different segments of cspA and cspD were constructed to determine if parts of cspA could confer cold-responsive properties to cspD to improve its expression. The activities of these chimeric mRNAs in translation and in partial steps of translation initiation such as formation of 30S initiation complexes and 50S subunits docking to 30S complexes to yield 70S initiation complexes were analyzed. We show that the 5′ untranslated region (5′UTR) of cspA mRNA is sufficient to improve the translation of cspD mRNA at 37 °C whereas both the 5′UTR and the region immediately downstream the cspA mRNA initiation triplet are essential for translation at low temperature. Furthermore, the translational apparatus of cold-stressed cells contains trans-active elements targeting both 5′UTR and downstream regions of cspA mRNA, thereby improving translation of specific chimeric constructs at both 15 and 37 °C.