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.
Highlights
The life style of enterobacteriaceae like Escherichia coli entails frequent shuffling between the homeostatic environment of the gastrointestinal tract of the animal host and highly erratic external conditions
Upon a temperature downshift from 37 to 20 ◦C or less, a large number of modifications occur in the E. coli cells which undergo a cold adaptation phase during which gene expression is reprogrammed as a result of both transcriptional and post-transcriptional regulatory events, whereby only a small set of cold-shock proteins is synthesized whereas bulk protein synthesis stops [7,8,9,10,11]
An important role is played by a modification of the translational apparatus, which allows selective translation of cold-shock mRNAs and inhibition of non-cold-shock mRNAs translation during cold acclimation [12,13,14,15]
Summary
The life style of enterobacteriaceae like Escherichia coli entails frequent shuffling between the homeostatic environment of the gastrointestinal tract of the animal host and highly erratic external conditions. Upon a temperature downshift from 37 to 20 ◦C or less, a large number of modifications occur in the E. coli cells which undergo a cold adaptation phase during which gene expression is reprogrammed as a result of both transcriptional and post-transcriptional regulatory events, whereby only a small set of cold-shock proteins is synthesized whereas bulk protein synthesis stops [7,8,9,10,11] In this connection, an important role is played by a modification of the translational apparatus, which allows selective translation of cold-shock mRNAs and inhibition of non-cold-shock mRNAs translation during cold acclimation [12,13,14,15]. This phenomenon, designated “cold-shock translational bias” is due in part to trans-acting factors such as initiation factors IF3 and IF1, whose levels increase with respect to ribosomes [16,17] whose synthesis slows down [18] and in part to cis-acting elements present in the cold-shock transcripts [15,19]
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