Teamwork Makes The Dream Work: A Cyanobacterial RNA Helicase Is Regulated by Multiple Cis‐Regulatory Elements in Response to Low Temperature

RNA helicases are enzymes involved in, and necessary for, the proper functioning of all aspects of RNA metabolism, working as molecular motors by interacting with, and modifying, RNA and ribonucleoprotein secondary structures. Thus, they are ideally situated to function as regulators of gene expression. In bacteria, the expression of a DEAD‐box RNA helicase is often responsive to particular abiotic stress(es), which allows them to fulfill vital roles in coordinating the cell's genetic response to altered environmental conditions. The freshwater cyanobacterium Synechocystis sp. PCC 6803 encodes a single DEAD‐box helicase, crhR , which accumulates in response to multiple abiotic stresses, including low temperature (20°C). While crhR deletion does not affect cell viability at its optimal growth temperature, 30°C, crhR mutants exhibit an extreme cold sensitive phenotype. Although the accumulation of transcript and protein during cold stress have been characterized, we predicted that the temperature regulatory mechanism would involve a combination of DNA and RNA cis‐acting elements. crhR is encoded within the dicistronic rimO ‐ crhR operon. Previous analysis has indicted the presence of two promoters, an operonic rimO promoter (P rimO ) and an internal promoter (P crhR ). We hypothesized that crhR transcript abundance could therefore be modulated by the promoter‐associated 5’ untranslated regions (UTRs), the 3’ UTR, and an RNA processing site between the two open reading frames. In order to characterize the regulatory effects of these RNA sequences, plasmids were constructed in which these elements were deleted from the wild type operon or inserted downstream of a constitutive promoter. These constructs were analyzed in a complete crhR deletion strain to determine how low temperature stress (20°C) and return to normal growth temperature (30°C) alters protein and transcript accumulation. We observed that expression of crhR is regulated in a complex manner involving temperature‐dependent modulation of both transcription and translation. P rimO is a cold‐inducible promoter while its 5’ UTR imposes a robust inhibitory effect which attenuates transcription at 30°C but is relieved at 20°C, together resulting in 5‐ and 15‐fold increases in transcript and protein abundance, respectively. P crhR , on the other hand, is constitutively active and its 5’ UTR does not alter transcript abundance, regardless of temperature. Instead, the crhR 5’ UTR appears to modulate translation, resulting in a 5‐fold enhancement of protein accumulation during cold stress. Deletion of this 5’ UTR severely diminishes CrhR accumulation, without a corresponding loss of transcript accumulation, suggesting that its role is to ensure efficient translation of crhR during cold stress. Taken together, these findings demonstrate that crhR expression is tightly regulated by a series of DNA‐ and RNA‐derived regulatory elements which cooperate to control this integral component of the Synechocystis abiotic stress response, maintaining CrhR abundance at basal levels at 30°C or initiating rapid accumulation at lower temperatures.

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