Conditional, Temperature-Induced Proteolytic Regulation of Cyanobacterial RNA Helicase Expression

Abstract

Conditional proteolysis is a crucial process regulating the abundance of key regulatory proteins associated with the cell cycle, differentiation pathways, or cellular response to abiotic stress in eukaryotic and prokaryotic organisms. We provide evidence that conditional proteolysis is involved in the rapid and dramatic reduction in abundance of the cyanobacterial RNA helicase, CrhR, in response to a temperature upshift from 20 to 30°C. The proteolytic activity is not a general protein degradation response, since proteolysis is only present and/or functional in cells grown at 30°C and is only transiently active at 30°C. Degradation is also autoregulatory, since the CrhR proteolytic target is required for activation of the degradation machinery. This suggests that an autoregulatory feedback loop exists in which the target of the proteolytic machinery, CrhR, is required for activation of the system. Inhibition of translation revealed that only elongation is required for induction of the temperature-regulated proteolysis, suggesting that translation of an activating factor was already initiated at 20°C. The results indicate that Synechocystis responds to a temperature shift via two independent pathways: a CrhR-independent sensing and signal transduction pathway that regulates induction of crhR expression at low temperature and a CrhR-dependent conditional proteolytic pathway at elevated temperature. The data link the potential for CrhR RNA helicase alteration of RNA secondary structure with the autoregulatory induction of conditional proteolysis in the response of Synechocystis to temperature upshift.

Association of the Cold Shock DEAD-Box RNA Helicase RhlE to the RNA Degradosome in Caulobacter crescentus

“…The mechanisms of regulation of the cyanobacterial genes for RNA helicases have been studied in more detail; the reports showed that crhC from Anabaena sp. is regulated at the levels of transcription, mRNA stability, and translation ( 35 ) and that the CrhR RNA helicase from Synechocystis is regulated by proteolysis ( 39 ). The utilization of several levels of control in the expression of RNA helicases seems to be a recurrent feature in bacteria from distant phylogenetic lineages, indicating the important role of these enzymes at low temperature.…”

Inactivation of the RNA helicase CrhR impacts a specific subset of the transcriptome in the cyanobacterium Synechocystis sp. PCC 6803

“…The partial crhR deletion also removed the 3ʹ end of the downstream gene, argC which is transcribed in the opposite direction [30]; however, the resulting mutant cells are not auxotrophs [30]. The completely segregated crhR TR strain constitutively produces a 27 kDa truncated CrhR polypeptide, CrhR TR , the abundance of which is not repressed at 30°C [26,27] and which is inactive at both gene expression level [26,27] and at physiological and morphological level [30]. Microarray analysis was performed using the CrhR TR partial deletion mutant since this strain was utilized in previous investigations and provided crucial insights into the regulation of crhR expression that would not have been obtained with a complete deletion strain.…”

“…Expression of crhR is regulated by light-driven reduction of the electron transport chain [24], levels which are augmented by conditions that increase reduction of the chain, including salt and cold stress [25,26]. Temperature-downshift induction of crhR expression is a complex, auto-regulatory process involving both CrhR-dependent and -independent mechanisms [26], while conditional proteolysis contributes to the temperature upshift repression [27]. Induction of RNA helicases upon cold stress has been observed in several bacteria, conditions under which helicase activity has been proposed to be required to overcome the thermodynamically enhanced stability of RNA secondary structures at temperatures below the growth optimum [18].…”

Cyanobacterial RNA Helicase CrhR Localizes to the Thylakoid Membrane Region and Cosediments with Degradosome and Polysome Complexes in Synechocystis sp. Strain PCC 6803

“…The crhR TR mutant was created by insertion of a spectinomycin-streptomycin resistance cassette at the PmlI site halfway between motifs III (SAT) and IV (FVRTK), thereby removing the second RecA domain and C-terminal extension from crhR (33). CrhR TR , the 27-kDa truncated version of the CrhR polypeptide expressed in this strain (24,32), is biochemically inactive (D. Chamot and G. W. Owttrim, unpublished data). The crhR TR mutant strain was grown on BG-11 medium containing sodium thiosulfate (0.3%) that was buffered with tricine (10 mM; pH 8.0) and supplemented with 50 g/ml each of spectinomycin and streptomycin (34).…”

“…The autoregulatory, CrhR-dependent checkpoints include temperature regulation of crhR transcript and protein half-life (24). CrhR protein half-life is controlled by conditional, temperature-upshift-induced proteolysis that generates the reduction in CrhR abundance observed at the optimal growth temperature, 30°C (24,32). A truncated mutant of crhR, crhR TR , causes severe morphological and physiological aberrations in Synechocystis, including decreased photosynthetic electron transport, carbon fixation, and oxygen evolution, as well as significant disorganization of internal cell structures, including the thylakoid membrane (33).…”