Dual posttranscriptional regulation via a cofactor-responsive mRNA leader.

Riboswitches are cis-acting mRNA elements that regulate gene expression in response to ligand binding. Recently, a class of riboswitches was proposed to respond to the molybdenum cofactor (Moco), which serves as a redox center for metabolic enzymes. The 5' leader of the Escherichia coli moaABCDE transcript exemplifies this candidate riboswitch class. This mRNA encodes enzymes for Moco biosynthesis, and moaA expression is feedback inhibited by Moco. Previous RNA-seq analyses showed that moaA mRNA copurified with the RNA binding protein CsrA (carbon storage regulator), suggesting that CsrA binds to this RNA in vivo. Among its global regulatory roles, CsrA represses stationary phase metabolism and activates central carbon metabolism. Here, we used gel mobility shift analysis to determine that CsrA binds specifically and with high affinity to the moaA 5' mRNA leader. Northern blotting and studies with a series of chromosomal lacZ reporter fusions showed that CsrA posttranscriptionally activates moaA expression without altering moaA mRNA levels, indicative of translation control. Deletion analyses, nucleotide replacement studies and footprinting with CsrA-FeBABE identified two sites for CsrA binding. Toeprinting assays suggested that CsrA binding causes changes in moaA RNA structure. We propose that the moaA mRNA leader forms an aptamer, which serves as a target of posttranscriptional regulation by at least two different factors, Moco and the protein CsrA. While we are not aware of similar dual posttranscriptional regulatory mechanisms, additional examples are likely to emerge.

Inactivation of a low temperature-induced RNA helicase in Synechocystis sp. PCC 6803: physiological and morphological consequences.

Inactivation of the DEAD box RNA helicase, crhR, has dramatic effects on the physiology and morphology of the photosynthetic cyanobacterium, Synechocystis sp. PCC 6803. These effects are observed at both normal growth temperature (30°C) and under cold stress (20°C), indicating that CrhR performs crucial function(s) at all temperatures. A major physiological effect is the rapid cessation of photosynthesis upon temperature downshift from 30 to 20°C. This defect does not originate from an inability to transport or accumulate inorganic carbon or a deficiency in photosynthetic capacity as the mutant has sufficient electron transport and enzymatic capacity to sustain photosynthesis at 30°C and inorganic carbon (Ci) accumulation at 20°C. Oxygen consumption in the presence of methyl viologen indicated that while electron transport capacity is sufficient to accumulate Ci, the mutant does not possess sufficient activity to sustain carbon fixation at maximal rates. These defects are correlated with severely impaired cell growth and decreased viability, cell size and DNA content at low temperature. The ΔcrhR mutant also progressively accumulates structural abnormalities at low temperature that cannot be attributed solely to reactive oxygen species (ROS)-induced photooxidative damage, suggesting that they are manifestations of pre-existing defects that are amplified over time. The data indicate that the observed physiological and morphological effects are intimately related to crhR mutation, implying that the lack of CrhR RNA unwinding/annealing activity results in the inability to execute one or more vital steps in photosynthesis that are required at all temperatures but are crucial at low temperature.

Circuitry linking the Csr and stringent response global regulatory systems.

CsrA protein regulates important cellular processes by binding to target mRNAs and altering their translation and/or stability. In Escherichia coli, CsrA binds to sRNAs, CsrB and CsrC, which sequester CsrA and antagonize its activity. Here, mRNAs for relA, spoT and dksA of the stringent response system were found among 721 different transcripts that copurified with CsrA. Many of the transcripts that copurified with CsrA were previously determined to respond to ppGpp and/or DksA. We examined multiple regulatory interactions between the Csr and stringent response systems. Most importantly, DksA and ppGpp robustly activated csrB/C transcription (10-fold), while they modestly activated csrA expression. We propose that CsrA-mediated regulation is relieved during the stringent response. Gel shift assays confirmed high affinity binding of CsrA to relA mRNA leader and weaker interactions with dksA and spoT. Reporter fusions, qRT-PCR and immunoblotting showed that CsrA repressed relA expression, and (p)ppGpp accumulation during stringent response was enhanced in a csrA mutant. CsrA had modest to negligible effects on dksA and spoT expression. Transcription of dksA was negatively autoregulated via a feedback loop that tended to mask CsrA effects. We propose that the Csr system fine-tunes the stringent response and discuss biological implications of the composite circuitry.

A Synechocystis LexA-orthologue binds direct repeats in target genes.

Although evidence for LexA-orthologues, which do not regulate DNA damage repair, is accumulating, identification of binding sites and regulon members remains poorly characterized. In the cyanobacterium, Synechocystis sp. strain PCC 6803, we have recently identified a LexA-related protein that regulates expression of the crhR RNA helicase gene. Here we show that the Synechocystis LexA-orthologue binds as a dimer to 12 bp direct repeats containing a CTA-N9-CTA sequence conserved in two target genes, lexA and crhR. Characterization of this site provides the basis for identification of additional LexA targets and further evidence for LexA's divergence during evolution.

A LexA-related protein regulates redox-sensitive expression of the cyanobacterial RNA helicase, crhR.

Expression of the cyanobacterial DEAD-box RNA helicase, crhR, is regulated in response to conditions, which elicit reduction of the photosynthetic electron transport chain. A combination of electrophoretic mobility shift assay (EMSA), DNA affinity chromatography and mass spectrometry identified that a LexA-related protein binds specifically to the crhR gene. Transcript analysis indicates that lexA and crhR are divergently expressed, with lexA and crhR transcripts accumulating differentially under conditions, which respectively oxidize and reduce the electron transport chain. In addition, expression of the Synechocystis lexA gene is not DNA damage inducible and its amino acid sequence lacks two of three residues required for activity of prototypical LexA proteins, which repress expression of DNA repair genes in a range of prokaryotes. A direct effect of recombinant LexA protein on crhR expression was confirmed from the observation that LexA reduces crhR expression in a linear manner in an in vitro transcription/translation assay. The results indicate that the Synechocystis LexA-related protein functions as a regulator of redox-responsive crhR gene expression, and not DNA damage repair genes.