Oscillations in supercoiling drive circadian gene expression in cyanobacteria

Abstract

The cyanobacterium Synechococcus elongatus PCC 7942 exhibits oscillations in mRNA transcript abundance with 24-h periodicity under continuous light conditions. The mechanism underlying these oscillations remains elusive-neither cis nor trans-factors controlling circadian gene expression phase have been identified. Here, we show that the topological status of the chromosome is highly correlated with circadian gene expression state. We also demonstrate that DNA sequence characteristics of genes that appear monotonically activated and monotonically repressed by chromosomal relaxation during the circadian cycle are similar to those of supercoiling-responsive genes in Escherichia coli. Furthermore, perturbation of superhelical status within the physiological range elicits global changes in gene expression similar to those that occur during the normal circadian cycle.circadian clock ͉ gene expression ͉ supercoiling ͉ cyanobacteria C ircadian rhythms in gene expression have been identified in many organisms. In general, 5-15% of an organism's transcriptome oscillates with 24-h periodicity in the absence of external cues such as light to dark or dark to light transitions (1). These transcriptional rhythms are controlled by an endogenous biological clock and allow organisms to schedule processes at appropriate times during the day and night cycle. The cyanobacterium Synechococcus elongatus PCC 7942 (hereafter, S. elongatus) is particularly striking because the majority of its gene expression is under circadian control in continuous light conditions. A ''promoter trap'' analysis using a bacterial luciferase reporter integrated at approximately 30,000 random loci showed circadian oscillations in bioluminescence at all 800 locations where bioluminescence signal was detected (2). A recent measurement of mRNA levels by microarray analysis demonstrated that at least 30% of transcript levels oscillated in circadian fashion (3). The discrepancy between promoter trap and microarray analysis is not surprising and is at least partially attributable to a combination of: (i) the limited time-resolution of microarrays and; (ii) the time-averaging of transcript levels observed in the bioluminescence output of promoter trap studies due to finite luciferase protein lifetime. The actual percentage of circadian transcripts in S. elongatus is likely between 30 and 100%; here we observe that 64% of transcripts oscillate with circadian periodicity (Fig. 1A).In S. elongatus, circadian oscillations in transcriptional activity require three genes, kaiA, kaiB, and kaiC, whose products comprise the core circadian oscillator. The proteins encoded by the kai genes interact with one another to generate circadian rhythms in KaiC phosphorylation at serine and threonine residues (4). Amazingly, an in vitro mixture of the Kai proteins and ATP reproduces in vivo oscillations in the phosphorylation state of KaiC (5). Inactivation of any kai gene abolishes circadian oscillations in both transcription and phosphorylation, and when kaiC is mutated such that p...

Torque measurements reveal sequence-specific cooperative transitions in supercoiled DNA

“…These constructs are readily prepared using standard molecular biology techniques, as described in SI Text. Accurate quantitative models of sequence-dependent structural transitions are needed to explain sophisticated cellular functions such as supercoiling-dependent regulation of gene expression profiles (5,6). In three divergent examples, we found that the behaviors of short regions of homogenous sequences were well described by a model for cooperative structural transitions in polymers, allowing the extraction of thermodynamic parameters relevant to sequence-dependent torsional responses (Fig.…”

“…The sequence dependence of stress-induced destabilization (2) has been hypothesized to affect the specific localization of replication initiation (3,4) and the variable response of transcriptional promoters (5). Supercoiling can lead to either activation or repression of transcription, and promoter-specific responses are exploited by organisms that use supercoiling as a global regulator of changing transcriptional programs (6). In addition to strand separation, superhelical tension may also be released by the formation of Z-DNA, a left-handed double-helical structure (7,8).…”

Robust circadian clocks from coupled protein-modification and transcription–translation cycles

“…Moreover, one would need to bring kaiBC expression under the control of a promoter that is constitutively active. However, most promoters of S. elongatus (37,43), and even many heterologous promoters from Escherichia coli (52), are influenced by the circadian clock. Nevertheless, a number of promoters have been reported that exhibit arrhythmic activity (43), and these might be possible candidates.…”

“…Although the TTC is much less well understood than the PPC, recently much progress has been made (37)(38)(39). In particular, several proteins important for transcriptional regulation of the kaiBC operon have now been identified.…”

Flexibility of the C-terminal, or CII, ring of KaiC governs the rhythm of the circadian clock of cyanobacteria

“…The cyanobacterial circadian oscillator (3), composed of only three proteins-KaiA, KaiB, and KaiC (4)-sustains an ordered pattern of KaiC autophosphorylation and autodephosphorylation (5). This oscillator governs circadian rhythms of gene expression (6,7) and chromosome compaction (8)(9)(10), and gates cell division (11)(12)(13).…”