kaiABC Gene Cluster & Clock Mutant C44a

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Questions and Answers

Match the following experimental observations with the most accurate conclusion about the kaiABC gene cluster's role in cyanobacterial circadian rhythms:

Wild-type Synechococcus exhibits robust bioluminescence rhythms (~25h period) = This establishes the baseline circadian rhythm and validates the bioluminescence reporter system. Deletion of the entire kaiABC cluster (ΔkaiABC) results in arrhythmia. = This demonstrates that the kaiABC cluster is essential for generating circadian rhythms. Reintroducing the kaiABC cluster into the ΔkaiABC strain restores normal ~25h rhythms. = This shows that kaiABC is both necessary and sufficient for circadian rhythm generation. Individual knockouts of kaiA, kaiB, or kaiC all lead to arrhythmia. = This confirms that each gene in the kaiABC cluster is individually essential for maintaining circadian rhythms.

Match the following experimental results concerning kai gene expression patterns with the most relevant conclusion about the circadian regulation of these genes:

kaiA and kaiB promoters fused to lux reporters exhibit rhythmic bioluminescence, while the kaiC promoter does not. = This suggests that kaiA and kaiB are transcriptionally regulated, while kaiC is controlled primarily through post-transcriptional mechanisms. A kaiABC::lux reporter fusion shows rhythmic bioluminescence. = This indicates that the entire kaiABC operon is subject to circadian regulation, although individual genes might be regulated differently. Northern blot analysis reveals that the kaiC transcript is larger than expected. = This supports the hypothesis that kaiC undergoes significant post-transcriptional modification or processing. Northern blot analysis demonstrates that kaiA and kaiC mRNA levels oscillate with ~25h periods. = This confirms that circadian regulation occurs at the transcriptional level for both kaiA and kaiC, despite other regulatory mechanisms affecting kaiC.

Match the observation to the corresponding conclusion regarding the role of KaiC phosphorylation in the circadian clock of cyanobacteria.

KaiC phosphorylation levels oscillate rhythmically over a 24-hour period. = KaiC phosphorylation state is a key indicator of circadian time and regulates downstream processes. Specific mutations in KaiC phosphorylation sites alter the period length of the circadian rhythm. = Phosphorylation of KaiC at specific residues is critical for determining the pace of the circadian clock. Deletion or mutation of kinases and phosphatases that regulate KaiC phosphorylation disrupts circadian rhythms. = The precise balance of KaiC phosphorylation and dephosphorylation is essential for maintaining circadian oscillations. In vitro reconstitution of the KaiABC complex demonstrates ATP-dependent KaiC autophosphorylation. = The KaiABC complex functions as a self-sustaining oscillator, with KaiC phosphorylation driven by ATP hydrolysis.

Match the specific experimental manipulation of kaiC expression to the resulting effect on circadian rhythms in Synechococcus:

Introduction of kaiC mutations (A30a, B22a, C28a). = The strain maintains ~25h rhythms, suggesting that these mutations do not disrupt core clock function. Introduction of the kaiC mutation CLAb. = The mutated strain becomes arrhythmic, proving the role of KaiC in clock function. Overexpression of KaiC via IPTG induction. = Circadian oscillations are abolished, indicating that KaiC dosage must be tightly regulated for proper clock function. KaiC overexpression via IPTG induction at different circadian phases. = The phase of circadian rhythms is shifted, supporting the role of KaiC in controlling circadian phase. Signup and view all the answers

Match each observation to the corresponding conclusion regarding the role of KaiA in the cyanobacterial circadian clock.

KaiA interacts directly with KaiC in vitro. = KaiA modulates the phosphorylation state and activity of KaiC. Deletion of kaiA results in arrhythmia. = KaiA is essential for maintaining sustained oscillations of the circadian clock. Overexpression of KaiA lengthens the period of the circadian rhythm. = KaiA influences the pace of the circadian clock. KaiA promotes the autophosphorylation of KaiC. = KaiA functions as an activator of KaiC, enhancing its ATPase activity and phosphorylation. Signup and view all the answers

Match the different methods to the corresponding descriptions.

Transformation of a long-period clock mutant (C44a) with a wild-type (WT) genomic DNA library = A method for restoring normal circadian rhythms, thus demonstrating that the kaiC gene mutation was responsible for the altered period. Homologous recombination of kaiABC = A method for deleting targeted genes in a genome. A kaiABC::lux reporter fusion = A technique to show that an entire operon is subject to circadian regulation, although individual genes might be regulated differently. Northern Blot Assays = A technique for confirming the transcription of certain genes. Signup and view all the answers

Match each domain with the corresponding description.

Walker A and B motifs = Regions that indicate ATP/GTP binding sites are present Catalytic glutamate residues = Regions that are important for ATP hydrolysis DXXG motifs = Regions that are common in GTP-binding proteins Promoter Region = A regulatory region upstream from a gene, key for transcription. Signup and view all the answers

Match the following proteins to their descriptions.

KaiA = A protein which does not have known enzymatic domains but is critical for rhythm regulation. KaiB = A protein which is involved in feedback for circadian rhythms, and lacks enzymatic domains. KaiC = An ATP-binding protein that has ATPase motifs and is structurally similar to clock proteins in other organisms, suggesting it is critically involved in regulation of circadian rhythms. Lux = The bioluminescence reporter system. Signup and view all the answers

Relate each of the proteins with its role in the circadian clock.

KaiA = Enhances KaiC phosphorylation. KaiB = Antagonizes KaiA. KaiC = Forms the core of the circadian oscillator. N/A = There is no evidence from the text to support a role of RNA Polymerase in Kai mediated timekeeping. Signup and view all the answers

Understanding the intricate relationship between gene structure and function is paramount in dissecting circadian rhythms. Correlate each genetic element or structural motif with its functional implication in the kaiABC operon:

The intergenic region between kaiB and kaiC = Potential regulatory elements that could affect translation efficiency. The Shine-Dalgarno sequence upstream of each kai gene = Ribosome binding, critical for translation initiation of each protein. The presence of a Rho-independent terminator downstream of kaiC = Ensures transcriptional termination of the entire operon. Codon usage bias within the kaiC gene = The speed and accuracy of translation and influences KaiC protein folding. Signup and view all the answers

Considering the broader implications of circadian clock research, match each discovery from the kaiABC system to its potential application in synthetic biology:

The KaiABC oscillator = Design of synthetic biological clocks with tunable periods and amplitudes. Post-translational modification of KaiC = Development of novel biosensors that respond to specific phosphorylation states. The role of KaiB in antagonizing KaiA activity = Creating synthetic feedback loops for robust and adaptive control of gene expression. The regulatory role of the kaiABC promoter region = Engineering of synthetic promoters that drive rhythmic gene expression in heterologous systems. Signup and view all the answers

Given the complex interplay of the Kai proteins, match the experimental condition with its potential effect on the period length of the circadian rhythm:

Increased concentration of KaiA protein = Elongation of the circadian period by increased kaiC phosphorylation. Elevated levels of KaiB protein = Shortening of the circadian period. Mutation in KaiC that impairs its ATPase activity = Lengthening of the circadian rhythm. Reduced expression of the kaiABC operon = We cannot definitively say, given the data, the period will drastically change, but it certainly could. Signup and view all the answers

Match the experimental assay with its purpose in elucidating the function of the kaiABC gene cluster:

Bioluminescence reporter assays = Measuring the rhythms by tracking promoter activity under different conditions. Northern blot analysis = Detecting mRNA expression patterns. Western blot analysis = Monitoring protein levels and modification states. Mass spectrometry = Analyzing protein-protein interactions. Signup and view all the answers

Match each experimental manipulation of the KaiC protein with its expected effect on the circadian period in Synechococcus:

Mutation of KaiC that impairs its autophosphorylation activity. = Lengthening of the circadian period due to slower cycling of the KaiC phosphorylation state. Overexpression of KaiC under a strong inducible promoter. = Arrhythmicity due to disruption of the precise stoichiometric balance of the KaiABC complex. Introduction of a phosphomimetic mutation in KaiC that constitutively mimics phosphorylation. = Shortening of the circadian period due to accelerated progression through the phosphorylation cycle. Knockdown of KaiC expression using antisense RNA. = Arrhythmia due to insufficient levels of KaiC protein to sustain the circadian oscillator. Signup and view all the answers

Considering the broader implications of circadian clock research, connect each finding from the study of the kaiABC system to its potential application in understanding human health.

The discovery of a post-translational oscillator (PTO) based on KaiC phosphorylation. = Provide a deeper understanding of sleep disorders. Insight on how kaiABC affects transcription. = Elucidating the molecular basis of seasonal affective disorder (SAD). Understanding the sensitivity of the circadian clock. = Chronotherapy. All of the Above = Offer strategies for improving synchronization and overall health. Signup and view all the answers

Flashcards

Rescue of Clock Mutant C44a

Clock mutant transformed with wild-type DNA to restore normal circadian rhythms.

kaiABC Gene Cluster

The gene cluster essential for circadian rhythms in cyanobacteria.