Podcast
Questions and Answers
Given the experimental setup in Figure 1A, what is the most rigorous control experiment to definitively conclude that the wild-type kaiC gene is solely responsible for rescuing the circadian rhythm in the C44a mutant?
Given the experimental setup in Figure 1A, what is the most rigorous control experiment to definitively conclude that the wild-type kaiC gene is solely responsible for rescuing the circadian rhythm in the C44a mutant?
- Transforming the C44a mutant with an empty plasmid vector lacking any _kai_ genes.
- Transforming the C44a mutant with a plasmid containing only the _kaiA_ and _kaiB_ genes.
- Site-directed mutagenesis of the rescued _kaiC_ gene in plasmid p44N, followed by transformation into C44a to assess whether the rescue phenotype is abolished, concomitant with expression analysis of _kaiA_ and _kaiB_. (correct)
- Transforming the wild-type strain with the same plasmid used to rescue the C44a mutant and observing any changes in its circadian period.
Considering Figure 1B, what is the most parsimonious explanation for the close proximity of kaiA, kaiB, and kaiC genes within a single cluster in Synechococcus?
Considering Figure 1B, what is the most parsimonious explanation for the close proximity of kaiA, kaiB, and kaiC genes within a single cluster in Synechococcus?
- The proximity facilitates frequent genetic recombination, allowing for rapid adaptation to environmental changes.
- This gene clustering is a vestigial remnant of horizontal gene transfer, where the genes were originally acquired from a different species and have not yet been separated by evolutionary processes.
- The clustered arrangement ensures co-regulation and coordinated expression of all three genes, crucial for the circadian clock's function whilst also preventing cryptic transcription. (correct)
- The spatial arrangement minimizes the energetic cost of transcription by using a single promoter for all three genes, thereby increasing the cell's fitness.
Given the identification of Walker A and B motifs in KaiC (Figure 1C), which of the following experimental approaches would provide the most direct evidence that ATP binding and hydrolysis are essential for KaiC's function in the circadian clock?
Given the identification of Walker A and B motifs in KaiC (Figure 1C), which of the following experimental approaches would provide the most direct evidence that ATP binding and hydrolysis are essential for KaiC's function in the circadian clock?
- Synthesize ATP analogs with varying phosphate chain lengths and test their binding affinity to KaiC using surface plasmon resonance (SPR).
- Perform in vitro phosphorylation assays using purified KaiC protein with mutated Walker A and B motifs.
- Measure the expression levels of _kaiA_ and _kaiB_ genes in a strain lacking functional Walker A and B motifs in KaiC.
- Generate site-directed mutations in the Walker A and B motifs of KaiC, express the mutated protein in a _kaiC_ knockout strain, and assess the restoration of circadian rhythms, in conjunction with in vitro ATPase activity assays. (correct)
Based on the data presented in Figure 1D, what evolutionary constraint might explain the higher frequency of mutations observed in kaiC compared to kaiA and kaiB in circadian rhythm mutants?
Based on the data presented in Figure 1D, what evolutionary constraint might explain the higher frequency of mutations observed in kaiC compared to kaiA and kaiB in circadian rhythm mutants?
Suppose a novel mutation is discovered in kaiC that results in a significantly shortened circadian period. Which biophysical property of the KaiC protein is most likely affected by this mutation?
Suppose a novel mutation is discovered in kaiC that results in a significantly shortened circadian period. Which biophysical property of the KaiC protein is most likely affected by this mutation?
If the kaiABC gene cluster were artificially introduced into a non-circadian bacterium, what additional genetic modifications would be necessary to observe functional circadian rhythms?
If the kaiABC gene cluster were artificially introduced into a non-circadian bacterium, what additional genetic modifications would be necessary to observe functional circadian rhythms?
Given that KaiC is an ATPase, how might its activity be allosterically regulated within the circadian clock system, and what experimental approach could best identify such regulators?
Given that KaiC is an ATPase, how might its activity be allosterically regulated within the circadian clock system, and what experimental approach could best identify such regulators?
Considering the complexity of circadian clock regulation, what emergent property might arise from the interactions between KaiA, KaiB, and KaiC that cannot be predicted from studying each protein in isolation?
Considering the complexity of circadian clock regulation, what emergent property might arise from the interactions between KaiA, KaiB, and KaiC that cannot be predicted from studying each protein in isolation?
What are the limitations of the presented data in fully explaining the entrainment of the Synechococcus circadian clock to environmental light-dark cycles?
What are the limitations of the presented data in fully explaining the entrainment of the Synechococcus circadian clock to environmental light-dark cycles?
Given the cyclic phosphorylation of KaiC, what are the implications for designing synthetic biological oscillators based on the kaiABC system, particularly concerning robustness and tunability?
Given the cyclic phosphorylation of KaiC, what are the implications for designing synthetic biological oscillators based on the kaiABC system, particularly concerning robustness and tunability?
Flashcards
Rescue of Clock Mutant C44a
Rescue of Clock Mutant C44a
Wild-type kaiC restores normal circadian rhythms in a clock mutant, correcting the altered period.
kaiABC Gene Cluster
kaiABC Gene Cluster
The kaiABC gene cluster encodes core components essential for regulating circadian rhythms in cyanobacteria.
KaiC Amino Acid Sequence
KaiC Amino Acid Sequence
KaiC has ATPase motifs similar to clock proteins in other organisms, suggesting an enzymatic role in rhythm regulation.
Clock Mutations in kaiABC
Clock Mutations in kaiABC
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kaiC Mutation Effect
kaiC Mutation Effect
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Amino Acid Sequences of Kai Proteins
Amino Acid Sequences of Kai Proteins
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Study Notes
Rescue of Clock Mutant C44a
- Mutant C44a which has a long-period clock (44 hours) was transformed using a wild-type (WT) genomic DNA library.
- The aim of the transformation was to restore normal circadian rhythms.
- The kaiC gene mutation was responsible for the altered period.
- Restoring a wild-type copy of kaiC corrected the rhythm.
- A rescued clone (plasmid p44N) was identified to restore a normal 25-hour rhythm.
- The rescued clones had a 25-hour period like the wild type.
- The correction indicates the mutant had a recessive loss-of-function mutation in kaiC.
- Wild-type kaiC expression restores circadian rhythms in a clock mutant.
Map of the kaiABC Gene Cluster
- The genetic map of the kaiABC gene cluster is essential for circadian rhythms in cyanobacteria.
- The three genes (kaiA, kaiB, kaiC) are responsible for generating the rhythm.
- Researchers mapped a 4.7 kb EcoRI segment of genomic DNA to rescue the mutant.
- DNA sequencing revealed six open reading frames (ORFs).
- kaiA, kaiB, kaiC form a single cluster which were identified as the three clock genes.
- These genes are adjacent in the cyanobacteria genome.
- The mutation in the mutant strain C44a was in kaiC.
- Southern blot analysis confirmed only one copy of this gene cluster exists in wild-type Synechococcus.
- The kaiABC gene cluster encodes core circadian clock components.
Deduced Amino Acid Sequences of Kai Proteins
- The amino acid sequences of KaiA, KaiB, and KaiC proteins shows key functional motifs.
- The predicted amino acid sequences of KaiA (284 residues), KaiB (102 residues), and KaiC (519 residues) were analysed.
- Functional motifs in KaiC were identified as Walker A & B motifs (ATP/GTP binding sites), catalytic glutamate residues (important for ATP hydrolysis), and DXXG motifs (common in GTP-binding proteins).
- KaiC is an ATP-binding protein, similar to clock proteins in other species.
- KaiA and KaiB do not have known enzymatic domains but are critical for rhythm regulation.
- The structural features suggest KaiC plays a central role in the feedback loop.
- KaiC has ATPase motifs and is structurally similar to clock proteins in other organisms.
Mapping of Clock Mutations
- The location of mutations in kaiA, kaiB, and kaiC in various circadian rhythm mutants is shown.
- Mutations in any of the three kai genes disrupt rhythms.
- The kai genes were sequenced in 19 clock mutants with long periods, short periods, or arrhythmic phenotypes.
- The exact mutation site in each mutant was mapped.
- 14 mutants had mutations in kaiC, suggesting it is the most important regulator.
- 3 mutants had mutations in kaiA, and 2 had mutations in kaiB.
- A single amino acid change in kaiC caused complete loss of rhythms.
- kaiABC is essential for the cyanobacterial clock.
- Most mutations occurred in kaiC (14/19).
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