Difference between revisions of "Team:Tianjin/Description"

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Revision as of 16:58, 13 October 2018

<!DOCTYPE html> Team:Tianjin - 2018.igem.org

Background

Many organisms contain a biological rhythm system to adapt to the Earth's circadian rhythm. The biological rhythm system in the cyanobacteria is designed to allow cyanobacterial cells to predict the time of daylighting in order to prepare for light reaction in advance.

In Synechococcus elongatus, the oscillator is composed of only three proteins KaiA, KaiB, and KaiC, which together generate the circadian rhythm of KaiC phosphorylation at residues serine 431 (S431) and threonine 432 (T432) in the CII domain. KaiA promotes KaiC (auto)phosphorylation during the subjective day, whereas KaiB provides negative regulation to inhibit KaiA and promotes KaiC (auto)dephosphorylation during the subjective night. [1]The 24-h KaiC phosphorylation pattern can be reconstituted in vitro by merely combining the three Kai proteins and ATP, suggesting that the oscillating system is a post-translational oscillation system. KaiA and KaiB are also involved in regulating two antagonistic clock-output proteins—SasA and CikA, which reciprocally control the final regulator of transcription, RpaA.

Molecular changes in the KaiABC circadian clock system: Stepwise binding of two KaiA dimers triggers KaiC autophosphorylation at Thr432 and Ser431. These phosphorylation events enable cooperative binding of fold-switched KaiB monomers to the KaiC-CI domain, forming the KaiBC complex. KaiBC provides a scaffold for the successive sequestration of KaiA in ternary KaiABC assemblies, concurring with a rearrangement of the KaiA PsR domains. KaiA sequestration promotes KaiC autodephosphorylation, resulting in the regeneration of free KaiC through release of KaiAB subcomplexes. [2]

Temporal information from the oscillator is transmitted to downstream genes via the histidine protein kinase SasA (Synechococcus adaptive sensor A), whose autophosphorylation is stimulated by interaction with KaiC. Phosphorylated SasA in turn transfers a phosphoryl group to RpaA (regulator of phycobilisome association A) , a transcription factor that directly regulates the expression of approximately 100 genes. Moreover, RpaA indirectly regulates the expression of nearly all genes in the genome. [3]Disruption of sasA also results in severely damped gene expression rhythms. CikA acquires the ability to dephosphorylate RpaA by interacting with the KaiBC complex and acts on downstream genes via RpaA to complete the subjective night biochemical reaction.

Our team hopes to build a cyanobacteria biological rhythm system in yeast. Since yeast is a eukaryote, prokaryotic promoters cannot be used directly, so we abandoned the strategy of verifying oscillations by controlling downstream genes by RpaA. Finally, we used the yeast two-hybrid system to reporter gene expression.We selected KaiC-SasA, KaiC-CikA, and KaiC-KaiB three pairs of proteins for the yeast two-hybrid system. During the subjectively day, KaiC binds to SasA, causing spatial proximity of AD and BD leading to the initiation of downstream reporter genes. The KaiC and KaiB, CikA and KaiBC complexes are combined on subjective night, and downstream reporter genes are activated by the yeast two-hybrid system respectively.

Yeast Two-Hybrid System

References

[1] Roger Tseng, Nicolette F. Goularte, Archana Chavan, Jansen Luu, Susan E. Cohen, Yong-Gang Chang, Joel Heisler, Sheng Li, Alicia K. Michael, Sarvind Tripathi, Susan S. Golden, Andy LiWang, Carrie L. Partch, Structural basis of the day-night transition in a bacterial circadian clock. Science, 1174-1180 (2017).

[2] Joost Snijder, Jan M. Schuller, Anika Wiegard, Philip Lössl, Nicolas Schmelling, Ilka M. Axmann, Jürgen M. Plitzko, Friedrich Förster, Albert J. R. Heck, Structures of the cyanobacterial circadian oscillator frozen in a fully assembled state. Science, 1181-1184 (2017).

[3] Joseph S.Markson, Joseph R.Piechura, Anna M.Puszynska, Erin K.O’Shea, Circadian Control of Global Gene Expression by the Cyanobacterial Master Regulator RpaA. Cell, 1396-1408 (2013).

[4]Jun O. Liu,et al.Everything you need to know about the yeast two-hybrid system.DOI:10.1038/788

[5]Bhaumik S R, Raha T, Aiello D P, et al. In vivo target of a transcriptional activator revealed by fluorescence resonance energy transfer.[J]. DOI:10.1101/gad.1148404

[6]Melcher K, Johnston S A. GAL4 interacts with TATA-binding protein and coactivators.[J]. DOI:10.1128/MCB.15.5.2839

[7]Wu Y, Reece R J, Ptashne M. Quantitation of putative activator-target affinities predicts transcriptional activating potentials.DOI:10.1002/j.1460-2075.1996.tb00769.x

[8]Hideo Iwasaki,* Stanly B. Williams,et al.A KaiC-Interacting Sensory Histidine Kinase, SasA, Necessary to Sustain Robust Circadian Oscillation in Cyanobacteria.Volume 101, Issue 2

[9]Anang, Saumya,Subramani, Chandru,et al. Identification of critical residues in Hepatitis E virus macro domain involved in its interaction with viral methyltransferase and ORF3 proteins .DOI:10.1038/srep25133

[10]Xiangui Lin & Xi Huang ,et al.Identification of JAZ-interacting MYC transcription factors involved in latex drainage in Hevea brasiliensis.DOI:10.1038/s41598-018-19206-3

[11]James R.Broach,et al.Galactose regulation in Saccharomyces cerevisiae. The enzymes encoded by the GAL7, 10, 1 cluster are co-ordinately controlled and separately translated.DOI:10.1016/0022-2836(79)90300-0

[12]Kristy M. Hawkins and Christina D. Smolke 1,et al.The Regulatory Roles of the Galactose Permease and Kinase in the Induction Response of the GAL Network in Saccharomyces cerevisiae.DOI:10.1074/jbc.M512317200

[13]M Johnston, J S Flick, T Pexton,et al.Multiple Mechanisms Provide Rapid and Stringent Glucose Repression of GAL Gene Expression in Saccharomyces cerevisiae.DOI:10.1128/MCB.14.6.3834

[14]Elena Frolova John Majors,et al.Binding of the glucose-dependent Mig1p repressor to the GAL1 and GAL4 promoters in vivo: regulationby glucose and chromatin structure.DOI:10.1093/nar/27.5.1350

[15]https://thebiogrid.org/

[16]DE ROBICHON-SZULMAJSTER H,et al. Induction of enzymes of the galactose pathway in mutants of Saccharomyces cerevisiae. Science 127(3288):28-9

[17]Platt A and Reece RJ,et al. The yeast galactose genetic switch is mediated by the formation of a Gal4p-Gal80p-Gal3p complex. EMBO J 17(14):4086-91

[18]Lohr D, et al. Transcriptional regulation in the yeast GAL gene family: a complex genetic network. FASEB J 9(9):777-87

[19]Peng G and Hopper JE,et al. Gene activation by interaction of an inhibitor with a cytoplasmic signaling protein. Proc Natl Acad Sci U S A 99(13):8548-53

[20]Mylin LM, et al. SIP1 is a catabolite repression-specific negative regulator of GAL gene expression. Genetics 137(3):689-700

[21]Horak J and Wolf DH,et al. Catabolite inactivation of the galactose transporter in the yeast Saccharomyces cerevisiae: ubiquitination, endocytosis, and degradation in the vacuole. J Bacteriol 179(5):1541-9

[22]Horak J and Wolf DH,et al. Glucose-induced monoubiquitination of the Saccharomyces cerevisiae galactose transporter is sufficient to signal its internalization. J Bacteriol 183(10):3083-8

[23]Horak J and Wolf DH,et al. The ubiquitin ligase SCF(Grr1) is required for Gal2p degradation in the yeast Saccharomyces cerevisiae. Biochem Biophys Res Commun 335(4):1185-90

[24]https://www.yeastgenome.org/locus/S000004071

[25]Roger Tseng, Nicolette F. Goularte,et al. Structural basis of the day-night transition in a bacterial circadian clock.Science, 1174-1180(2017)