Sandbox Reserved 973: Difference between revisions
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The two peptides involved in the dimere have very similar sequences . In ''mus musculus'' BMAL1 is 387 residues long and CLOCK is 361. | The two peptides involved in the dimere have very similar sequences . In ''mus musculus'' BMAL1 is 387 residues long and CLOCK is 361. | ||
Both of these subunits are basic helix-loop-helix-PAS proteins (bHLH-PAS) which contain the same 3 particular domains: a bHLH domain, a PAS-A domain and a PAS-B domain. They are involved in DNA binding and dimerization abilities. Mutations that affects the heterodimer interfaces can then disturb the activity of the complex and therefore the persistence and periodicity of the circadian cycle. | Both of these subunits are basic helix-loop-helix-PAS proteins (bHLH-PAS) which contain the same 3 particular domains: a bHLH domain, a PAS-A domain and a PAS-B domain. They are involved in DNA binding and dimerization abilities. Mutations that affects the heterodimer interfaces can then disturb the activity of the complex and therefore the persistence and periodicity of the circadian cycle. In deed the two subunits are tightly intertwined as each domain of CLOCK interact with the corresponding one of BMAL1. | ||
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This part deals with the structure of the 3 domains involved in the dimerization pattern. The transactivator domains of the two subunits which are both transcription factors working as positive elements in the circadian molecular clock will not be presented here.The data exposed below were obtained by crystallographic analysis with mouse CLOCK (residues 26–384) and BMAL1 (residues 62–447) | This part deals with the structure of the 3 domains involved in the dimerization pattern. The transactivator domains of the two subunits which are both transcription factors working as positive elements in the circadian molecular clock will not be presented here.The data exposed below were obtained by crystallographic analysis with mouse CLOCK (residues 26–384) and BMAL1 (residues 62–447) | ||
=== bHLH domain=== | === bHLH domain === | ||
Basic helix-loop-helix domains of CLOCK and BMAL1 are especially composed by 2 C-terminal helices called α1 and α2 that are involved in the formation of a canonical four-helical bHLH bundle. This bond between the HLH domains helps to stabilize the heterodimeric complex as the core of the the bundle is very hydrophobic. The spatial arrangement of this assembly has a major role in the the E-box recognition. The α1 helices are responseable for the DNA binding and the aminoacids sequence is crucial. Site-directed mutagenesis experiments showed that some hydrophobic residues, leucine in particular, were necessary in ordre to interact with the major grive of DNA duplex.In fact, when Leu57 and Leu74 of CLOCK, and Leu95 and Leu115 of BMAL1 are mutated to glutamate, mutants show no transactivation activity anymore bécasse the ability to form stable four-helice bundle is reduced as we can observe it through a bimolecular fluorescence complementation (BiFC) assay. in addition most of these mutations tend to unsettle the full length hétérodimeric complex. | Basic helix-loop-helix domains of CLOCK and BMAL1 are especially composed by 2 C-terminal helices called α1 and α2 that are involved in the formation of a canonical four-helical bHLH bundle. This bond between the HLH domains helps to stabilize the heterodimeric complex as the core of the the bundle is very hydrophobic. The spatial arrangement of this assembly has a major role in the the E-box recognition. The α1 helices are responseable for the DNA binding and the aminoacids sequence is crucial. Site-directed mutagenesis experiments showed that some hydrophobic residues, leucine in particular, were necessary in ordre to interact with the major grive of DNA duplex.In fact, when Leu57 and Leu74 of CLOCK, and Leu95 and Leu115 of BMAL1 are mutated to glutamate, mutants show no transactivation activity anymore bécasse the ability to form stable four-helice bundle is reduced as we can observe it through a bimolecular fluorescence complementation (BiFC) assay. in addition most of these mutations tend to unsettle the full length hétérodimeric complex. | ||
