Sandbox Reserved 933: Difference between revisions
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The ''LFY'' gene encodes a 424 amino acids protein that containing two domains. The N-terminal domain of LFY has been proved mediating homodimerization (ref) and it is also thought to be responsible for transcriptional activation (ref). The C-terminal consensus is highly conserved among land species and functioning as DNA-binding domain. Two DNA-protein binding structure for LEAFY were first published by Hame et al. 2008 (ref). These two structures include a recombinant C-terminal domain of LEAFY expressed by ''Escherichia coli'' strain RosettaBlue (DE3) and a short nucleotide structure from AP1 or AG promoter region. Final models of LFY-pAP1 and LFY-pAG were solved at 2.1 Å and 2.3 Å by X-ray diffraction and deposited as PDB entry <scene name='57/579703/2vy1/1'>2VY1</scene>/<scene name='57/579703/2vy2/1'>2VY2</scene>. | The ''LFY'' gene encodes a 424 amino acids protein that containing two domains. The N-terminal domain of LFY has been proved mediating homodimerization (ref) and it is also thought to be responsible for transcriptional activation (ref). The C-terminal consensus is highly conserved among land species and functioning as DNA-binding domain. Two DNA-protein binding structure for LEAFY were first published by Hame et al. 2008 (ref). These two structures include a recombinant C-terminal domain of LEAFY expressed by ''Escherichia coli'' strain RosettaBlue (DE3) and a short nucleotide structure from AP1 or AG promoter region. Final models of LFY-pAP1 and LFY-pAG were solved at 2.1 Å and 2.3 Å by X-ray diffraction and deposited as PDB entry <scene name='57/579703/2vy1/1'>2VY1</scene>/<scene name='57/579703/2vy2/1'>2VY2</scene>. | ||
=== Site specific DNA recognition is conducted by a HTH-like motif === | === Site specific DNA recognition is conducted by a HTH-like motif === | ||
The general structure of LEAFY DNA binding domain consists 2 <scene name='57/579703/Beta-strand/1'>β strands</scene> at the beginning followed by 7 <scene name='57/579703/Alpha-helices_color/2'> α helices</scene>. A <scene name='57/579703/Alpha-helices_color/3'>helix-turn-helix</scene> (HTH) motif can be found between α2 and α3 helices, which is recruited to the <scene name='57/579703/Major_groove/2'>major groove </scene>of the binding DNA. There are two amino acid at this motif, <scene name='57/579703/Major_groove_asn291/1'>Asn 291</scene> on α2 and <scene name='57/579703/Major_groove_asn291/2'>Lys 307</scene> on α3 directly mediate site specific recognition with <scene name='57/579703/Major_groove_detail/1'>two guanines</scene> at the DNA strand. These two recognition sites were further validated by electrophoresis mobility shift assay (EMSA): mutation at either Asn 291 or Lys 307 dramatically decrease binding affinity to pAP1. In the minor groove, site specific recognition is conducted by <scene name='57/579703/Arg_237/1'>Arg 237</scene>, which is at the beginning of this structure. ''Arabidopsis'' intermediate mutant ''lfy-4'' (P240L) and ''lfy-5'' (T244M) were located near this site and validate the function ''in planta''. The | [[Image:AtLFY_MajorGroove.jpg|300px|left|thumb| Recognition of DNA binding sites at major groove. ]] | ||
The general structure of LEAFY DNA binding domain consists 2 <scene name='57/579703/Beta-strand/1'>β strands</scene> at the beginning followed by 7 <scene name='57/579703/Alpha-helices_color/2'> α helices</scene>. A <scene name='57/579703/Alpha-helices_color/3'>helix-turn-helix</scene> (HTH) motif can be found between α2 and α3 helices, which is recruited to the <scene name='57/579703/Major_groove/2'>major groove </scene>of the binding DNA. There are two amino acid at this motif, <scene name='57/579703/Major_groove_asn291/1'>Asn 291</scene> on α2 and <scene name='57/579703/Major_groove_asn291/2'>Lys 307</scene> on α3 directly mediate site specific recognition with <scene name='57/579703/Major_groove_detail/1'>two guanines</scene> at the DNA strand. These two recognition sites were further validated by electrophoresis mobility shift assay (EMSA): mutation at either Asn 291 or Lys 307 dramatically decrease binding affinity to pAP1. In the minor groove, site specific recognition is conducted by <scene name='57/579703/Arg_237/1'>Arg 237</scene>, which is at the beginning of this structure. ''Arabidopsis'' intermediate mutant ''lfy-4'' (P240L) and ''lfy-5'' (T244M) were located near this site and validate the function ''in planta''. The super position of specific recognition sites is summaries at right figure produced by PyMol. | |||
=== DNA binding required cooperative dimerization === | === DNA binding required cooperative dimerization === | ||
Transcription factors tent to form homodimer or heterodimer to increase the binding specificity and affinity. Experimental evidence indicates a potential LFY dimer on the binding site. Crystal structure proved that LFY can form dimers at both <scene name='57/579703/Ap1-dimer/2'>pAP1</scene> and <scene name='57/579703/2vy2_assembly/2'>pAG</scene> sites. The binding affinity of LFY protein dimer binds increased by 90-fold compared to the first LFY monomer in EMSA assay. Detailed structure revealed that the contact of two dimerized protein is mediated by <scene name='57/579703/Ap1-dimer/8'>three residues</scene> located at on helix (<scene name='57/579703/Ap1-dimer/3'>α7</scene>) and one loop (<scene name='57/579703/Ap1-dimer/4'>loop12</scene>) at the other protein. Hydrogen bonds can be formed between <scene name='57/579703/Ap1-dimer/7'>Asn 290</scene> and <scene name='57/579703/Ap1-dimer/5'>His 387</scene>/<scene name='57/579703/Ap1-dimer/6'>Arg 390</scene> are essential for this dimeriation. The detailed interaction is shown in the right figure produced by Pymol. Mutation in any of these three amino acids abolished the binding in EMSA assay. Recently, another experiment showing that despite these three residues, the entire N-terminal consensus is critical important for stabilizing the homodimerization, where strong physical interaction can be found by GST-pull down, Y2H and BiFC experiment at ''in vitro'', ''in vivo'' and ''in planta'' level. | Transcription factors tent to form homodimer or heterodimer to increase the binding specificity and affinity. Experimental evidence indicates a potential LFY dimer on the binding site. Crystal structure proved that LFY can form dimers at both <scene name='57/579703/Ap1-dimer/2'>pAP1</scene> and <scene name='57/579703/2vy2_assembly/2'>pAG</scene> sites. The binding affinity of LFY protein dimer binds increased by 90-fold compared to the first LFY monomer in EMSA assay. Detailed structure revealed that the contact of two dimerized protein is mediated by <scene name='57/579703/Ap1-dimer/8'>three residues</scene> located at on helix (<scene name='57/579703/Ap1-dimer/3'>α7</scene>) and one loop (<scene name='57/579703/Ap1-dimer/4'>loop12</scene>) at the other protein. Hydrogen bonds can be formed between <scene name='57/579703/Ap1-dimer/7'>Asn 290</scene> and <scene name='57/579703/Ap1-dimer/5'>His 387</scene>/<scene name='57/579703/Ap1-dimer/6'>Arg 390</scene> are essential for this dimeriation. The detailed interaction is shown in the right figure produced by Pymol. Mutation in any of these three amino acids abolished the binding in EMSA assay. Recently, another experiment showing that despite these three residues, the entire N-terminal consensus is critical important for stabilizing the homodimerization, where strong physical interaction can be found by GST-pull down, Y2H and BiFC experiment at ''in vitro'', ''in vivo'' and ''in planta'' level. | ||
