arf1, a transcription factor that binds to auxin response
TRANSCRIPT
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ARF1, a Transcription Factor That Binds to Auxin Response Elements
Tim Ulmasov, Gretchen Hagen, Tom J. Guilfovle (1997)
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Introduction
AuxREAux/IAA
E1 likeAXR
Proteasom
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• Test for Identification ofthe consensussequence
• Palindromic repeats ofTGTCTC [P3(4x)] functioned as AuxREs
• Mutated P3(4x) wereinaktive
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Yeast-one-hybrid System
• Identification of cDNAencoding protein, thatbinds on targetsequence
• Five cDNA clones wereisolated that encodedsame protein
• ARF 1 was found
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Schematic diagrams of ARF1 andrelated proteins
• ARF sequences relatedto c-terminal regions ofVP1/ABI3
• Also to boxes III and IV in Aux/IAA
• Contains NLS (nuclearlocalization sequence)
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Gel mobility shift assay
• Southern Blot
• Binding proteindecreases mobility of a DNA-fragment
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• EMSA (electrophoresis mobility shift assay)
• To detect in vitro interaction of ARF1
• Effects of boxes III and IV by truncating
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Importance of the TGTCTC-sequence
• They detected the specificity of ARF1 to the consensus sequence of the AuxREby mutating it
Kontrolle
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In vivo assay of auxin responsiveness
• To verify the in vitro assays
• AuxREs were fused upstream of the minimal promotor GUS reporter gene
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DNase footprinting assay
• A method to discover specific DNA-protein interactions
• You can detect the DNA binding sequence
Molecular Biology of the Cell, Alberts et al, Garland Sience, 5th edition 2008, p. 430
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IR ER
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Biochemistry, Voet and Voet, Wiley VCH, 4th edition 2011, Fig. 31-34
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AuxRE
Aux/IAA
E1 likeAXR
Proteasom
ARF1
Conclusion
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AuxRE
Aux/IAA
E1 likeAXR
Proteasom
ARF1repressor
Ubi
Ubi
Ubi
Ubi
Ubi
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Ubi
Ubi
UbiUbi
Ubi
AuxRE
Aux/IAA
E1 likeAXR
Proteasom
ARF1