The anaerobic responsive element contains two GC-rich sequences essential for binding a nuclear protein and hypoxic activation of the maize Adh1 promoter.
We have identified a protein (GCBP-1) in nuclear extracts from maize suspension cell cultures that binds to specific sequences within the Anaerobic Responsive Element (ARE) of the maize Adh1 promoter. Competition analyses show that the GCBP-1 binding activity distinguishes ARE sequence motifs from other enhancer elements or pUC19 sequences. The binding activities of several mutant ARE sequences define two regions of the ARE important for GCBP-1 binding in vitro, between nucleotides -135 to -131 and nucleotides -120 to -112 of the maize Adh1 promoter. Both regions are required for efficient GCBP-1 binding to occur in vitro. The minimum consensus binding site for GCBP-1 is 5'-GC(G/C)CC-3'. This sequence is similar to a part of the binding site of the human transcription factor Sp1 (1). We demonstrate that maize GCBP-1 and human Sp1 have similar recognition properties. Using ARE mutants in a transient assay in maize protoplasts we have shown that mutation of the GCBP-1 binding sites prevents significant hypoxic activation of the maize Adh1 promoter. These results suggest a direct role for GCBP-1 in the hypoxic activation of Adh1 gene expression. GCBP-1 is present in both uninduced and induced nuclei, indicating that inducible gene expression is not dependent upon synthesis of GCBP-1 and suggesting that post-translational modification of bound GCBP-1 may be important for enhanced transcription to occur.
Olive
M R
MR
CSIRO Division of Plant Industry, Canberra, Australia.
Peacock
W J
WJ
Dennis
E S
ES
D000426:Alcohol Dehydrogenase / Q000235:genetics*; D000693:Anaerobiosis / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation / Q000235:genetics; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D011089:Polydeoxyribonucleotides / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D016329:Sp1 Transcription Factor / Q000235:genetics; D003313:Zea mays / Q000201:enzymology / Q000235:genetics*
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D010940:Plant Proteins; D011089:Polydeoxyribonucleotides; D016329:Sp1 Transcription Factor; C019530:poly(dC-dG); D000426:Alcohol Dehydrogenase