A novel HMG A-like protein binds differentially to the AT-rich regions located in the far distal and proximal parts of a soybean glutamine synthetase gene (GS15) promoter.
In soybean (Glycine max L.) ammonium provided externally or as the result of symbiotic nitrogen fixation stimulates the transcription of GS15, a gene encoding cytosolic glutamine synthetase. Strong constitutive positive expression (SCPE), silencer-like and organ-specific elements, located respectively in the distal, the central and the proximal region of the promoter are required to control the ammonium responsiveness of the gene expression [Tercé-Laforgue et al. (1999) Plant Mol. Biol. 39: 551]. It was hypothesized that the correct spatial conformation of the promoter permitted the cooperative action of these three cis-acting elements. Further investigations were therefore required to ascertain this hypothesis. A nodule nuclear protein, binding to a 66 bp AT-rich DNA fragment containing a 13 bp AT-rich repeated sequence (AT-1) and located just downstream of the SCPE element, was identified using a gel retardation assay. A cDNA clone likely to code for this protein was isolated using the yeast one-hybrid system. It encodes a novel DNA binding protein (AT-1SNBP) similar to HMG A proteins but exhibiting a higher molecular weight. AT-1SNBP appears to be encoded by a single gene that is expressed in roots, root nodules and leaves of soybean. Since two other 13 bp AT-rich repeated sequences (AT-2 and AT-3) were localized in the organ-specific element, we have quantified the binding affinity of AT-1SNBP to these sequences. We demonstrate that AT-1SNBP binds differentially to DNA fragments containing AT-1, AT-2 and AT-3 and that its binding affinity depends on the presence of adjacent sequences. This result suggests that AT-1SNBP may be an architectural protein involved in maintaining the spatial conformation of the GS15 promoter, thus facilitating the interaction between the distal and proximal regulatory elements.
D020856:AT Rich Sequence / Q000235:genetics*; D024761:AT-Hook Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D020369:Bradyrhizobium / Q000254:growth & development; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D018744:DNA, Plant / Q000737:chemistry / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D017343:Genes, Plant / Q000235:genetics; D005974:Glutamate-Ammonia Ligase / Q000235:genetics* / Q000378:metabolism; D024003:HMGA Proteins / Q000235:genetics* / Q000378:metabolism; D000070116:Lotus / Q000201:enzymology / Q000235:genetics / Q000382:microbiology; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D012231:Rhizobium / Q000254:growth & development; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion; D030262:Soybean Proteins / Q000235:genetics* / Q000378:metabolism; D013025:Glycine max / Q000201:enzymology / Q000235:genetics* / Q000382:microbiology; D014157:Transcription Factors / Q000235:genetics; D020798:Two-Hybrid System Techniques
D002352:Carrier Proteins; D018076:DNA, Complementary; D018744:DNA, Plant; D004268:DNA-Binding Proteins; D024003:HMGA Proteins; D000644:Quaternary Ammonium Compounds; D030262:Soybean Proteins; D014157:Transcription Factors; D005974:Glutamate-Ammonia Ligase