The trans-acting protein interacting with the DNA motif proximal to the transcriptional start site of plant L-asparaginase is bacterial sarcosine oxidase.
A trans-acting protein interacting with a specific sequence motif proximal to the transcriptional start site of the L-asparaginase promoter has been observed previously (E. Vincze, J. M. Reeves, E. Lamping, K. J. F. Farnden, and P. H. S. Reynolds, Plant Mol. Biol. 26:303-311, 1994). Gel retardation experiments in which protein extracts of Mesorhizobium loti and developing nodules were used suggested a bacterial origin for the repressor binding protein (rep2037). Nodulation tests were performed by using different Fix(-) Tn5 mutants of M. loti. Analyses of these mutants revealed a correlation between the presence of Mesorhizobium in the nodule-like structures and the ability of nodule protein extracts to bind the repressor binding domain (RBD). Through the use of mutated RBD sequences, the RBD sequence was identified as CTAAAAT. The repressor protein was isolated from M. loti NZP2037 by multiple chromatographic procedures and affinity separation by using concatemers of RBD attached to magnetic beads. Sequencing of the recovered protein resulted in identification of the repressor protein as the sarcosine oxidase alpha subunit. This was confirmed by expression of the gene encoding the M. loti alpha subunit of sarcosine oxidase in Escherichia coli. When the expressed peptide was bound to RBD, the gel retardation result was identical to the result obtained with rep2037 from M. loti strain NZP2037.
Jones
William T
WT
Horticultural Research Institute of New Zealand, Palmerston North, New Zealand. wjones@hortresearch.co.nz
Al-Samarrai
Taha
T
Reeves
Janice M
JM
Ryan
Gordon B
GB
Kirk
Christopher A
CA
Vincze
Eva
E
Harvey
Dawn
D
McCambridge
Marie
M
Greenwood
David
D
Reynolds
Paul H S
PH
D000595:Amino Acid Sequence; D001215:Asparaginase / Q000235:genetics*; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D010089:Oxidoreductases, N-Demethylating / Q000737:chemistry / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D021122:Protein Subunits; D012097:Repressor Proteins / Q000302:isolation & purification / Q000378:metabolism*; D050879:Sarcosine Oxidase; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D021122:Protein Subunits; D012097:Repressor Proteins; D004247:DNA; D010089:Oxidoreductases, N-Demethylating; D050879:Sarcosine Oxidase; D001215:Asparaginase