Figure 5.

The TAG1 DNA Binding Domain Contains a Putative Zinc Finger.

(A) Amino acid alignment of TAG1 with tobacco 3AF1 and tomato E4/E8BP sequences. The alignment was performed using the ClustalW program. Shown are TAG1 residues 39 to 76, 3AF1 residues 33 to 72, and E4/E8BP residues 213 to 252. Putative metal binding amino acids are indicated with asterisks.

(B) Chelator inhibition of DNA binding. Approximately 300 ng (0.1 μL) of affinity-purified ΔC-133 (BL21 expressed) was incubated with increasing quantities of EDTA or 1,10-phenanthroline in 2% ethanol. Although 1 mM EDTA was present in all EMSA binding reactions (see Methods), only the additional EDTA quantities are indicated (lanes 3 to 5); the reaction in lane 2 contained 1 mM EDTA and is labeled as containing no chelator merely as a reference for the inhibition by excess chelator. Approximately 24,000 cpm of ³²P-labeled 5′ STR probe (∼1.4 ng) was added, and complexes were resolved on a 4% polyacrylamide gel; the gel was dried and exposed to film at −80°C.