Characterization of a gene encoding a DNA binding protein with specificity for a light-responsive element

Area of Science:

• Molecular Biology
• Plant Science
• Genetics

Background:

• The rbcS-3A gene in pea contains a light-responsive element with a regulatory sequence element known as box II.
• Nuclear protein GT-1 was previously identified as a DNA-binding activity that interacts with box II.

Purpose of the Study:

• To isolate and characterize cDNA sequences encoding a DNA-binding protein specific for the box II element.
• To investigate the structural and functional relationship between the isolated protein and known GT-binding proteins.

Main Methods:

• Isolation and characterization of cDNA sequences.
• Recombinant protein expression and DNA-binding assays using mutated box II elements.
• Immunological assays to compare nuclear GT-1 and recombinant tobacco GT-1a.
• Sequence analysis to predict protein structure and compare with other GT-binding proteins.
• Genomic and RNA gel blot analyses to study gene family and transcript distribution.

Main Results:

• A cDNA encoding tobacco GT-1a, a DNA-binding protein specific for box II, was isolated.
• Recombinant tobacco GT-1a exhibited similar DNA-binding sequence requirements to nuclear GT-1 and was immunologically related.
• The protein structure prediction revealed a novel helix-helix-turn-helix (HHTH) motif, conserved in other GT-binding proteins and involved in DNA interaction.
• Tobacco contains a small gene family for related sequences, and transcripts are found in various tissues.

Conclusions:

• Tobacco GT-1a is a novel DNA-binding protein that recognizes the light-responsive box II element.
• The HHTH motif is critical for the interaction of GT-binding proteins with box II.
• The gene family and tissue-specific expression suggest a role in light-regulated gene expression in tobacco.