A split binding site for transcription factor tau on the tRNA3Glu gene

Area of Science:

• Molecular Biology
• Gene Regulation
• Yeast Genetics

Background:

• Transcription factors play crucial roles in regulating gene expression.
• Transfer RNA (tRNA) genes have unique regulatory elements.
• The yeast transcription factor tau is known to interact with tRNA genes.

Purpose of the Study:

• To characterize the binding interaction of yeast transcription factor tau with the tRNA3Glu gene.
• To identify the specific DNA regions and contact points involved in this interaction.
• To propose a model for tau factor's role in tRNA gene transcription.

Main Methods:

• Purification of tau factor using a specific tRNA gene affinity column.
• DNA footprinting assays to determine the protected regions of the tRNA3Glu gene.
• Exonuclease digestion to map the 5' and 3' boundaries of the tau-tRNA complex.
• Dimethyl sulfate protection experiments to identify guanine contact points.

Main Results:

• Tau factor forms a stable complex protecting the yeast tRNA3Glu gene from position -8 to +81.
• A DNase-sensitive site was observed in the middle of the gene.
• The 5' boundary was initially mapped at -11, but prolonged digestion revealed binding to +30 to +38.
• Dimethyl sulfate protection identified contact points within the A and B block regions, with predominant binding at the B block.
• All invariant GC pairs within the A and B blocks showed strong contacts with tau factor.

Conclusions:

• Yeast transcription factor tau interacts with both the 5' and 3' halves of the tRNA3Glu gene.
• The B block region represents the predominant binding site for tau factor.
• The dual binding site suggests a model where tau factor facilitates transcription by RNA polymerase C through alternate binding at A and B blocks.