Identification of a transcription factor IIIA-interacting protein

Area of Science:

• Molecular Biology
• Gene Regulation
• Protein-Protein Interactions

Background:

• Transcription Factor IIIA (TFIIIA) is crucial for 5S ribosomal RNA gene transcription in eukaryotes.
• Vertebrate TFIIIA contains nine Cys(2)His(2) zinc fingers for nucleic acid binding and a C-terminal region for transcription activation.

Purpose of the Study:

• To identify novel protein partners interacting with the C-terminal region of Xenopus TFIIIA.
• To characterize the function of identified TFIIIA-interacting proteins in gene transcription.

Main Methods:

• Yeast two-hybrid screens using Xenopus TFIIIA C-terminus and a rat cDNA library.
• Sequencing of identified cDNA clones to determine human homolog (TFIIIA-intP).
• In vitro immuno-precipitation and co-chromatography assays to confirm protein interactions.
• In vitro transcription assays using HeLa cell nuclear extracts.

Main Results:

• A rat cDNA clone encoding a TFIIIA-interacting protein (TFIIIA-intP) was identified.
• Recombinant human TFIIIA-intP demonstrated in vitro interaction with Xenopus TFIIIA.
• Interaction between rat TFIIIA and rat TFIIIA-intP was confirmed via co-chromatography.
• TFIIIA-intP significantly stimulated TFIIIA-dependent transcription of the Xenopus 5S rRNA gene.

Conclusions:

• TFIIIA-intP is a novel interaction partner for TFIIIA.
• TFIIIA-intP plays a role in enhancing TFIIIA-mediated 5S rRNA gene transcription.