Two polypeptide chains in yeast transcription factor τ interact with DNA

• Published in: The Journal of biological chemistry 1989-05, Vol.264 (13), p.7505-7511
• Main Authors: Gabrielsen, O S, Marzouki, N, Ruet, A, Sentenac, A, Fromageot, P
• Format: Article
• Language: English
• Subjects: Antigen-Antibody Reactions; Biological and medical sciences; Blotting, Western; Chromatography, Affinity; Cross-Linking Reagents; DNA Polymerase III; DNA, Fungal - metabolism; DNA-Binding Proteins - immunology; DNA-Binding Proteins - isolation & purification; DNA-Binding Proteins - metabolism; Fundamental and applied biological sciences. Psychology; Genes, Fungal; In Vitro Techniques; Macromolecular Substances; Molecular and cellular biology; Molecular genetics; Molecular Weight; RNA, Transfer, Glu - genetics; Saccharomyces cerevisiae - physiology; Transcription Factors - immunology; Transcription Factors - isolation & purification; Transcription Factors - metabolism; Transcription, Genetic; Transcription. Transcription factor. Splicing. Rna processing; Ultraviolet Rays
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)83263-X

Yeast transcription factor τ interacts with the A and B blocks of the intragenic promoter of tRNA genes. The structure of τ was investigated by identifying the polypeptide chains specifically complexed to the tRNA3Glu gene. Highly purified factor, obtained by an improved purification procedure, contained several polypeptide chains, four of which (Mr = 145,000, 135,000, 100,000 and 65,000) comigrated with τ-DNA complex by polyacrylamide gel electrophoresis. Antibodies raised against the 145- and 100-kDa components altered the migration of τ-DNA complexes in band shift assays and inhibited tRNA synthesis in a reconstituted transcription system. These components are immunologically unrelated proteins. By UV cross-linking to 32P-body-labeled tDNA followed by extensive DNase treatment, two polypeptides of the same size (145 and 100 kDa) were found to be radioactively labeled. Factor τ, therefore, appears to be a multisubunit DNA-binding protein with two distinct polypeptides contributing to DNA recognition. Limited proteolysis of τ generated a protease-resistant τB (τB) domain that binds solely to the B block. τB-tDNA complexes were recognized by anti-145 IgG and contained a 120-kDa polypeptide that could originate from the 145-kDa component by proteolysis. These results strongly suggest that the 145-kDa polypeptide belongs to τB and is responsible for B block binding.