Transcription of eukaryotic transfer RNA genes involves, as a primary event, the stable binding of a protein factor to the intragenic promoter. The internal control region is composed of two non-contiguous conserved sequence elements, the A and B blocks. These are variably spaced depending on the genes. tau, a large transcription factor purified from yeast cells, interacts with these two control elements as shown by DNase I footprinting, exonuclease digestion, dimethyl sulphate protection experiments and by analysis of point mutations. Here we used a limited proteolysis treatment to obtain a smaller form of tau with drastically altered DNA binding properties. A protease-resistant domain interacts solely with the B block region of tRNA genes.
Yeast transcription factor X interacts with the intragenic promoter of tRNA genes, binding to both the A and B block elements. Affinity-purified r factor and r-tDNA complexes were examined by scanning transmission electron microscopy to analyze the structural features of free and DNA bound factor. The free factor appeared as two tightly associated globular domains of roughly similar size (10 nm in diameter) and mass ( -300 kd).A combination of these two domains results in a mass for the factor of 510-670 kd. When r was allowed to interact with recombinant tRNA3Leu genes with variable A block-B block spacing, different structures were observed. With short genes, the two globular domains were not resolved and T appeared as a large particle covering the A and B block region. On the other hand, with genes having a larger A-B distance (53 or 74 bp), mostly dumb-bell-shaped complexes were formed with individualized factor domains bound separately to the A and B blocks. A smaller proportion of the complexes appeared to consist of a large particle bound at only one site, essentially on the B block. Mapping of the binding domains in the DNA showed a good correlation with the respective positions of the A and B promoter elements. Factor binding did not induce a noticeable DNA bending, although with extended genes apparent DNA shortening and cases of DNA looping were observed. Upon cleavage of the tRNA3 uu gene between the A and B blocks after or prior to complex formation, the two factor domains remained attached to the same DNA fragment (mostly the B-DNA fragment). In addition, images of proteinlinked, reconstituted full-length genes were also observed. These different conformational states of the r-tDNA complexes probably reflect the dynamic aspect of the interaction of the factor with its DNA target.
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