The two DNA-binding domains of yeast transcription factor tau as observed by scanning transmission electron microscopy.
Yeast transcription factor tau interacts with the intragenic promoter of tRNA genes, binding to both the A and B block elements. Affinity-purified tau factor and tau-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 (approximately 300 kd). A combination of these two domains results in a mass for the factor of 510-670 kd. When tau was allowed to interact with recombinant tRNA(3Leu) genes with variable A block-B block spacing, different structures were observed. With short genes, the two globular domains were not resolved and tau 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 tRNA(3Leu) 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 protein-linked, reconstituted full-length genes were also observed. These different conformational states of the tau-tDNA complexes probably reflect the dynamic aspect of the interaction of the factor with its DNA target.
Schultz
P
P
Laboratoire de Génétique Moléculaire des Eucaryotes, Strasbourg, France.
Marzouki
N
N
Marck
C
C
Ruet
A
A
Oudet
P
P
Sentenac
A
A
D004258:DNA Polymerase III; D004268:DNA-Binding Proteins / Q000648:ultrastructure*; D005800:Genes, Fungal; D008855:Microscopy, Electron, Scanning; D009698:Nucleoproteins / Q000648:ultrastructure; D011401:Promoter Regions, Genetic; D012356:RNA, Transfer, Leu / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D014157:Transcription Factors / Q000302:isolation & purification / Q000378:metabolism
D004268:DNA-Binding Proteins; D009698:Nucleoproteins; D012356:RNA, Transfer, Leu; D014157:Transcription Factors; C041273:transcription factor tau; D004258:DNA Polymerase III