Proteolytic footprinting of transcription factor TFIIIA reveals different tightly binding sites for 5S RNA and 5S DNA.

Abstract

Transcription factor MIIA (TFIA) employs an array of nine N-terminal zinc fingers to bind specifically to both 5S RNA and 5S DNA. The binding of TFIIIA to 5S RNA and 5S DNA was studied by using a protease footprinting technique. Brief treatment of free or bound TFIIIA with trypsin or chymotrypsin generated fragments which were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fragments retaining the N terminus of TFIIIA were identified by immunoblotting with an antibody directed against the N terminus of TFIIIA. Proteolytic footprinting of TFIIIA complexed with 5S DNA derivatives reinforced other evidence that the three N-terminal zinc fingers of TFIIIA bind most tightly to 5S DNA. Proteolytic footprinting of TFIIIA in reconstituted 7S ribonucleoprotein particles revealed different patterns of trypsin sensitivity for TFIA bound to oocyte versus somatic 5S RNA. Trypsin cleaved TFIA between zinc fingers 3 and 4 more readily when the protein was bound to somatic 5S RNA than when it was bound to oocyte 5S RNA. A tryptic fragment of TFIIIA containing zinc fingers 4 through 7 remained tightly associated with somatic SS RNA. Zinc fingers 4 through 7 may represent a tightly binding site for 5S RNA in the same sense that fingers 1 through 3 represent a tightly binding site for 5S DNA.TFIIIA is a unique example of a zinc finger transcription factor that binds specifically to 5S RNA as well as to 5S DNA (20,30). TFIIIA is typically prepared from an abundant 7S storage particle from immature Xenopus laevis ovaries. Smith et al. (37) showed that TFIIIA within the 7S particle could be cleaved with proteases such as papain and trypsin to yield fragments of the protein that retained the ability to bind to the intragenic control region (ICR) of the 5S RNA gene. A 30-kDa fragment that contains nine zinc fingers provided essentially the same DNase I footprint over residues 46 to 95 of the 5S RNA gene obtained with intact TFIIIA. A 20-kDa fragment that contains the N-terminal 220 residues of TFIIIA (seven fingers) protected only the distal portion of the ICR of the 5S RNA gene from digestion by DNase I. This work provided the first clear evidence that TFIIIA binds to the ICR in an extended conformation with zinc fingers 1 through 9 aligned from the 3' to the 5' end of the ICR. This alignment leaves the carboxyl-terminal nonzinc finger domain of TFIIIA near the 5' portion of the ICR, where it appears to interact with other transcription factors (19, 37). Two groups have recently shown that aminoterminal fragments of TFIIIA containing as few as three zinc fingers bind tightly to the distal portion of the ICR (5, 25). Indeed, fingers 1 through 3 bind to gene residues 77 through 92 nearly as tightly as intact TFIIIA binds to the complete ICR. This implies that specific contacts of fingers 4 through 9 of TFIIIA with gene sequences located 5' to this tightly binding site are not as energetically favorable. Current models suggest that fingers 4 and 6 may not make tight contacts in the major groove of DNA (6,11,18 (40...

Systematic analysis and evolution of 5S ribosomal DNA in metazoans

“…The internal 5S rRNA boxes (Figure 1) are essential for transcription of 5S rRNA itself Bogenhagen, 1993;Hall, 2005). For instance, the C2H2 zinc finger protein TFIIIA binds Xenopus 5S rRNA internal promoters (named Box A, intermediate element and Box C ), see Bogenhagen (1993) and Hall (2005).…”

“…Green motifs were used as additional filters in some species (H. sapiens, P. pygmaeus, M. mulatta, B. taurus, P. vampyrus and S. kowalevskii) because the number of candidates was too large. Whereas function of U1 boxes are diverse (Box A recognizes 5 0 -splice site in mRNA precursors (Zhuang and Weiner, 1986, and references therein); Box B is part of the U1-70K protein-binding site (Query et al, 1989); Box C is part of the U1-A protein-binding region (Scherly et al, 1989) and the Sm protein-binding region, named 'domain A' (Branlant et al, 1982)), those 5S rRNA boxes are essential for transcription of 5S rRNA itself Bogenhagen, 1993;Hall, 2005). A full color version of this figure is available at the Heredity journal online.…”

“…For instance, the C2H2 zinc finger protein TFIIIA binds Xenopus 5S rRNA internal promoters (named Box A, intermediate element and Box C ), see Bogenhagen (1993) and Hall (2005). To analyze the occurrence of these essential boxes in our set of 5S rRNAs, we obtained consensus sequences for each block of 5S rRNAs taking into account the IUPAC coding system.…”

Probing the assembly of transcription initiation complexes through changes in σ N protease sensitivity

“…Both contacts between protein and ligand (30,32) and conformational changes in the proteins (34,35) have been inferred. We have used partial proteolysis of 32 P end-labeled N [labeled through introduction of a heart muscle protein kinase (HMK) site, ϪHMK] as a means of examining the interactions of with core, DNA, and in open complexes.…”

“…Partial proteolysis, or ''protease footprinting,'' has been used to detect protein-protein, protein-DNA, and proteinligand interactions (29)(30)(31)(32)(33). Both contacts between protein and ligand (30,32) and conformational changes in the proteins (34,35) have been inferred.…”