Comparison of the sequence and structure of transcription factor IIIA from Bufo americanus and Rana pipiens.

Amino acid (aa) sequences of transcription factor IIIA (TFIIIA) from the toad, Bufo americanus, and the grass frog, Rana pipiens, were determined by cDNA cloning and DNA sequencing. The 3'-untranslated regions of the cDNAs reveal that the TFIIIA gene polyadenylation signal is ATTAAA, rather than the conventional AATAAA. The B. americanus and R. pipiens proteins share about 60% aa sequence homology with each other and with Xenopus laevis TFIIIA. Although these results indicate that TFIIIA has more sequence variation than other DNA-binding proteins, a number of conserved features are evident and of likely functional significance. These include potential guanine nucleotide-binding sites at arginines in zinc fingers (ZnF) II, V, and IX, acidic residues between metal-coordinating cysteines, and a basic region in the C-terminal tail possibly involved in transcription promotion. Sequence similarity also exists in an aa stretch bridging the ninth ZnF and C-terminal tail of both TFIIIA and the 5S RNA-binding protein, p43. DNase I protection analyses demonstrate that B. americanus and R. pipiens TFIIIA interact with the internal control region (ICR) of the Xenopus borealis 5S RNA-encoding gene (5S) in different manners: the B. americanus interaction is similar to X. laevis TFIIIA, protecting the entire 5S gene ICR (nt +96 to +43) from DNase I digestion, whereas the R. pipiens TFIIIA strongly protects the ICR from nt +96 up to +78 and less strongly from +78 to +43. Possibly accounting for the binding differences observed, R. pipiens and R. catesbeiana oocyte 5S RNAs (and by inference 5S genes) were found to contain a G or U at nt position 50 while B. americanus, X. laevis, and other eukaryotic 5S RNAs have an A in the analogous position (nt 53 in generalized eukaryotic structure).

Sequence variation in transcription factor IIIA.

Previous studies characterized macromolecular differences between Xenopus and Rana transcription factor IIIA (TFIIIA) (Gaskins et al., 1989, Nucl. Acids Res. 17, 781-794). In the present study, cDNAs for TFIIIA from Xenopus borealis and Rana catesbeiana (American bullfrog) were cloned and sequenced in order to gain molecular insight into the structure, function, and species variation of TFIIIA and the TFIIIA-type zinc finger. X. borealis and R. catesbeiana TFIIIAs have 339 and 335 amino acids respectively, 5 and 9 fewer than X. laevis TFIIIA. X. borealis TFIIIA exhibited 84% sequence homology (55 amino acid differences) with X. laevis TFIIIA and R. catesbeiana TFIIIA exhibited 63% homology (128 amino acid changes) with X. laevis TFIIIA. This sequence variation is not random; the C-terminal halves of these TFIIIAs contain substantially more non-conservative changes than the N-terminal halves. In particular, the N-terminal region of TFIIIA (that region forming strong DNA contacts) is the most conserved and the C-terminal tail (that region involved in transcription promotion) the most divergent. Hydropathy analyses of these sequences revealed zinc finger periodicity in the N-terminal halves, extreme hydrophilicity in the C-terminal halves, and a different C-terminal tail hydropathy for R. catesbeiana TFIIIA. Although considerable sequence variation exists in these TFIIIA zinc fingers, the Cys/His, Tyr/Phe and Leu residues are strictly conserved between X. laevis and X. borealis. Strict conservation of only the Cys/His motif is observed between X. laevis and R. catesbeiana TFIIIA. Overall, Cys/His zinc fingers in TFIIIA are much less conserved than Cys/Cys fingers in erythroid transcription factor (Eryf 1) and also less conserved than homeo box domains in segmentation genes. The collective evidence indicates that TFIIIA evolved from a common precursor containing up to 12 finger domains which subsequently evolved at different rates.

Zinc finger structure of a ribosomal gene-specific transcription factor.

Xenopus transcription factor IIIA (TFIIIA) or TFIIIA mutants with internal deletions were expressed in E.coli, isolated from E.coli cell extracts, and identified by SDS PAGE and immunoblotting with rabbit antiserum against native TFIIIA. Specific DNA binding of intact or internally deleted TFIIIA was compared by analyzing their abilities to protect the internal control region (ICR) of the Xenopus 5S ribosomal RNA gene from DNase I digestion. Intact protein bound specifically to the entire ICR (+96 to +43). One TFIIIA deletion mutant, expressed from cDNA lacking the coding sequence for the putative fourth zinc finger protected the ICR from DNase I digestion from nucleotide positions +96 to +78. A second TFIIIA mutant resulting from fusion of putative zinc fingers 7 and 8 protected the 5S gene ICR from positions +96 to +63. The regions of the protein comprising the N-terminal 3 fingers and N-terminal six fingers appear to be in contact with approximately 18 and 33 bp of DNA respectively on the 3' side of the 5S gene ICR.

Internal deletion mutants of Xenopus transcription factor IIIA.

Xenopus transcription factor IIIA (TFIIIA) or TFIIIA mutants with internal deletions were expressed in E. coli utilizing a bacteriophage T7 RNA polymerase system. TFIIIA or deletion mutant TFIIIAs, isolated from E.coli cell extracts, were identified by SDS PAGE and immunoblotting with rabbit antiserum against native TFIIIA purified from Xenopus immature oocytes. Specific DNA binding of intact or internally deleted TFIIIA was compared by analyzing their abilities to protect the internal control gene (ICR) of the Xenopus 5S RNA gene from DNase I digestion. Intact protein, synthesized from a full-length TFIIIA cDNA, bound specifically to the entire ICR (+96 to +43) and promoted 5S RNA gene transcription in vitro. One TFIIIA deletion mutant, expressed from cDNA lacking the coding sequence for the putative fourth zinc finger (designated from the N-terminus, amino acids 103-132) protected the ICR from DNase I digestion from nucleotide positions +96 to +78. A second TFIIIA mutant resulting from fusion of putative zinc fingers 7 and 8 (deletion of amino acids 200-224) protected the 5S gene ICR from positions +96 to +63. The DNase I protection patterns of these mutant proteins are consistent with the formation of strong ICR contacts by those regions of the protein on the N-terminal side of the mutation but not by those regions on the C-terminal side of the mutation. The regions of the protein comprising the N-terminal 3 fingers and N-terminal six fingers appear to be in contact with approximately 18 and 33 bp of DNA respectively on the 3' side of the 5S gene ICR. These internal deletion mutants promoted 5S RNA synthesis in vitro and DNA renaturation.

Conformation states of Xenopus transcription factor IIIA.

The conformation of Xenopus transcription factor IIIA (TFIIIA) free in solution, bound to 5S RNA in the 7S particle, depleted of zinc, or bound to plasmid DNA was analyzed by (1) trypsin digestion and electrophoretic analysis of proteolytic fragments or (2) measurement of the fluorescence of TFIIIA mildly derivatized with N-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonic acid (IAEDANS). TFIIIA free or complexed with 5S RNA has a similar conformation as judged (a) by trypsin-dependent generation of similar metastable 20-kDa domains (corresponding to the N-terminal half of the protein) or (b) by the negligible change in AEDANS-TFIIIA fluorescence when free or bound to 5S RNA. When TFIIIA binds plasmid DNA, its N-terminal half becomes hypersensitive to trypsin digestion, indicating a structural change in this region of the protein upon interaction with DNA. Quenching of AEDANS-TFIIIA fluorescence is observed upon interaction of the protein with plasmid DNA, a result also indicative of a conformational change upon protein-DNA interaction. Removal of zinc from TFIIIA by EDTA chelation results in (a) increased proteolysis of this 20-kDa domain, indicating a structural change in the N-terminal half of the protein upon zinc removal, and (b) large enhancement of AEDANS-TFIIIA fluorescence. EDTA chelation of TFIIIA bound to 5S RNA in the 7S particle, a procedure which does not deplete all zinc from the protein, neither increases the trypsin sensitivity of the 20-kDa domain nor alters appreciably the fluorescence of AEDANS-TFIIIA. These results indicate that zinc is involved in maintaining the native conformation of at least the N-terminal half of the protein.

Species variation in transcription factor IIIA.

Species variation in transcription factor IIIA (TFIIIA) was examined by comparing the abilities of TFIIIAs isolated from different Xenopus and Rana species to 1) bind rabbit anti-Xenopus laevis TFIIIA IgG, 2) specifically interact with the Xenopus borealis somatic 5S RNA gene, and 3) promote transcription of the Xenopus borealis 5S RNA gene in vitro. In immunoblot assays, Rana catesbeiana or Rana pipiens TFIIIA did not react readily with rabbit anti-Xenopus laevis TFIIIA IgG (assayed with anti-rabbit F(ab')2 fragment conjugated with alkaline phosphatase) whereas Xenopus borealis TFIIIA exhibited similar reactivity with this IgG as Xenopus laevis TFIIIA. When compared to Xenopus TFIIIAs, Rana TFIIIAs exhibited similar interactions with the 3' portion of the intragenic control region of the Xenopus 5S RNA gene (to residue +78 on the coding strand and up to and including +74 on the non-coding strand, nucleotides protected from DNase I digestion by the N-terminal half of Xenopus TFIIIA) and incomplete interactions with the remaining 5' portion of the control region (nucleotides protected from DNase I digestion by the C-terminal half of Xenopus TFIIIA). In a Xenopus laevis unfertilized egg extract, Rana catesbeiana and Rana pipiens TFIIIAs promoted transcription of the Xenopus borealis somatic 5S RNA gene less efficiently than Xenopus laevis and Xenopus borealis TFIIIAs.