Minor groove DNA-protein contacts upstream of a tRNA gene detected with a synthetic DNA binding ligand.

Transcription factor IIIB (TFIIIB) is composed of the TATA box binding protein (TBP) and class III gene-specific TBP-associated factors (TAFs). TFIIIB is brought to a site centered approximately 35 bp upstream from the transcription start site of tRNA genes via protein-protein interactions with the intragenic promoter-recognition factor TFIIIC. Since TBP interacts with TATA elements through the minor groove of DNA, we asked whether TFIIIB interacts with DNA in the minor groove. Polyamides containing pyrrole (Py) and imidazole (Im) amino acids are synthetic DNA ligands that bind to predetermined sequences in the minor groove of double helical DNA. These small molecules have been shown to interfere with protein-DNA interactions in the minor groove. A series of DNA constructs was generated in which the binding site for a Py-Im polyamide was placed at various distances upstream from a tRNA gene transcription start site. We find that a match polyamide will effectively inhibit tRNA gene transcription when its binding site is located within 33 bp of the transcription start site of the Xenopus TyrD tRNA gene. Moreover, in the presence of polyamide, RNA polymerase III is redirected to a new transcription initiation site located approximately one DNA helical turn downstream from the native start site. Our results suggest that a subunit of TFIIIB, possibly TBP, makes an essential minor groove DNA contact centered approximately 30 bp upstream from the tRNA gene.

Differential kinetics of transcription complex assembly distinguish oocyte and somatic 5S RNA genes of Xenopus.

Differential transcription of the Xenopus gene families encoding the oocyte and somatic 5S ribosomal RNAs can be reproduced in vitro with cell-free extracts prepared from Xenopus oocytes and unfertilized eggs. The transcriptional activities of these genes as assayed in these in vitro systems are a consequence of large differences in the rates of assembly of active transcription complexes. The somatic 5S genes sequester limiting transcription factors much more rapidly than the corresponding oocyte 5S genes and, as a consequence, are far more active. However, once transcription complexes are formed, these complexes are stable on both of these genes. Previous studies have established that transcription factors IIIA and IIIC are sufficient to form a stable protein-DNA complex on the somatic 5S gene. The rate of formation of the stable TFIIIA+C complex for the oocyte gene is far slower than that for the somatic 5S gene. Insertion of the DNA binding site for TFIIIC2 (the B-block promoter element from tRNA genes) into the 3' flanking region of a synthetic oocyte 5S gene increases the transcription efficiency and rate of transcription complex assembly of this gene relative to the parent gene lacking the B-block element. Our results support a model in which competition for limiting transcription factors plays a pivotal role in establishing differential transcription of the two classes of 5S genes during early embryogenesis.

TATA-box DNA binding activity and subunit composition for RNA polymerase III transcription factor IIIB from Xenopus laevis.

The RNA polymerase III transcription initiation factor TFIIIB contains the TATA-box-binding protein (TBP) and polymerase III-specific TBP-associated factors (TAFs). Previous studies have shown that DNA oligonucleotides containing the consensus TATA-box sequence inhibit polymerase III transcription, implying that the DNA binding domain of TBP is exposed in TFIIIB. We have investigated the TATA-box DNA binding activity of Xenopus TFIIIB, using transcription inhibition assays and a gel mobility shift assay. Gel shift competition assays with mutant and nonspecific DNAs demonstrate the specificity of the TFIIIB-TATA box DNA complex. The apparent dissociation constant for this protein-DNA interaction is approximately 0.4 nM, similar to the affinity of yeast TBP for the same sequence. TFIIIB transcriptional activity and TATA-box binding activity cofractionate during a series of four ion-exchange chromatographic steps, and reconstituted transcription reactions demonstrate that the TATA-box DNA-protein complex contains TFIIIB TAF activity. Polypeptides with apparent molecular masses of 75 and 92 kDa are associated with TBP in this complex. These polypeptides were renatured after elution from sodium dodecyl sulfate-gels and tested individually and in combination for TFIIIB TAF activity. Recombinant TBP along with protein fractions containing the 75- and 92-kDa polypeptides were sufficient to reconstitute TFIIIB transcriptional activity and DNA binding activity, suggesting that Xenopus TFIIIB is composed of TBP along with these polypeptides.

Assessment of major and minor groove DNA interactions by the zinc fingers of Xenopus transcription factor IIIA.

Zinc finger proteins of the Cys2His2 class are DNA sequence-specific transcription factors. Previous structural studies of zinc finger protein-DNA complexes have shown that amino acids in the finger tip and alpha-helix regions within individual finger domains make base-specific contacts with the major groove of DNA. The nine finger protein transcription factor IIIA (TFIIIA) from Xenopus oocytes binds a 43 base pair region of the 5S RNA gene through major groove interactions with two sets of three fingers (fingers 1-3 and 7-9) and with finger 5. Previous studies have suggested that zinc fingers 4 and 6 each bind in or across the minor groove to bridge these major groove-binding zinc fingers. Here it is shown that a polypeptide containing zinc fingers 1-5 (zf1-5) binds oligonucleotides with modifications in the major groove of the finger 4 binding site with wild-type affinity. Mutagenesis and binding site selection studies were performed to determine whether high affinity DNA binding by zf1-5 requires a particular sequence in the binding site for finger 4. Several mutations in this region of the 5S gene reduced the DNA-binding affinity of zf1-5; however, selection and amplification binding assays did not recover the wild-type finger 4 binding site sequence from a pool of mixed sequence oligonucleotides. Rather, a purine-rich sequence on the top strand was highly selected within the finger 4 binding site. We suggest that high affinity DNA binding by zinc finger 4 may be dictated by a sequence-specific DNA structure rather than by a unique DNA sequence. Deletion of finger 4 from zf1-5 results in a protein with poor binding affinity, demonstrating the importance of finger 4 in proper alignment of neighboring fingers with the DNA, and/or the importance of correct protein-protein interactions between fingers.

Repression of vertebrate RNA polymerase III transcription by DNA binding proteins located upstream from the transcription start site.

Derivatives of yeast tRNA and Xenopus tRNA and 5 S RNA genes have been constructed in which natural 5' flanking sequences have been replaced by the binding sites for either the yeast transcription activator protein GCN4 or the three amino-terminal zinc fingers of the Xenopus factor TFIIA (zf1-3). The binding sites for these proteins have been placed at various distances upstream from the start site for transcription initiation in the parent genes. Each of these plasmid DNAs is actively transcribed in both an unfractionated transcription extract prepared from unfertilized Xenopus eggs and in a reconstituted Xenopus transcription system. Binding of the test proteins to plasmid DNAs harboring the cognate binding sites severely represses transcription when these binding sites are located less than approximately 40 base-pairs upstream from the transcription start site. The DNA-binding proteins are without effect on the transcription of plasmids lacking binding sites or when the binding sites are located further upstream. Assembly of DNA templates into a complete transcription complex prior to addition of the DNA-binding proteins prevents repression. Proteins present in a fraction containing TFIIIB are necessary for this reversal of repression. These data suggest that vertebrate TFIIIB binds upstream from class III genes and this binding can be prevented by occlusion of the TFIIIB binding site by the test proteins GCN4 and zf1-3.

Interaction of the RNA binding fingers of Xenopus transcription factor IIIA with specific regions of 5 S ribosomal RNA.

Zinc fingers 4 to 7 of Xenopus transcription factor IIIA (TFIIIA) represent the minimal polypeptide necessary for high-affinity binding to 5 S RNA. Mutations covering the entire 5 S RNA structure have been compared for their effects on the binding affinity of full-length TFIIIA and a polypeptide consisting of fingers 4 to 7 of TFIIIA (zf4-7). In addition, ribonuclease footprinting was used to compare the binding sites of TFIIIA and zf4-7 on 5 S RNA. The consistency between the data obtained from these two approaches provided a clear indication that zinc fingers 4 to 7 of TFIIIA bind to a central core region on the 5 S RNA molecule consisting of loop B/helix II/loop A/helix V/region E. This information was used to design a truncated 75-nucleotide-long RNA molecule that retains high affinity for zf4-7. Therefore, we conclude that the specific interaction of TFIIIA with 5 S RNA can be represented by a complex formed between a four zinc finger polypeptide and a truncated 5 S RNA molecule.

Relative contributions of the zinc fingers of transcription factor IIIA to the energetics of DNA binding.

We have expressed and purified a series of recombinant zinc finger polypeptides derived from the cDNA for the Xenopus 5 S gene-specific transcription factor TFIIIA. Dissociation constants for the interaction of each of the truncated polypeptides with the 5 S gene promoter have been measured using gel mobility shift assays. DNase I footprinting and proteolysis experiments provide additional insights into the interactions of individual fingers within complexes of the truncated proteins. These results are discussed in terms of recently proposed models for the TFIIIA-DNA interaction. The effects of mutations in two of the strongly binding proteins, zf1-3 and zf1-7, on DNA binding affinity have been investigated. Mutations have been made both in putative DNA-contact residues and in the linker regions between zinc fingers. The observed decreases in binding affinity cannot be explained simply in terms of loss of protein-DNA contacts. Our results support a model in which DNA binding is accomplished through sets of interacting zinc fingers that make different energetic contributions to the overall binding of the protein and different contacts with the DNA.

Molecular basis for specific recognition of both RNA and DNA by a zinc finger protein.

Transcription factor IIIA (TFIIIA) from Xenopus oocytes binds both the internal control region of the 5S ribosomal RNA genes and the 5S RNA transcript itself. The nucleic acid binding domain of TFIIIA contains nine tandemly repeated zinc finger motifs. A series of precisely truncated forms of this protein have been constructed and assayed for 5S RNA and DNA binding. Different sets of zinc fingers were found to be responsible for high affinity interactions with RNA and with DNA. These results explain how a single protein can exhibit equal affinities for these two very different nucleic acids.