Abstract
Transcription factor (TF) IIIA, which contains nine zinc finger motifs, binds to the internal control region of the 5S RNA gene as the first step in the assembly of a multifactor complex that promotes accurate initiation of transcription by RNA polymerase III. We have monitored the interaction of wild-type and truncated forms of yeast TFIIIA with the 5 S RNA gene. The DNase I footprints obtained with full-length TFIIIA and a polypeptide containing the amino-terminal five zinc fingers (TF5) were indistinguishable, extending from nucleotides +64 to +99 of the 5 S RNA gene. This suggests that fingers 6 through 9 of yeast TFIIIA are not in tight association with DNA. The DNase I footprint obtained with a polypeptide containing the amino-terminal four zinc fingers (TF4) was 14 base pairs shorter than that of TF5, extending from nucleotides +78 to +99 on the nontranscribed strand and from nucleotides +79 to +98 on the transcribed strand of the 5 S RNA gene. Protection provided by a polypeptide containing the first three zinc fingers (TF3) was similar to that provided by TF4, with the exception that protection on the nontranscribed strand ended at nucleotide +80, rather than nucleotide +78. Methylation protection analysis indicated that finger 5 makes major groove contacts with guanines +73 and +74. The amino-terminal four zinc fingers make contacts that span the internal control region, which extends from nucleotides +81 to +94 of the 5 S RNA gene, with finger 4 appearing to contact guanine +82. Measurements of the apparent Kd values of the TFIIIA.DNA complexes indicated that the amino-terminal three zinc fingers of TFIIIA have a binding energy that is similar to that of the full-length protein.
Highlights
Transcription factor (TF)1 IIIA, a sequence-specific DNAbinding protein, binds to the 5 S RNA gene as the first step in the assembly of a multifactor transcription complex
Using wild-type and truncated forms of TFIIIA that had been purified from bacteria, we studied the interaction of the protein with the 5 S RNA gene using DNase I and dimethyl sulfate as probes
Studies of the Xenopus TFIIIA-DNA interaction (Clemens et al, 1992; Fairall and Rhodes, 1992; Hayes and Clemens, 1992; Hayes and Tullius, 1992; Del Rio et al, 1993) suggest that the carboxyl-terminal three fingers of Xenopus TFIIIA wrap around the major groove of the DNA helix at the 5Ј-end of the amphibian internal control region (ICR) and immediately upstream of this element
Summary
Plasmids for Bacterial Expression of Wild-type and Truncated Forms of Yeast TFIIIA—A plasmid for bacterial expression of wild-type TFIIIA was obtained by inserting the 2.3-kilobase pair NcoI-BamHI fragment of pJA454 (Archambault et al, 1992) between the corresponding sites of pET-11d (Studier et al, 1990), which places the coding region of TFIIIA under the control of a bacteriophage T7 RNA polymerase promoter. The NcoI-BamHI fragment of each of the resultant plasmids, containing TFIIIA-coding sequence, was subcloned between the corresponding sites of the pET-11d expression vector. This cloning procedure introduced the non-TFIIIA, polylinker-encoded amino acids EFLQPLAS, SNSCSP, LISNSCSP, and SNSCSP at the carboxyl terminus of TF8, TF7*, TF4*, and TF3*, respectively. After end filling of the digested DNA with the Klenow form of DNA polymerase I in the presence of [␣-32P]dCTP, the fragment containing the 5 S RNA gene was purified by gel electrophoresis and recovered by electroelution. Methylation Protection Analysis—Standard 20-l gel retardation reactions containing 2.5 ng of uniquely end-labeled 5 S DNA (see above) were set up in triplicate and incubated with protein for 5 min at room temperature. Quantification of relative intensities of bands was carried out using IPLab Gel software (Signal Analytics Corp.) to analyze phosphorimages of dried gels obtained with a Molecular Dynamics PhosphorImager
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