JMB—MS 442 Cust. Ref. No. PEW 147/94 [SGML] J. Mol. Biol. (1995) 248, 44–57 Interaction of the RNA Binding Fingers of Xenopus Transcription Factor IIIA with Specific Regions of 5 S Ribosomal RNA Steven J. McBryant 1 , Nik Veldhoen 2 , Ben Gedulin 1 , Anne Leresche 1 Mark P. Foster 1 , Peter E. Wright 1 , Paul J. Romaniuk 2 * and Joel M. Gottesfeld 1 * Zinc fingers 4 to 7 of Xenopus transcription factor IIIA (TFIIIA) represent the 1 Department of Molecular Biology, The Scripps Research minimal polypeptide necessary for high-affinity binding to 5 S RNA. Mutations covering the entire 5 S RNA structure have been compared for Institute, 10666 North Torrey their effects on the binding affinity of full-length TFIIIA and a polypeptide Pines Road, La Jolla consisting of fingers 4 to 7 of TFIIIA (zf4-7). In addition, ribonuclease CA 92037, U.S.A. footprinting was used to compare the binding sites of TFIIIA and zf4-7 2 Department of Biochemistry on 5 S RNA. The consistency between the data obtained from these and Microbiology, University two approaches provided a clear indication that zinc fingers 4 to 7 of TFIIIA of Victoria, PO Box 3055 bind to a central core region on the 5 S RNA molecule consisting of loop Victoria, British Columbia B/helix II/loop A/helix V/region E. This information was used to design Canada V8W 3P6 a truncated 75-nucleotide-long RNA moleculer 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. Keywords: TFIIIA; 5 S RNA; RNA; protein; zinc finger protein *Corresponding authors Introduction In addition to its role as a positive transcription factor for the expression of 5 S ribosomal RNA (5 S RNA) genes, Xenopus transcription factor IIIA (TFIIIA) performs a second essential function by binding to 5 S RNA in the cytoplasm of immature oocytes (Picard & Wegnez, 1979; Honda & Roeder, 1980). The resulting ribonucleoprotein storage particle (7 S RNP) stabilizes the RNA until it is required for ribosome assembly. The nucleic acid binding domain of TFIIIA consists of nine zinc fingers (Brown et al ., 1985; Miller et al ., 1985) and there has been considerable interest in determining how this protein interacts specifically with the coding region of the 5 S RNA gene, and with the transcript of this gene. The binding site for TFIIIA on the 5 S RNA gene spans a 50 base-pair region within the coding sequence of the gene. This internal control region (ICR) is organized into box A and box C elements at opposite ends with a small intermediate element in the middle (Bogenhagen et al ., 1980; Engelke et al ., 1980; Sakonju et al ., 1980; Sakonju & Brown, 1982; Pieler et al ., 1985, 1987; You et al ., 1991; Veldhoen et al ., 1994). Nucleotide sequences within these elements in some cases are critical for TFIIIA binding, and in others facilitate the assembly of a complete preinitiation complex of factors on the 5 S RNA gene (Bogenhagen, 1985; Majowski et al ., 1987; McConkey & Bogenhagen, 1987; You et al ., 1991; Keller et al ., 1992; Veldhoen et al ., 1994). It has recently been demonstrated that the first three amino-terminal fingers of TFIIIA constitute the minimum polypep- tide necessary for high-affinity binding to the 5 S RNA gene, and that these three fingers specifically interact with the box C element of the ICR (Christensen et al ., 1991; Darby & Joho, 1992; Liao et al ., 1992; Bogenhagen, 1993). The effects of scanning and point mutagenesis of the ICR on TFIIIA binding affinity support this result: mutations in box C result in large decreases in TFIIIA binding affinity, while mutations in the intermediate element or box A have only small effects on TFIIIA binding affinity (Pieler et al ., 1987; Sands & Bogenhagen, 1987; You et al ., 1991; Veldhoen et al ., 1994). Other studies have provided information on the location of fingers 4 to 9 of TFIIIA within the ICR on 5 S DNA (Fairall et al ., 1986; Vrana et al ., 1988; Churchill et al ., 1990; Clemens et al ., 1992; Hayes & Clemens, 1992; Hayes & Tullius, Abbreviations used: TFIIIA, Xenopus transcription factor IIIA; ICR, internal control region; zf, zinc finger; PCR, polymerase chain reaction. 0022–2836/95/160044–14 $08.00/0  1995 Academic Press Limited