Binding of TFIIIA to derivatives of 5S RNA containing sequence substitutions or deletions defines a minimal TFIIIA binding site

The repetitive zinc finger domain of transcription factor IIIA binds 5S DNA and 5S RNA with similar affinity. Site directed mutagenesis of the Xenopus borealis somatic 5S RNA gene has been used to produce a series of derivatives of 5S RNA containing local sequence substitutions or sequence deletions. Gel mobility shift analyses of the binding of TFIIIA to these altered 5S RNAs revealed that all three of the helical stems of the 5S RNA secondary structure are required for binding. TFIIIA was observed to bind with normal affinity to RNAs lacking 12 nucleotides at either the loop c or loop e/hellx V regions of 5S RNA, as well as to a double mutant containing both deletions. The secondary structure of the resulting 96-nucleotide RNA, studied using structure-specific ribonucleases, was found to resemble the central portion of 5S RNA.