Defining the binding site ofXenopustranscription factor IIIA on 5S RNA using truncated and chimeric 5S RNA molecules

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Abstract
The interaction of TFIIIA with deletion fragments of Xenopus 5S RNA has been quantified using a nitrocellulose filter binding assay. TFIIIA binding was found to be more sensitive to the deletion of nucleotides from the 5' terminus of the 5S RNA as opposed to the 3' terminus. These effects have been correlated to the changes in RNA secondary structure resulting from the deletions. Nucleotides 11-108 of the intact 5S RNA provide the necessary sequence and conformational information required for the binding of TFIIIA. Synthetic 5S RNA genes have been constructed so that in vitro transcription with T7 RNA polymerase yields mature 5S RNA. The transcription factor has a higher affinity for somatic vs. oocyte 5S RNA, similar to the differential affinity of TFIIIA for the two genes. Binding studies with chimeric 5S RNA molecules indicated that the increased binding strength of somatic 5S RNA is conferred by nucleotide substitutions in the 5' half of the molecule.