Defining the binding site of Xenopus transcription factor IIIA on 5S RNA using truncated and chimeric 5S RNA molecules

摘要

The Interaction of TFIIIA with deletion fragments of Xenopus 55 RNA has been quantified using a nitrocellulose filter binding assay. TFIIIA binding was found to be more sensitive to the deletion of nucleotldes from the 5' terminus of the 55 RNA as opposed to the 3' terminus. These effects have been correlated to the changes In RNA secondary structure resulting from the deletions. Nucleotldes 11 -108 of the Intact 5S RNA provide the necessary sequence and conformatlonal Information required for the binding of TFIIIA. Synthetic 5S RNA genes have been constructed so that in vttro transcription with T7 RNA polymerese 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 chimertc 5S RNA molecules Indicated that the Increased binding strength of somatic 55 RNA Is conferred by nucleotide substitutions In the 5' half of the molecule