Romaniuk P J, de Stevenson I L, Wong H H
Nucleic Acids Res. 1987 Mar 25;15(6):2737-55. doi: 10.1093/nar/15.6.2737.
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.
已使用硝酸纤维素滤膜结合试验对TFIIIA与非洲爪蟾5S RNA缺失片段的相互作用进行了定量分析。结果发现，与3'末端相比，TFIIIA结合对5S RNA 5'末端核苷酸的缺失更为敏感。这些效应与缺失导致的RNA二级结构变化相关。完整5S RNA的核苷酸11 - 108提供了TFIIIA结合所需的必要序列和构象信息。已构建合成5S RNA基因，以便用T7 RNA聚合酶进行体外转录可产生成熟的5S RNA。该转录因子对体细胞5S RNA的亲和力高于卵母细胞5S RNA，类似于TFIIIA对这两个基因的差异亲和力。对嵌合5S RNA分子的结合研究表明，体细胞5S RNA结合强度的增加是由分子5'端一半的核苷酸取代所致。
