5S RNA structure and interaction with transcription factor A. 2. Ribonuclease probe of the 7S particle from Xenopus laevis immature oocytes and RNA exchange properties of the 7S particle.
The 5S RNA complexed in the 7S particle of immature Xenopus laevis oocytes was 32P labeled at its 3' end and then subjected in situ to partial digestion using ribonucleases T1, T2, A, and V1 in order to study the conformation of the complexed RNA and its interaction with the transcription factor A (TFIIIA). Digested samples were gel purified to retrieve 5S RNA that was still complexed with the transcription factor protein, and the cleavages in these RNAs were analyzed on sequencing gels. The RNA associated with the 7S particle is very susceptible to ribonuclease activity despite the presence of the protein. Also, the 5S RNA in the 7S particle is in a different conformation from renatured Xenopus laevis (Xlo) 5S RNA and appears to have less secondary structure predominantly in the stem that includes helices IV and V. A species of native Xlo 5S RNA which was isolated from 7S particle preparations under nondenaturing conditions revealed a conformation that was more similar to the 5S RNA in the 7S particle than to renatured 5S RNA. Comparison of data from partial ribonuclease digestions performed on renatured 5S RNA, on the native 5S RNA, and on the complexed 5S RNA allowed us to approximate sites of protein-induced structural change in the complexed 5S RNA, which may signal protein interaction domains. These sites include the 5' side of helices III and V. In another approach to the study of 5S RNA-TFIIIA interactions, we have observed that incubation of 32P-labeled Xlo 5S RNA with 7S particles results in the incorporation of labeled RNA into 7S particles.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004722:Endoribonucleases / Q000378:metabolism; D005260:Female; D009690:Nucleic Acid Conformation; D009695:Nucleic Acid Renaturation; D009865:Oocytes / Q000032:analysis*; D012313:RNA / Q000378:metabolism*; D006163:Ribonuclease T1 / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism*; D015534:Trans-Activators; D014157:Transcription Factors / Q000378:metabolism*; D029867:Xenopus Proteins; D014982:Xenopus laevis
C044544:TFAM protein, Xenopus; D015534:Trans-Activators; D014157:Transcription Factors; C097078:XL-MTTFA protein, Xenopus; D029867:Xenopus Proteins; D012313:RNA; D004722:Endoribonucleases; D012260:Ribonucleases; C032825:ribonuclease V(1); C022543:ribonuclease T(2); D006163:Ribonuclease T1; D012259:Ribonuclease, Pancreatic