Structural studies on site-directed mutants of domain 3 of Xenopus laevis oocyte 5 S ribosomal RNA.

Base substitutions have been introduced into the highly conserved sequences of loops D and E within domain 3 of Xenopus laevis oocyte 5 S rRNA. The effects of these mutations on the solution structure of this 5 S rRNA have been studied by means of probing with nucleases, and with chemical reagents under native and semi-denaturing conditions. The data obtained with these mutants support the graphic model of Xenopus oocyte 5 S rRNA proposed by Westhof et al. In particular, our results rule out the existence of long-range base-pairing interactions between loop C and either loop D or loop E. The data also confirm that loops D and E in the wild-type 5 S RNA adopt unusual secondary structures and illustrate the importance of nucleotide sequence in the formation of intrinsic local loop conformations via non-canonical base-pairs and specific base-phosphate contacts. Consistent with this conclusion is our observation that the domain 3 fragment of Xenopus oocyte 5 S rRNA adopts the same conformation as the corresponding region in the full-length 5 S rRNA.

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

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.

A comparison of the solution structures and conformational properties of the somatic and oocyte 5S rRNAs of Xenopus laevis.

The secondary and tertiary structures of Xenopus oocyte and somatic 5S rRNAs were investigated using chemical and enzymatic probes. The accessibility of both RNAs towards single-strand specific nucleases (T1, T2, A and S1) and a helix-specific ribonuclease from cobra venom (RNase V1) was determined. The reactivity of nucleobase N7, N3 and N1 positions towards chemical probes was investigated under native (5 mM MgCl2, 100 mM KCl, 20 degrees C) and semi-denaturing (1 mM EDTA, 20 degrees C) conditions. Ethylnitrosourea was used to identify phosphates not reactive towards alkylation under native conditions. The results obtained confirm the presence of the five helical stems predicted by the consensus secondary structure model of 5S rRNA. The chemical reactivity data indicate that loops C and D are involved in a number of tertiary interactions, and loop E folds into an unusual secondary structure. A comparison of the data obtained for the two types of Xenopus 5S rRNA indicates that the conformations of the oocyte and somatic 5S rRNAs are very similar. However, the data obtained with nucleases under native conditions, and chemical probes under semi-denaturing conditions, reveal that helices III and IV in the somatic 5S rRNA are less stable than the same structures in oocyte 5S rRNA. Using chimeric 5S rRNAs, it was possible to demonstrate that the relative resistance of oocyte 5S rRNA to partial denaturation in 4 M urea is conferred by the five oocyte-specific nucleotide substitutions in loop B/helix III. In contrast, the superior stability of oocyte 5S rRNA in the presence of EDTA is related to a single C substitution at position 79.