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

Abstract

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.

A 3' exonuclease activity degrades the pseudogene 5S RNA transcript and processes the major oocyte 5S RNA transcript in Xenopus oocytes.

“…Although no eukaryotic 3' exonuclease has been purified yet, a number of prokaryotic RNases are known to act in such a manner (Deutscher 1985). The 142-base-long major oocyte 5S RNA precursor is folded in Figure 8a in the characteristic and universal 5S RNA secondary structure (see, Romaniuk et al 1988;Wolters and Erdmann 1988). In this folding pattern, the 22-baselong overhanging tail is a good substrate for a 3' exonuclease, and the 9-bp helix formed by the complementary 5' and 3' ends of the mature 5S RNA is a good candidate for the structure that prevents further RNA breakdown.…”

SeqFold: Genome-scale reconstruction of RNA secondary structure integrating high-throughput sequencing data

“…Precise mapping of the RNA structure is essential for understanding the functions of RNAs, especially for the large set of functionally uncharacterized noncoding RNAs (ncRNAs) (Wan et al 2011). Experimental methods for RNA structure determination include X-ray crystallography (Guo et al 2004), NMR (Latham et al 2005), cryo-electron microscopy (Mueller et al 2000), and chemical and enzymatic probing (Romaniuk et al 1988;Brenowitz et al 2002;Alkemar and Nygard 2006;Das et al 2008;Mitra et al 2008). Although quite accurate, these methods are traditionally only applicable to analyze a single RNA per experiment and limited in the length of the probed RNA.…”

Analysis of the Binding of Xenopus Transcription Factor IIIA to Oocyte 5 S rRNA and to the 5 S rRNA Gene

“…Helix IV contains a U:U mismatch and a bulged adenosine flanked by a G:U pair that could be potentially utilized by TFIIIA. Experiments with chemical and enzymatic probes indicate that the bulged nucleotide at position 83 is external to helix IV, as are the other bulged nucleotides at positions 49, 50, and 63 (8,55). Additionally, the metal complex Rh(phen) 2 (phi) 3ϩ , which targets widened major grooves in RNA helices such as occurs at base triples and mismatched pairs, does not cleave at A 83 (56).…”

“…The N3 positions of both U 80 and U 96 do not react with CMCT (8,55), suggesting a 2-carbonyl-N3, 4-carbonyl-N3 mismatched pair between these two bases. Cleavage by Rh(phen) 2 (phi) 3ϩ at positions U 80 and G 81 establishes that the major groove is accessible at the site of this mismatch (56).…”

“…Changes in the DNase I cleavage patterns of several mutants relative to the wild type gene exemplify the potential importance of sequence-generated conformation. For example, inverting the G:C pair at position 81 mitigates the strong cutting that normally occurs at this site, while changing T 55 to C abolishes the internal hypersensitive site on the coding strand at position 51 (Fig. 4).…”