The primary structure of oocyte and somatic 5S rRNAs from the loach Misgurnus fossilis.

Somatic and oocyte 5S rRNAs from the liver and unfertilized eggs of the loach (Misgurnus fossilis have been sequenced and found to differ in six nucleotides. All the substitutions are confined to the 5'-half of the molecules; 4 of them are pyrimidine-pyrimidine substitutions, and 2 are purine-pyrimidine ones. Considerable differences, both in the position and the character of substitutions, have been established when these 5S rRNAs were compared with somatic and oocyte 5S rRNAs from Xenopus borealis and Xenopus laevis. Among the known primary structures, somatic 5S rRNA of M. fossilis is most similar to trout 5S rRNA.     

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.     

Secondary structure of mouse 28S rRNA and general model for the folding of the large rRNA in eukaryotes.

We present a secondary structure model for the entire sequence of mouse 28S rRNA (1) which is based on an extensive comparative analysis of the available eukaryotic sequences, i.e. yeast (2, 3), Physarum polycephalum (4), Xenopus laevis (5) and rat (6). It has been derived with close reference to the models previously proposed for yeast 26S rRNA (2) and for prokaryotic 23S rRNA (7-9). Examination of the recently published eukaryotic sequences confirms that all pro- and eukaryotic large rRNAs share a largely conserved secondary structure core, as already apparent from the previous analysis of yeast 26S rRNA (2). These new comparative data confirm most features of the yeast model (2). They also provide the basis for a few modifications and for new proposals which extend the boundaries of the common structural core (now representing about 85% of E. coli 23S rRNA length) and bring new insights for tracing the structural evolution, in higher eukaryotes, of the domains which have no prokaryotic equivalent and are inserted at specific locations within the common structural core of the large subunit rRNA.     

Oocyte and somatic 5S ribosomal RNA and 5S RNA encoding genes in Xenopus tropicalis.

We have investigated the structure of oocyte and somatic 5S ribosomal RNA and of 5S RNA encoding genes in Xenopus tropicalis. The sequences of the two 5S RNA families differ in four positions, but only one of these substitutions, a C to U transition in position 79 within the internal control region of the corresponding 5S RNA encoding genes, is a distinguishing characteristic of all Xenopus somatic and oocyte 5S RNAs characterized to date, including those from Xenopus laevis and Xenopus borealis. 5S RNA genes in Xenopus tropicalis are organized in clusters of multiple repeats of a 264 base pair unit; the structural and functional organization of the Xenopus tropicalis oocyte 5S gene is similar to the somatic but distinct from the oocyte 5S DNA in Xenopus laevis and Xenopus borealis. A comparative sequence analysis reveals the presence of a strictly conserved pentamer motif AAAGT in the 5'-flanking region of Xenopus 5S genes which we demonstrate in a separate communication to serve as a binding signal for an upstream stimulatory factor.     

The secondary structure of the protein L1 binding region of ribosomal 23S RNA. Homologies with putative secondary structures of the L11 mRNA and of a region of mitochondrial 16S rRNA.

An heterologous complex was formed between E. coli protein L1 and P. vulgaris 23S RNA. We determined the primary structure of the RNA region which remained associated with protein L1 after RNase digestion of this complex. We also identified the loci of this RNA region which are highly susceptible to T1, S1 and Naja oxiana nuclease digestions respectively. By comparison of these results with those previously obtained with the homologous regions of E. coli and B. stearothermophilus 23S RNAs, we postulate a general structure for the protein L1 binding region of bacterial 23S RNA. Both mouse and human mit 16S rRNAs and Xenopus laevis and Tetrahymena 28S rRNAs contain a sequence similar to the E. coli 23s RNS region preceding the L1 binding site. The region of mit 16S rRNA which follows this sequence has a potential secondary structure bearing common features with the L1-associated region of bacterial 23S rRNA. The 5'-end region of the L11 mRNA also has several sequence potential secondary structures displaying striking homologies with the protein L1 binding region of 23S rRNA and this probably explains how protein L1 functions as a translational repressor. One of the L11 mRNA putative structures bears the features common to both the L1-associated region of bacterial 23S rRNA and the corresponding region of mit 16S rRNA.