Identical 3′-terminal octanucleotide sequence in 18S ribosomal ribonucleic acid from different eukaryotes. A proposed role for this sequence in the recognition of terminator codons

The 3'-terminal sequence of 18S ribosomal RNA from Drosophila melanogaster and Saccharomyces cerevisiae was determined by stepwise degradation from the 3'-terminus and labelling with [(3)H]isoniazid. The sequence G-A-U-C-A-U-U-A(OH) was found at the 3'-terminus of both 18S rRNA species. Less extensive data for 18S RNA from a number of other eukaryotes are consistent with the same 3'-terminal sequence, and an identical sequence has previously been reported for the 3'-end of rabbit reticulocyte 18S rRNA (Hunt, 1970). These results suggest that the base sequence in this region is strongly conserved and may be identical in all eukaryotes. As the 3'-terminal hexanucleotide is complementary to eukaryotic terminator codons we discuss the possibility that the 3'-end of 18S rRNA may have a direct base-pairing role in the termination of protein synthesis.     

Studies on the 3′-terminal sequences of the large ribosomal ribonucleic acid of different eukaryotes and those associated with `hidden'' breaks in heat-dissociable insect 26S ribonucleic acid

The 3'-terminal sequences associated with the large rRNA complex from a range of eukaryotes were determined after pancreatic or T(1)-ribonuclease digestion of RNA terminally labelled with [(3)H]isoniazid. In all higher eukaryotes examined except Drosophila melanogaster, the 3'-terminal sequences Y-G-U(OH) and G-C-U(OH) were demonstrated for the large RNA component(s) and for 6S RNA respectively. The 3'-terminal sequence of Saccharomyces cerevisiae 26S RNA was Y-G-U(OH) and that of 6S RNA Y-A-U-U-U(OH). Three 3'-terminal sequences were found in equimolar amounts in the heat-dissociable 26S rRNA characteristic of insect ribosomes. These were Y-G-U-G-U(OH), Y-C-G-U(OH) and G-C-U(OH) for cultured Antheraea eucalypti cells, Y-G-U(OH), Y-G-U(OH) and G-C-U(OH) for Galleria mellonella larvae and Y-C-G-A(OH), Y-G-U-A(OH) and G-Y-U-G(OH) for Drosophila melanogaster flies. Thus the introduction of the central scission in insect 26S rRNA results in the generation of a unique 3'-terminus and does not arise from random cleavage of the polynucleotide chain.     

Isolation from rat liver and sequence of a RNA fragment containing 32 nucleotides from position 5 to 36 from the 3'' end of ribosomal 18S RNA.

Crude tRNA isolated from rat liver by the method of Rogg et al. (Biochem. Biophys. Acta 195, 13-15 1969) contains N6-dimethyladenosine (m6-2A) and was therefore fractionated in order to identify the m6-2A-containing RNAs. A unique species of RNA was purified which contained all the m62A present in the crude tRNA. Sequence analysis by postlabeling with gamma-32p-ATP and polynucleotide kinase revealed that this RNA represents the 32 nucleotides AAGGUUUC(C)U GUAGGUGm62Am62ACCUGCGGAAGGAUC from position 5 to 36 of the 3' terminus of ribosomal 18S RNA. The 36 nucleotide long sequence from the 3' end of rat liver 18S rRNA exhibits extensive homology with the corresponding sequence of E. coli 16S rRNA and with the 21 nucleotide long 3' terminal sequence so far known from Saccharomyces carlsbergensis 17S rRNA. A heterogeneity in this sequence provides the first evidence on the molecular level for the existence of (at least) two sets of redundant ribosomal 18S RNA genes in the rat.     

Electron microscopy of secondary structure in partially denatured precursor and mature Escherichia coli 16 S and 23 S rRNA

The secondary structure of 16 S and 23 s rRNA sequences in 30 S preribosomal RNA of Escherichia coli was analyzed by electron microscopy after partial denaturation and compared to mature 16 S and 23 S rRNA examined under the same conditions. The sequences in the pre-rRNA notably lack the specific loops that dominate the 5'-terminal regions of mature 16 S and 23 S rRNA. In other respects, the sizes and locations of loops in the 23 S rRNA sequence are qualitatively very similar in mature and pre-rRNA. Eleven of 12 loops outside of the 5'-terminal domain correspond, with the most frequent features in the 3'-half of the molecule. In contrast, the sizes and locations of loops in the 16 S rRNA sequence differ between precursor and mature forms. In the pre-rRNA, instead of the 370-nucleotide 5'-terminal loop of mature rRNA, some 1000-nucleotide terminal loops are observed. The pre-rRNA also shows a frequent 610-nucleotide central loop and a large 1240-nucleotide loop not seen in the mature rRNA. Also, in the 3'-region of the sequence, the largest loops in pre-rRNA are 120 nucleotides shorter than in mature rRNA. We suggest that the structure of pre-rRNA may promote some alternate conformational features, and that these could be important during ribosome formation or function.     

5' -and 3'-terminal nucleotide sequences of Tetrahymena pyriformis 17S rRNA.

Ribonuclease T1 oligonucleotides arising from the 5' and 3' termini of the 17S rRNA of Tetrahymena pyriformis were isolated by the diagonal method of Dahlberg (Dahlberg, J. E. (1968), Nature (London) 220, 548), and their nucleotide sequences were determined. The base sequence of the 3'-terminal fragment is (G)AUCAUUAoh, which is identical to that found in other 17S-18S eucaryotic rRNA species. The nucleotide sequence of the 5'-terminal oligonucleotide is pAACCUGp, which is identical in length to that found in other eucaryotes and shows a partial but significant sequence homology to the 5' RNase TI oligonucleotides isolated from other eucaryotic species.     

Intermolecular hybridization of 5S rRNA with 18S rRNA: identification of a 5'-terminally-located nucleotide sequence in mouse 5S rRNA which base-pairs with two specific complementary sequences in 18S rRNA.

Eukaryotic 5S rRNA hybridizes specifically with 18S rRNA in vitro to form a stable intermolecular RNA:RNA hybrid. We have used 5S rRNA/18S rRNA fragment hybridization studies coupled with ribonuclease digestion and primer extension/chain termination analysis of 5S rRNA:18S rRNA hybrids to more completely map those mouse 5S rRNA and 18S rRNA sequences responsible for duplex formation. Fragment hybridization analysis has defined a 5'-terminal region of 5S rRNA (nucleotides 6-27) which base-pairs with two independent sequences in 18S rRNA designated Regions 1 (nucleotides 1157-1180) and 2 (nucleotides 1324-1339). Ribonuclease digestion of isolated 5S rRNA:18S rRNA hybrids with both single-strand- and double-strand-specific nucleases supports the involvement of this 5'-terminal 5S rRNA sequence in 18S rRNA hybridization. Primer extension/chain termination analysis of isolated 5S rRNA:18S rRNA hybrids confirms the base-pairing of 5S rRNA to the designated Regions 1 and 2 of 18S rRNA. Using these results, 5S rRNA:18S rRNA intermolecular hybrid structures are proposed. Comparative sequence analysis revealed the conservation of these hybrid structures in higher eukaryotes and the same but smaller core hybrid structures in lower eukaryotes and prokaryotes. This suggests that the 5S rRNA:16S/18S rRNA hybrids have been conserved in evolution for ribosome function.     

Discrete poly(A)-RNA species from rat liver mitochondria are fragments of 16S mitochondrial rRNA carrying its 5′-termini

During electrophoresis in polyacrylamide gels containing 7M urea the major discrete components of preparations of rat liver mitochondrial poly(A)+ and poly(A)- RNA species have similar mobilities. Poly(A)- RNA components hybridize to the 16S rRNA gene of mtDNA. Analysis of 5'-terminal sequences of these components revealed their identity to the 5'-terminal sequence of 16S rRNA. These results show that poly(A)- RNA components are fragmentation products of 16S rRNA. Fragmentation occurs nonrandomly from the 3'-end of the original rRNA molecules and lead to formation of products with electrophoretic mobilities similar to those of poly(A)+ RNA components.     

The 3'-terminal primary structure of five eukaryotic 18S rRNAs determined by the direct chemical method of sequencing. The highly conserved sequences include an invariant region complementary to eukaryotic 5S rRNA.

The 3'-terminal sequences of 18S rRNA from chicken reticulocyte, mouse sarcoma, rat liver, rabbit reticulocyte and barley embryo were determined by the direct chemical sequencing method. The regions sequenced show complete homology for the first 73 nucleotides. A sequence 5'-proximal to the m6(2)Am6(2)A residues that is complementary to eukaryotic 5S RNAs is totally conserved. This supports the hypothesis that base-paired interaction between 5S and 18S rRNA, which are present in the large and small ribosomal subunits respectively, may be involved in the reversible association of ribosomal subunits during protein synthesis.     

Sequence analysis and in vitro maturation of five precursor 5S RNAs from Bacillus Q.

Bacillus Q, which is closely related to B. subtilis, contains at least six different precursors of 5S rRNA. The complete nucleotide sequences of four of these precursors, as well as the major part of the sequence of a fifth one, have been determined. They all contain the same 5'-terminal non-conserved segment which is to a large degree homologous with the corresponding segment of the B. subtilis p5S RNAs (Sogin, M.L., Pace, N.R., Rosenberg, M., Weissman, S.M. (1976) J. Biol. Chem. 251, 3480-3488). On the other hand the 3'-terminal non-conserved sequences of the various Bacillus Q precursors show considerable differences both in length and in nucleotide sequence, while there is also little or no homology with the 3'-terminal non-conserved sequence of the B. subtilis precursors. Bacillus Q p5S RNAs do not possess tetranucleotide repeats around the sites which are cleaved during maturation, as does B. subtilis p5S RNA. Like in B. subtilis, however, the cleavage sites are contained within a double-helical region of the precursor molecules. Crude RNAse M5 isolated from various Bacillus strains can maturate the Bacillus Q p5S RNAs with high efficiency. Despite considerable differences in primary structure between the precursors from the various strains, each RNAs M5 preparation can maturate all these precursors with about the same efficiency.     

Probable involvement of 3'-terminal segment of 18S rRNA in translation initiation of uncapped mRNAs in plants

A possibility of involvement of 3'-terminal 18S rRNA segment in the cap-independent initiation of translation on plant ribosomes was studied. It was shown that 3-terminal segment (nucleotides 1777-1811) of 18S rRNA including the last hairpin 45 is accessible for complementary interactions in 40S ribosomal subunits. Oligonucleotides complementary to this segment of rRNA when added to wheat germ cell-free protein synthesizing system were found to specifically inhibit translation of uncapped reporter mRNA coding for beta-glucuronidase, which bears in the 5'-untranslated region (UTR) a leader sequence of potato virus Y (PVY) genomic RNA possessing fragments complementary to the region 1777-1811. It was shown that a sequence corresponding to nucleotides 291-316 of PVY, which is complementary to a major portion of the 3-terminal 18S rRNA segment 1777-1808, when placed into 5'-UTR, is able to enhance translational efficiency of the reporter mRNAs. The results obtained suggest that complementary interactions between mRNA 5'-UTR and 18S rRNA 3'-terminal segment can take place in the course of cap-independent translation initiation.     

The 3''-terminal region of bacterial 23S ribosomal RNA: structure and homology with the 3''-terminal region of eukaryotic 28S rRNA and with chloroplast 4.5s rRNA.

The sequence of the 110 nucleotide fragment located at the 3'-end of E.coli, P.vulgaris and A.punctata 23S rRNAs has been determined. The homology between the E.coli and P.vulgaris fragments is 90%, whereas that between the E.coli and A.punctate fragments is only 60%. The three rRNA fragments have sequences compatible with a secondary structure consisting of two hairpins. Using chemical and enzymatic methods recently developed for the study of the secondary structure of RNA, we demonstrated that one of these hairpins and part of the other are actually present in the three 3'-terminal fragments in solution. This supports the existence of these two hairpins in the intact molecule. Indeed, results obtained upon limited digestion of intact 23S RNA with T1 RNase were in good agreement with the existence of these two hairpins. We observed that the primary structures of the 3'-terminal regions of yeast 26S rRNA and X.laevis 28S rRNA are both compatible with a secondary structure similar to that found at the 3'-end of bacterial 23S rRNAs. Furthermore, both tobacco and wheat chloroplast 4.5S rRNAs can also be folded in a similar way as the 3'-terminal region of bacterial 23S rRNA, the 3'-end of chloroplast 4.5S rRNAs being complementary to the 5'-end of chloroplast 23S rRNA. This strongly reinforces the hypothesis that chloroplast 4.5S rRNA originates from the 3'-end of bacterial 23S rRNA and suggests that this rRNA may be base-paired with the 5'-end of chloroplast 23S rRNA. Invariant oligonucleotides are present at identical positions in the homologous secondary structures of E.coli 23S, yeast 26S, X.laevis 28S and wheat and tobacco 4.5S rRNAs. Surprisingly, the sequences of these oligonucleotides are not all conserved in the 3'-terminal regions of A.punctata or even P.vulgaris 23S rRNAs. Results obtained upon mild methylation of E.coli 50S subunits with dimethylsulfate strongly suggest that these invariant oligonucleotides are involved in RNA tertiary structure or in RNA-protein interactions.     

Chemical synthesis of capped RNA fragments and their ability to complex with eukaryotic ribosomes

In other to examine the binding ability of the 5'-terminal part of eukaryotic mRNA to 80S ribosome, several kinds of oligoribonucleotides, pA-U-G, m7G5'pppA-U-G, m7G5'pppG-U, m7G5'pppA-U-G-A-C-C, were synthesized chemically. The binding experiments of oligonucleotides to 80S ribosome showed that the capped structure as well as AUG are essential for ribosome binding, and the efficiency is enhanced by the 5'-leader sequence if it would include complementary sequence to the 3'-terminal part of 18S rRNA.     

Terminal nucleotide sequences of 17-S ribosomal RNA and its immediate precursor 18-S RNA in yeast.

The 5' and 3'-terminal nucleotide sequences of 17-S rRNA and its immediate precursor 18-S RNA from the yeast Saccharomyces carlsbergensis have been analysed. Identification of the terminal oligonucleotides, as present in Ti ribonuclease digests, was performed by diagonal procedures. The major (molar yield 0.9) 5'-terminal oligonucleotide (molar yield 0.15) with the overall composition pU (U2,C2)G was observed. 18-S precursor RNA was found to contain the same 5'-terminal sequences as 17-S rRNA. However, the 3'-terminal sequences of the two types of RNA appeared to be different. The 17-S rRNA yields the oligonucleotide A-U-C-A-U-U-AOH while at least half of the 18-S RNA molecules contain the sequence U-U-U-C-A-A-U-AOH. In addition 18-S RNA yields several minor 3'-terminal oligonucleotides which appear to be structurally related to the major 3'-terminal sequence. These results demonstrate that the extra nucleotides in 18-S RNA relative to 17-S RNA are located exclusively at the 3'-terminus of the 18-S RNA molecule. The possibility that the 3'-terminal nucleotide sequence of 18-S RNA plays a role in the maturation process is discussed.     

Nucleotide sequence of a Euglena gracilis chloroplast gene coding for the 16S rRNA: homologies to E. coli and Zea mays chloroplast 16S rRNA

The nucleotide sequence of 16S rDNA from Euglena gracilis chloroplasts has been determined representing the first complete sequence of an algal chloroplast rRNA gene. The structural part of the 16S rRNA gene has 1491 nucleotides according to a comparative analysis of our sequencing results with the published 5'- and 3'-terminal "T1-oligonucleotides" from 16S rRNA from E. gracilis. Alignment with 16S rDNA from Zea mays chloroplasts and E. coli reveals 80 to 72% sequence homology, respectively. Two deletions of 9 and 23 nucleotides are found which are identical in size and position with deletions observed in 16S rDNA of maize and tobacco chloroplasts and which seem to be characteristic for all chloroplast rRNA species. We also find insertions and deletions in E. gracilis not seen in 16S rDNA of higher plant chloroplasts. The 16S rRNA sequence of E. gracilis chloroplasts can be folded by base pairing according to the general 16S rRNA secondary structure model.     

Sequence requirements for maturation of the 5' terminus of human 18 S rRNA in vitro.

Creation of the mature 5' terminus of human 18 S rRNA in vitro occurs via a two-step processing reaction. In the first step, an endonucleolytic activity found in HeLa cell nucleolar extract cleaves an rRNA precursor spanning the external transcribed spacer-18 S boundary at a position 3 bases upstream from the mature 18 S terminus leaving 2',3'-cyclic phosphate, 5' hydroxyl termini. In the second step, a nucleolytic activity(s) found in HeLa cell cytoplasmic extract removes the 3 extra bases and creates the authentic 5'-phosphorylated terminus of 18 S rRNA. Here we have examined the sequence requirements for the trimming reaction. The trimming activity(s), in addition to requiring a 5' hydroxyl terminus, prefers the naturally occurring adenosine as the 5'-terminal base. By a combination of deletion, site-directed mutagenesis, and chemical modification interference approaches we have also identified a region of 18 S rRNA spanning bases +6 to +25 (with respect to the mature 5' end) which comprises a critical recognition sequence for the trimming activity(s).     

Independent binding sites in mouse 5.8S ribosomal ribonucleic acid for 28S ribosomal ribonucleic acid

Limited digestion of mouse 5.8S ribosomal RNA (rRNA) with RNase T2 generates 5'- and 3'-terminal "half-molecules". These fragments are capable of independently and specifically binding to 28S rRNA, so there exist at least two contacts in the 5.8S rRNA for the 28S rRNA. The dissociation constants for the 5.8S/28S, 5' 5.8S fragment/28S, and 3' 5.8S fragment/28S complexes are 9 x 10(-8) M, 6 x 10(-8) M, and 13 x 10(-8) M, respectively. Thus, each of the fragment binding sites contributes about equally to the overall binding energy of the 5.8S/28S rRNA complex, and the binding sites act independently, rather than cooperatively. The dissociation constants suggest that the 5.8S rRNA termini from short, irregular helices with 28S rRNA. Thermal denaturation data on complexes containing 28S rRNA and each of the half-molecules of 5.8S rRNA indicate that the 5'-terminal binding site(s) exist(s) in a single conformation while the 3'-terminal site exhibits two conformational alternatives. The functional significance of the different conformational states is presently indeterminate, but the possibility they may represent alternative forms of a conformational switch operative during ribosome function is discussed.