Role of the 5.8S rRNA in ribosome translocation.

Studies on the inhibition of protein synthesis by specific anti 5.8S rRNA oligonucleotides have suggested that this RNA plays an important role in eukaryotic ribosome function. Mutations in the 5. 8S rRNA can inhibit cell growth and compromise protein synthesis in vitro . Polyribosomes from cells expressing these mutant 5.8S rRNAs are elevated in size and ribosome-associated tRNA. Cell free extracts from these cells also are more sensitive to antibiotics which act on the 60S ribosomal subunit by inhibiting elongation. The extracts are especially sensitive to cycloheximide and diphtheria toxin which act specifically to inhibit translocation. Studies of ribosomal proteins show no reproducible changes in the core proteins, but reveal reduced levels of elongation factors 1 and 2 only in ribosomes which contain large amounts of mutant 5.8S rRNA. Polyribosomes from cells which are severely inhibited, but contain little mutant 5.8S rRNA, do not show the same reductions in the elongation factors, an observation which underlines the specific nature of the change. Taken together the results demonstrate a defined and critical function for the 5.8S rRNA, suggesting that this RNA plays a role in ribosome translocation.     

Differentiated inhibition of DNA, RNA and protein synthesis in L1210 cells by 8-methoxypsoralen

The synthesis of DNA, RNA and protein was measured in L1210 cells following treatment with 8-methoxypsoralen in combination with long wavelength ultraviolet irradiation. The results show that the DNA synthesis is strongly inhibited (approximately 95%) at 200 ng/ml reaching a minimum within 2 hours while RNA synthesis is only weakly affected at this concentration (approximately 40% inhibition). At 2 micrograms/ml the RNA synthesis is inhibited approximately 90%. Even at this concentration only a moderate effect is seen on the protein synthesis. These results strongly indicate that the phototoxic action of 8-methoxypsoralen is primarily due to inhibition of DNA synthesis.     

Topography of 5.8 S rRNA in rat liver ribosomes. Identification of diethyl pyrocarbonate-reactive sites

The topography of 5.8 rRNA in rat liver ribosomes has been examined by comparing diethyl pyrocarbonate-reactive sites in free 5.8 S RNA, the 5.8 S-28 rRNA complex, 60 S subunits, and whole ribosomes. The ribosomal components were treated with diethyl pyrocarbonate under salt and temperature conditions which allow cell-free protein synthesis; the 5.8 S rRNA was extracted, labeled in vitro, chemically cleaved with aniline, and the fragments were analyzed by rapid gel-sequencing techniques. Differences in the cleavage patterns of free and 28 S or ribosome-associated 5.8 S rRNA suggest that conformational changes occur when this molecule is assembled into ribosomes. In whole ribosomes, the reactive sites were largely restricted to the "AU-rich" stem and an increased reactivity at some of the nucleotides suggested that a major change occurs in this region when the RNA interacts with ribosomal proteins. The reactivity was generally much less restricted in 60 S subunits but increased reactivity in some residues was also observed. The results further indicate that in rat ribosomes, the two -G-A-A-C- sequences, putative binding sites for tRNA, are accessible in 60 S subunits but not in whole ribosomes and suggest that part of the molecule may be located in the ribosomal interface. When compared to 5 S rRNA, the free 5.8 S RNA molecule appears to be generally more reactive with diethyl pyrocarbonate and the cleavage patterns suggest that the 5 S RNA molecule is completely restricted or buried in whole ribosomes.     

Phylogenetic relationships and altered genome structures among Tetrahymena mitochondrial DNAs.

Tetrahymena thermophila mitochondrial DNA is a linear molecule with two tRNAs, large subunit beta (LSU beta) rRNA (21S rRNA) and LSU alpha rRNA (5.8S-like RNA) encoded near each terminus. The DNA sequence of approximately 550 bp of this region was determined in six species of Tetrahymena. In three species the LSU beta rRNA and tRNA(leu) genes were not present on one end of the DNA, demonstrating a mitochondrial genome organization different from that of T. thermophila. The DNA sequence of the LSU alpha rRNA was used to construct a mitochondrial phylogenetic tree, which was found to be topologically equivalent to a phylogenetic tree based on nuclear small subunit rRNA sequences (Sogin et al. (1986) EMBO J. 5, 3625-3630). The mitochondrial rRNA gene was found to accumulate base-pair substitutions considerably faster than the nuclear rRNA gene, the rate difference being similar to that observed for mammals.     

Properties of Escherichia coli 16S ribosomal ribonucleic acid treated with 4,5',8-trimethylpsoralen and light.

16S rRNA reacted with the furocoumarin 4,5',8-trimethylpsoralen (trioxsalen) and 360-nm light showed a number of chemical and physical differences from untreated RNA. After extensive irradiation, five molecules of trioxsalen were bound per molecule of RNA. The trioxsalen-treated RNA had an altered ultraviolet absorption spectrum and a distinctive fluorescence emission spectrum. The modified RNA was significantly more resistant to T1 ribonuclease digestion than was control RNA. Treated RNA, when mixed with purified ribosomal proteins, was not functional in the in vitro reconstitution of 30S subunits and yielded more slowly sedimenting particles which were inactive in protein synthesis assays. By contrast, 16S rRNA within the 30S subunit structure did not exhibit these changes when reacted with the same dose of trioxsalen and light, suggesting that the ribosomal proteins were effective in protecting the RNA from interaction with the drug.     

Different mechanisms for pseudouridine formation in yeast 5S and 5.8S rRNAs

The selection of sites for pseudouridylation in eukaryotic cytoplasmic rRNA occurs by the base pairing of the rRNA with specific guide sequences within the RNA components of box H/ACA small nucleolar ribonucleoproteins (snoRNPs). Forty-four of the 46 pseudouridines (Psis) in the cytoplasmic rRNA of Saccharomyces cerevisiae have been assigned to guide snoRNAs. Here, we examine the mechanism of Psi formation in 5S and 5.8S rRNA in which the unassigned Psis occur. We show that while the formation of the Psi in 5.8S rRNA is associated with snoRNP activity, the pseudouridylation of 5S rRNA is not. The position of the Psi in 5.8S rRNA is guided by snoRNA snR43 by using conserved sequence elements that also function to guide pseudouridylation elsewhere in the large-subunit rRNA; an internal stem-loop that is not part of typical yeast snoRNAs also is conserved in snR43. The multisubstrate synthase Pus7 catalyzes the formation of the Psi in 5S rRNA at a site that conforms to the 7-nucleotide consensus sequence present in other substrates of Pus7. The different mechanisms involved in 5S and 5.8S rRNA pseudouridylation, as well as the multiple specificities of the individual trans factors concerned, suggest possible roles in linking ribosome production to other processes, such as splicing and tRNA synthesis.     

Differential effects of cordycepin triphosphate and 9 beta-D-arabinofuranosyladenine triphosphate on tRNA and 5 S RNA synthesis in isolated nuclei.

Although cordycepin 5'-triphosphate (3'-dATP), at low concentrations, preferentially inhibits chromatin-associated poly(A) synthesis in isolated nuclei, higher levels of the inhibitor prevent both rRNA (RNA polymerase I activity) and hnRNA (RNA polymerase II activity) synthesis in vitro (Rose, K.M., Bell, L.E. and Jacob, S.T. (1977) Nature 267, 178-180). The present studies demonstrate that this nucleotide can also inhibit tRNA and 5 S RNA synthesis (RNA polymerase III activity). At 50-200 microgram/ml, 3'-dATP inhibits incorporation of [3H]UTP into tRNA and 5 S RNA by approximately 65%, whereas the syntheses of these RNAs were completely blocked when [3H]GTP was used as the substrate. These data suggest the formation of poly(U) in the tRNA and 5 S RNA regions, which is resistant to 3'-dATP. In contrast, another ATP analog, Ara-ATP, which selectively inhibits poly(A) synthesis, does not block tRNA and 5 S RNA synthesis in isolated nuclei. The production of these RNA species in isolated nuclei is also insensitive to Ara-CTP and 2'-dATP. These data suggest that 3'-dATP exerts general inhibitory effects on RNA synthesis and further substantiate the conclusion that Ara-ATP is a selective inhibitor of the polyadenylation reaction in vitro.     

Stable low molecular weight RNA analyzed by staircase electrophoresis, a molecular signature for both prokaryotic and eukaryotic microorganisms

Low-molecular weight RNA (LMW RNA) analysis using staircase electrophoresis was performed for several species of eukaryotic and prokaryotic microorganisms. According to our results, the LMW RNA profiles of archaea and bacteria contain three zones: 5S RNA, class 1 tRNA and class 2 tRNA. In fungi an additional band is included in the LMW RNA profiles, which correspond to the 5.8S RNA. In archaea and bacteria we found that the 5S rRNA zone is characteristic for each genus and the tRNA profile is characteristic for each species. In eukaryotes the combined 5.8S and 5S rRNA zones are characteristic for each genus and, as in prokaryotes, tRNA profiles are characteristic for each species. Therefore, stable low molecular weight RNA, separated by staircase electrophoresis, can be considered a molecular signature for both prokaryotic and eukaryotic microorganisms. Analysis of the data obtained and construction of the corresponding dendrograms afforded relationships between genera and species; these were essentially the same as those obtained with 16S rRNA sequencing (in prokaryotes) and 18S rRNA sequencing (in eukaryotes).     

Location of single-stranded and double-stranded regions in rat liver ribosomal 5S RNA and 5.8S RNA.

Rat liver 5S rRNA and 5.8S rRNA were end-labelled with 32P at 5'-end or 3'-end of the polynucleotide chain and partially digested with single-strand specific S1 nuclease and double-strand specific endonuclease from the cobra Naja naja oxiana venom. The parallel use of these two structure-specific enzymes in combination with rapid sequencing technique allowed the exact localization of single-stranded and double-stranded regions in 5S RNA and 5.8 S RNA. The most accessible regions to S1 nuclease in 5S RNA are regions 33-42, 74-78, 102-103 and in 5.8 S RNA 16-20, 26-29, 34-36, 74-80 and a region around 125-130. The cobra venom endonuclease cleaves the following areas in 5S RNA: 7-8, 17-20, 28-30, 49-51, 56-57, 60-64, 69-70, 81-82, 95-97, 106-112. In 5.8S RNA the venom endonuclease cleavage sites are 4-7, 10-13, 21-22, 33-35, 43-45, 51-55, 72-74, 85-87, 98-99, 105-106, 114-115, 132-135. According to these results the tRNA binding sequences proposed by Nishikawa and Takemura [(1974) FEBS Lett. 40, 106-109], in 5S RNA are located in partly single-stranded region, but in 5.8S RNA in double-stranded region.     

Photo-induced protein cross-linking to 5S RNA and 28-5.8S RNA within rat-liver 60S ribosomal subunits

Rat liver 60S ribosomal subunits were irradiated with 254-nm ultraviolet light (1.26 X 10(4) quanta/subunit), under conditions which preserved their functional activity. Cross-linked RNA-protein complexes were recovered after unreacted proteins had been removed by repeated acetic acid extractions. Proteins linked to the whole rRNA, to 5S RNA and to 28-5.8 S RNAs were identified by two-dimensional gel electrophoresis after RNA hydrolysis by ribonucleases T1 and A. Our results showed that numerous proteins interact with rRNAs (at least ten with 28-5.8 S RNA, eight with 5S RNA and among these three are common to both) and have been discussed in the light of all the available data.     

Effects of ribosome dissociation on the structure of the ribosome-associated 5.8S RNA

Diethyl pyrocarbonate reactivity and thermal denaturation were used to probe potential ribosomal interactions between tRNA and the small 5.8S and 5S rRNAs. Puromycin, an analogue of the terminal aminoacyl-adenosine portion of aminoacyl-tRNA, was observed to increase the accessibility of the 5.8S rRNA, including the highly conserved GAACp sequences. EDTA which releases both tRNA and the 5S rRNA-protein complex resulted in an even greater accessibility in the 5.8S rRNA. The thermal dissociation of whole ribosomes resulted in the release of all three RNAs, with a striking similarity in the denaturation profiles. These results strongly suggest an interdependence in the ribosome-associated structures of the small rRNAs and provide in situ evidence for the various 5S rRNA, 5.8S rRNA, and tRNA containing ribonucleoprotein complexes previously reconstituted through affinity chromatography.     

The conserved 5 S rRNA complement to tRNA is not required for translation of natural mRNA

We have tested a putative base-paired interaction between the conserved GT psi C sequence of tRNA and the conserved GAAC47 sequence of 5 S ribosomal RNA by in vitro protein synthesis using ribosomes containing deletions in this region of 5 S rRNA. Ribosomes reconstituted with 5 S rRNA possessing a single break between residues 41 and 42, deletion of residues 42-46, or deletion of residues 42-52 were tested for their ability to translate phage MS2 RNA. Initiator tRNA binding, aminoacyl-tRNA binding, ppGpp synthesis, and miscoding were also tested. All of the measured functions could be carried out by ribosomes carrying the deleted 5 S rRNAs. The sizes and relative amounts of the polypeptides synthesized by MS2 RNA-programmed ribosomes were identical whether or not the 5 S RNA contained deletions. Aminoacyl-tRNA binding and miscoding were essentially unaffected. Significant reduction in ApUpG (but not poly(A,U,G) or MS2 RNA)-directed fMet-tRNA binding and ppGpp synthesis were observed, particularly in the case of the larger (residues 42-52) deletion. We conclude that if tRNA and 5 S rRNA interact in this fashion, it is not an obligatory step in protein synthesis.     

The 5S and 5.8S ribosomal RNA sequences of Tetrahymena thermophila and T. pyriformis

The nucleotide sequences of the 5S rRNAs of Tetrahymena thermophila and two strains of T. pyriformis have been determined to be identical. The 5.8S rRNA sequences have also been determined; these sequences correct several errors in an earlier report. The 5.8S rRNAs of the two species differ at a single position. The sequencing results indicate that the species are of recent common ancestry. Molecular evidence that has been interpreted in the past as suggestive of an ancient divergence has been reviewed and found to be consistent with a T. pyriformis complex radiation beginning approximately 30-40 million years ago.     

5S rRNA Gene Arrangements in Protists: A Case of Nonadaptive Evolution

Given their high copy number and high level of expression, one might expect that both the sequence and organization of eukaryotic ribosomal RNA genes would be conserved during evolution. Although the organization of 18S, 5.8S and 28S ribosomal RNA genes is indeed relatively well conserved, that of 5S rRNA genes is much more variable. Here, we review the different types of 5S rRNA gene arrangements which have been observed in protists. This includes linkages to the other ribosomal RNA genes as well as linkages to ubiquitin, splice-leader, snRNA and tRNA genes. Mapping these linkages to independently derived phylogenies shows that these diverse linkages have repeatedly been gained and lost during evolution. This argues against such linkages being the primitive condition not only in protists but also in other eukaryote species. Because the only characteristic the diverse genes with which 5S rRNA genes are found linked with is that they are tandemly repeated, these arrangements are unlikely to provide any selective advantage. Rather, the observed high variability in 5S rRNA genes arrangements is likely the result of the fact that 5S rRNA genes contain internal promoters, that these genes are often transposed by diverse recombination mechanisms and that these new gene arrangements are rapidly homogenized by unequal crossingovers and/or by gene conversions events in species with short generation times and frequent founder events.     

Inhibition of protein synthesis by anti-5.8 S rRNA oligodeoxyribonucleotides

To examine the role of the 5.8 S rRNA in ribosome function, oligodeoxyribonucleotides, complementary to chemically accessible sequences, were incubated with rabbit reticulocyte or wheat germ extracts undergoing protein synthesis in vitro. Significant and reproducible inhibitions were observed with several different oligonucleotides, the most inhibitory being specific for the universally conserved GAAC sequence. Mutant or heterologous sequences were substantially less inhibitory, results which clearly implicate the 5.8 S rRNA in the inhibitory process and are consistent with the possibility that the 5.8 S rRNA plays an important role in the binding of tRNA.     

Possible interaction sites of mRNA, tRNA, translation factors and the nascent peptide in 5S, 5.8S and 28S rRNA in in vivo assembled eukaryotic ribosomal complexes.

We have investigated possible interaction sites for mRNA, tRNA, translation factors and the nascent peptide on 5S, 5.8S and 28S rRNA in in vivo assembled translational active mouse ribosomes by comparing the chemical footprinting patterns derived from native polysomes, salt-washed polysomes (mainly lacking translational factors) and salt-washed runoff ribosomes (lacking mRNA, tRNA and translational factors). Several ligand-induced footprints were observed in 28S rRNA while no reactivity changes were seen in 5S and 5.8S rRNA. Footprints derived from mRNA, tRNA and/or the nascent peptide chain were observed in domain I of 28S rRNA (hairpin 23), in domain II (helix 37/38 and helices 42 and 43 and in the eukaryotic expansion segment 15), in domain IV (helices 67 and 74) and in domain V (helices 94 and 96 and in the peptidyl transferase ring). Some of the protected sites were homologous to sites previously suggested to be involved in mRNA, tRNA and/or peptide binding in in vitro assembled prokaryotic complexes. Additional footprints were located in regions that have not previously been found involved in ligand binding. Part of these sites could derive from the nascent peptide in the exit channel of the ribosome.     

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.