Mutations in helix 27 of the yeast Saccharomyces cerevisiae 18S rRNA affect the function of the decoding center of the ribosome

A dynamic structural rearrangement in the phylogenetically conserved helix 27 of Escherichia coli 16S rRNA has been proposed to directly affect the accuracy of translational decoding by switching between "accurate" and "error-prone" conformations. To examine the function of helix 27 in eukaryotes, random and site-specific mutations in helix 27 of the yeast Saccharomyces cerevisiae 18S rRNA have been characterized. Mutations at positions of yeast 18S rRNA corresponding to E. coli 886 (rdn8), 888 (rdn6), and 912 (rdn4) increased translational accuracy in vivo and in vitro, and caused a reduction in tRNA binding to the A-site of mutant ribosomes. The double rdn4rdn6 mutation separated the killing and stop-codon readthrough effects of the aminoglycoside antibiotic, paromomycin, implicating a direct involvement of yeast helix 27 in accurate recognition of codons by tRNA or release factor eRF1. Although our data in yeast does not support a conformational switch model analogous to that proposed for helix 27 of E. coli 16S rRNA, it strongly suggests a functional conservation of this region in tRNA selection.     

Translational fidelity mutations in 18S rRNA affect the catalytic activity of ribosomes and the oxidative balance of yeast cells

The function of mutations rdn1A, rdn1T, and rdn2 in 18S rRNA of Saccharomyces cerevisiae is investigated. The mutations correspond to substitutions C1054A, C1054U in helix 34, and G517A in helix 18 of 16S rRNA in Escherichia coli, respectively, in which the first and third mutations caused nonsense suppression, while C1054U caused no suppression. In yeast, rdn1A caused phenotypic suppression at nonsense codons, whereas rdn1T and rdn2 caused antisuppression. We provide in vitro evidence that, in addition, rdn1A decreases translational accuracy at sense codons as well, by a factor of 8, accompanied by extreme sensitivity to paromomycin, compatible with its error-prone character. Mutations rdn1T andrdn2 exhibit hyperaccuracy and paromomycin resistance. Thus, mutations in conserved rRNA regions may affect the same functions in the various species but in opposite directions. Mutation rdn1A, but not rdn1T or rdn2, affected also the catalytic activity of the ribosome, a 60S subunit activity. The rate of peptide bond formation was reduced to half its normal value, indicating a communication between the two subunits. Moreover, error-prone mutation rdn1A was less susceptible to oxidative modifications than wild type, indicated by decreased lipid peroxidation and nonprotein/protein disulfides, as well as by increased protein thiols. In contrast, hyperaccurate mutations rdn1T and rdn2 displayed increased oxidative stress. Our results suggest that the cells may consume more energy to achieve hyperaccuracy leading to increased oxidative modifications.     

A translational fidelity mutation in the universally conserved sarcin/ricin domain of 25S yeast ribosomal RNA.

Recent evidence suggests that ribosomal RNAs have functional roles in translation. We describe here a new ribosomal RNA mutation that causes translational suppression and antibiotic resistance in eukaryotic cells. Using random mutagenesis of the cloned ribosomal RNA gene and in vivo selection, we isolated a C --> U mutation in the universally conserved sarcin/ricin domain in Saccharomyces cerevisiae 25S ribosomal RNA. This mutation changes the putative CG pair, which closes the GAGA tetraloop in the sarcin/ricin domain, into a weaker UG pair without eliminating ribosomal sensitivity to ricin. We show that suppression of several UGA, UAG, and frameshift mutations is evident when a portion of the cellular ribosomal RNA contains the C --> U mutation. Cells that contain essentially all mutant ribosomal RNA grow only 10% slower than the wild-type, but show increased suppression as well as resistance to paramomycin, G418, and hygromycin, and sensitivity to cycloheximide. Our results provide genetic evidence for the participation of the sarcin/ricin loop in maintaining translational accuracy and are discussed in terms of a hypothesis that this ribosomal RNA region normally undergoes a conformational change during translation.     

Genetic interaction between yeast Saccharomyces cerevisiae release factors and the decoding region of 18 S rRNA

Functional and structural similarities between tRNA and eukaryotic class 1 release factors (eRF1) described previously, provide evidence for the molecular mimicry concept. This concept is supported here by the demonstration of a genetic interaction between eRF1 and the decoding region of the ribosomal RNA, the site of tRNA-mRNA interaction. We show that the conditional lethality caused by a mutation in domain 1 of yeast eRF1 (P86A), that mimics the tRNA anticodon stem-loop, is rescued by compensatory mutations A1491G (rdn15) and U1495C (hyg1) in helix 44 of the decoding region and by U912C (rdn4) and G886A (rdn8) mutations in helix 27 of the 18 S rRNA. The rdn15 mutation creates a C1409-G1491 base-pair in yeast rRNA that is analogous to that in prokaryotic rRNA known to be important for high-affinity paromomycin binding to the ribosome. Indeed, rdn15 makes yeast cells extremely sensitive to paromomycin, indicating that the natural high resistance of the yeast ribosome to paromomycin is, in large part, due to the absence of the 1409-1491 base-pair. The rdn15 and hyg1 mutations also partially compensate for inactivation of the eukaryotic release factor 3 (eRF3) resulting from the formation of the [PSI+] prion, a self-reproducible termination-deficient conformation of eRF3. However, rdn15, but not hyg1, rescues the conditional cell lethality caused by a GTPase domain mutation (R419G) in eRF3. Other antisuppressor rRNA mutations, rdn2(G517A), rdn1T(C1054T) and rdn12A(C526A), strongly inhibit [PSI+]-mediated stop codon read-through but do not cure cells of the [PSI+] prion. Interestingly, cells bearing hyg1 seem to enable [PSI+] strains to accumulate larger Sup35p aggregates upon Sup35p overproduction, suggesting a lower toxicity of overproduced Sup35p when the termination defect, caused by [PSI+], is partly relieved.     

Two nucleotide substitutions in the A-site of yeast 18S rRNA affect translation and differentiate the interaction of ribosomes with aminoglycoside antibiotics

Two mutations in the A-site of 18S rRNA of Saccharomyces cerevisiae were investigated. The first, A1491G (rdn15), creates in yeast the same C1409-G1491 base pair as in Escherichia coli and has behaved as an antisuppressor in genetic studies. Ribosomes from rdn15 are error-restrictive but their peptidyltransferase activity remains unchanged. The second mutation, U1495C (rdnhyg1), was initially isolated as a hygromycin-resistant mutation in Tetrahymena thermophila. We show that rdnhyg1 ribosomes are slightly error prone. Mutation rdnhyg1 does not affect catalytic activity, but it affects translocation, confirming the importance of nucleotide 1495 in the ratchet-like movement of the two subunits during translation. Paromomycin, an aminoglycoside antibiotic that binds to the ribosomal A-site, induces translational misreading and causes sensitivity to yeast cells. Mutation rdn15 is shown to be highly sensitive to both effects of paromomycin, while mutation rdnhyg1 is relatively resistant. Tobramycin, another aminoglycoside, does not affect the growth of yeast cells. Like paromomycin, however, it increases the error rate in rdn15 ribosomes relative to wild-type and decreases it in rdnhyg1 ribosomes. These mutations help define the role of two crucial sites in ribosome function and distinguish the modes of action of two aminoglycosides, a useful fact in the search for new strategies in drug design.     

Endogenous RNA cleavages at the ribosomal SRL site likely reflect miRNA (miR) mediated translational suppression

We previously suggested a mechanism whereby the RNA induced silencing complex (RISC) brings about a specific cleavage at the sarcin–ricin loop (SRL) of 28S ribosomal RNA thereby eliciting translational suppression. Here we experimentally show that endogenous cleavages take place at the SRL site, in both mammalian cells and in Caenorhabditis elegans. Furthermore we demonstrate that bulged and looped-out residues present in the imperfect miRNA–[mRNA target site] duplexes, are complementary to the SRL site. These results support, and are compatible with, our described mechanism whereby microRNAs mediate cleavage of the highly conserved 28S rRNA sarcin/ricin loop leading to translational suppression.     

The ribosomal RNA identity elements for ricin and for alpha-sarcin: mutations in the putative CG pair that closes a GAGA tetraloop.

alpha-Sarcin is a ribonuclease that cleaves the phosphodiester bond on the 3' side of G4325 in 28S rRNA; ricin A-chain is a RNA N-glycosidase that depurinates the 5' adjacent A4324. These single covalent modifications inactivate the ribosome. An oligoribonucleotide that reproduces the structure of the sarcin/ricin domain in 28S rRNA was synthesized and mutations were constructed in the 5' C and the 3' G that surround a GAGA tetrad that has the sites of toxin action. Covalent modification of the RNA by ricin, but not by alpha-sarcin, requires a Watson-Crick pair to shut off a putative GAGA tetraloop. Either the recognition elements for the two toxins are different despite their catalyzing covalent modification of adjacent nucleotides in 28S rRNA or there are transitions in the conformation of the alpha-sarcin/ricin domain in 28S rRNA and one conformer is recognized by alpha-sarcin and the other by ricin A-chain.     

Eukaryotic elongation factor 2 can bind to the synthetic oligoribonucleotide that mimics sarcin/ricin domain of rat 28S ribosomal RNA

Eukaryotic elongation factor 2 (eEF2) catalyzes the translocation of peptidyl-tRNA from the A site to P site by binding to the ribosome. In this work, the complex formation of rat liver eEF2 with a synthetic oligoribonucleotide (SRD RNA) that mimics sarcin/ricin domain of rat 28S ribosomal RNA is invested in vitro. Purified eEF2 can specifically bind SRD RNA to form a stable complex. tRNA competes with SRD RNA in binding to eEF2 in a less extent. Pretreatment of eEF2 with GDP or ADP-ribosylation of eEF2 by diphtheria toxin can obviously reduce the ability of eEF2 to form the complex with the synthetic oligoribonucleotide. These results indicate that eEF2 is likely to bind directly to the sarcin/ricin domain of 28S ribosomal RNA in the process of protein synthesis.     

A new class of enzyme acting on damaged ribosomes: ribosomal RNA apurinic site specific lyase found in wheat germ

A new enzyme, which we named ribosomal RNA apurinic site specific lyase (RALyase), is described. The protein was found in wheat embryos and has a molecular weight of 50 625 Da. The enzyme specifically cleaves the phosphodiester bond at the 3' side of the apurinic site introduced by ribosome-inactivating proteins into the sarcin/ricin domain of 28S rRNA. The 3' and 5' ends of wheat 28S rRNA at the cleavage site are 5'-GUACG-alpha-hydroxy-alpha, beta-unsaturated aldehyde and pGAGGA-3', demonstrating that the enzyme catalyzes a beta-elimination reaction. The substrate specificity of the enzyme is extremely high: it acts only at the apurinic site in the sarcin/ricin domain of intact ribosomes, not on deproteinized rRNA or DNA containing apurinic sites. The amino acid sequences of five endopeptidase LysC-liberated peptides from the purified enzyme were determined and used to obtain a cDNA sequence. The open reading frame encodes a protein of 456 amino acids, and a homology search revealed a related rice protein. Similar enzyme activities were also found in other plants that express ribosome-inactivating proteins. We believe that RALyase is part of a complex self-defense mechanism.     

Mechanism of ribosome RNA apurinic site specific lyase.

A new enzyme, which we named ribosome RNA apurinic site specific lyase (RALyase), has been characterized. The enzyme specifically cleaves a phosphodiester bond at the apurinic site in the sarcin/ricin domain of 28S rRNA in ribosomes. The cut ends of wheat 28S rRNA were determined as 5'---GUACG-alpha-hydroxy-alpha, beta-unsaturated aldehyde and pGAGGA---3' for the 3' fragment, demonstrating that the enzyme catalyzes the beta-elimination reaction.     

Mutation of a highly conserved base in the yeast mitochondrial 21S rRNA restricts ribosomal frameshifting

A mutation shown to cause resistance to chloramphenicol inSaccharomyces cerevisiae was mapped to the central loop in domain V of the yeast mitochondrial 21S rRNA. The mutant 21S rRNA has a base pair exchange from U₂₆₇₇ (corresponding to U₂₅₀₄ inEscherichia coli) to C₂₆₇₇, which significantly reduces rightward frameshifting at a UU UUU UCC A site in a + 1 U mutant. There is evidence to suggest that this reduction also applies to leftward frameshifting at the same site in a – 1 U mutant. The mutation did not increase the rate of misreading of a number of mitochondrial missense, nonsense or frameshift (of both signs) mutations, and did not adversely affect the synthesis of wild-type mitochondrial gene products. It is suggested here that ribosomes bearing either the C₂₆₇₇ mutation or its wild-type allele may behave identically during normal decoding and only differ at sites where a ribosomal stall, by permitting non-standard decoding, differentially affects the normal interaction of tRNAs with the chloramphenicol resistant domain V. Chloramphenicol-resistant mutations mapping at two other sites in domain V are described. These mutations had no effect on frameshifting.     

Mutations in eukaryotic 18S ribosomal RNA affect translational fidelity and resistance to aminoglycoside antibiotics.

Mutations have been created in the Saccharomyces cerevisiae 18S rRNA gene that correspond to those known to be involved in the control of translational fidelity or antibiotic resistance in prokaryotes. Yeast strains, in which essentially all chromosomal rDNA repeats are deleted and all cellular rRNAs are encoded by plasmid, have been constructed that contain only mutant 18S rRNA. In Escherichia coli, a C-->U substitution at position 912 of the small subunit rRNA causes streptomycin resistance. Eukaryotes normally carry U at the corresponding position and are naturally resistant to streptomycin. We show that a U-->C transition (rdn-4) at this position of the yeast 18S rRNA gene decreases resistance to streptomycin. The rdn-4 mutation also increases resistance to paromomycin and G-418, and inhibits nonsense suppression induced by paromomycin. The same phenotypes, as well as a slow growth phenotype, are also associated with rdn-2, whose prokaryotic counterpart, 517 G-->A, manifests itself as a suppressor rather than an antisuppressor. Neither rdn-2- nor rdn-4-related phenotypes could be detected in the presence of the normal level of wild-type rDNA repeats. Our data demonstrate that eukaryotic rRNA is involved in the control of translational fidelity, and indicate that rRNA features important for interactions with aminoglycosides have been conserved throughout evolution.     

Specific contacts between protein S4 and ribosomal RNA are required at multiple stages of ribosome assembly

Assembly of bacterial 30S ribosomal subunits requires structural rearrangements to both its 16S rRNA and ribosomal protein components. Ribosomal protein S4 nucleates 30S assembly and associates rapidly with the 5′ domain of the 16S rRNA. In vitro, transformation of initial S4–rRNA complexes to long-lived, mature complexes involves refolding of 16S helix 18, which forms part of the decoding center. Here we use targeted mutagenesis of Geobacillus stearothermophilus S4 to show that remodeling of S4–rRNA complexes is perturbed by ram alleles associated with reduced translational accuracy. Gel mobility shift assays, SHAPE chemical probing, and in vivo complementation show that the S4 N-terminal extension is required for RNA binding and viability. Alanine substitutions in Y47 and L51 that interact with 16S helix 18 decrease S4 affinity and destabilize the helix 18 pseudoknot. These changes to the protein–RNA interface correlate with no growth (L51A) or cold-sensitive growth, 30S assembly defects, and accumulation of 17S pre-rRNA (Y47A). A third mutation, R200A, over-stabilizes the helix 18 pseudoknot yet results in temperature-sensitive growth, indicating that complex stability is finely tuned by natural selection. Our results show that early S4–RNA interactions guide rRNA folding and impact late steps of 30S assembly.     

A role for the 30S subunit E site in maintenance of the translational reading frame

The exit (E) site has been implicated in several ribosomal activities, including translocation, decoding, and maintenance of the translational reading frame. Here, we target the 30S subunit E site by introducing a deletion in rpsG that truncates the β-hairpin of ribosomal protein S7. This mutation (S7ΔR77–Y84) increases both −1 and +1 frameshifting but does not increase miscoding, providing evidence that the 30S E site plays a specific role in frame maintenance. Mutation S7ΔR77–Y84 also stimulates +1 programmed frameshifting during prfB′-lacZ translation in many synthetic contexts. However, no effect is seen when the E codon of the frameshift site corresponds to those found in nature, suggesting that E-tRNA release does not normally limit the rate of prfB frameshifting. Ribosomes containing S7ΔR77–Y84 exhibit an elevated rate of spontaneous reverse translocation and an increased K_1/2 for E-tRNA. These effects are of similar magnitude, suggesting that both result from destabilization of E-tRNA. Finally, this mutation of the 30S E site does not inhibit EF-G-dependent translocation, consistent with a primary role for the 50S E site in the mechanism.     

All three functional domains of the large ribosomal subunit protein L25 are required for both early and late pre-rRNA processing steps in Saccharomyces cerevisiae

Mutational analysis has shown that the integrity of the region in domain III of 25S rRNA that is involved in binding of ribosomal protein L25 is essential for the production of mature 25S rRNA in the yeast Saccharomyces cerevisiae. However, even structural alterations that do not noticeably affect recognition by L25, as measured by an in vitro assay, strongly reduced 25S rRNA formation by inhibiting the removal of ITS2 from the 27S_B precursor. In order to analyze the role of L25 in yeast pre-rRNA processing further we studied the effect of genetic depletion of the protein or mutation of each of its three previously identified functional domains, involved in nuclear import (N-terminal), RNA binding (central) and 60S subunit assembly (C-terminal), respectively. Depletion of L25 or mutating its (pre-)rRNA-binding domain blocked conversion of the 27S_B precursor to 5.8S/25S rRNA, confirming that assembly of L25 is essential for ITS2 processing. However, mutations in either the N- or the C-terminal domain of L25, which only marginally affect its ability to bind to (pre-)rRNA, also resulted in defective ITS2 processing. Furthermore, in all cases there was a notable reduction in the efficiency of processing at the early cleavage sites A₀, A₁ and A₂. We conclude that the assembly of L25 is necessary but not sufficient for removal of ITS2, as well as for fully efficient cleavage at the early sites. Additional elements located in the N- as well as C-terminal domains of L25 are required for both aspects of pre-rRNA processing.     

A functional relationship between helix 1 and the 900 tetraloop of 16S ribosomal RNA within the bacterial ribosome

The conserved 900 tetraloop that caps helix 27 of 16S ribosomal RNA (rRNA) interacts with helix 24 of 16S rRNA and also with helix 67 of 23S rRNA, forming the intersubunit bridge B2c, proximal to the decoding center. In previous studies, we investigated how the interaction between the 900 tetraloop and helix 24 participates in subunit association and translational fidelity. In the present study, we investigated whether the 900 tetraloop is involved in other undetected interactions with different regions of the Escherichia coli 16S rRNA. Using a genetic complementation approach, we selected mutations in 16S rRNA that compensate for a 900 tetraloop mutation, A900G, which severely impairs subunit association and translational fidelity. Mutations were randomly introduced in 16S rRNA, using either a mutagenic XL1-Red E. coli strain or an error-prone PCR strategy. Gain-offunction mutations were selected in vivo with a specialized ribosome system. Two mutations, the deletion of U12 and the U12C substitution, were thus independently selected in helix 1 of 16S rRNA. This helix is located in the vicinity of helix 27, but does not directly contact the 900 tetraloop in the crystal structures of the ribosome. Both mutations correct the subunit association and translational fidelity defects caused by the A900G mutation, revealing an unanticipated functional interaction between these two regions of 16S rRNA.     

Non-specific deadenylation and deguanylation of naked RNA catalyzed by ricin under acidic condition.

Ricin A-chain catalyzes the hydrolysis of the N-glycosidic bond of a conserved adenosine residue at position 4324 in the sarcin/ricin domain of 28S RNA of rat ribosome. The GAGA tetraloop closed by C-G pairs is required for recognition of the cleavage site on 28S ribosomal RNA by ricin A-chain. In this study, ricin A-chain (reduced ricin) exhibits specific depurination on a synthetic oligoribonucleotide (named SRD RNA) mimic of the sarcin/ricin domain of rat 28S ribosomal RNA under neutral and weak acidic conditions. Furthermore, the activity of intact ricin is also similar to that of ricin A-chain. However, under more acidic conditions, both enzymes lose their site specificity. The alteration in specificity of depurination is not dependent on the GAGA tetraloop of SRD RNA. A higher concentration of KCl inhibits the non-specific N-glycosidase activity much more than the specific activity of ricin A-chain. In addition, characterization of depurination sites by RNA sequencing reveals that under acidic conditions ricin A-chain can release not only adenines, but also guanines from SRD RNA or 5S ribosomal RNA. This is the first report of the non-specific deadenylation and deguanylation activity of ricin A-chain to the naked RNA under acidic conditions.     

Mutation K42R in Ribosomal Protein S12 Does Not Affect Susceptibility of Mycobacterium smegmatis 16S rRNA A-Site Mutants to 2-Deoxystreptamines

Recent studies have suggested that ribosomal protein S12 modulates 16S rRNA function and susceptibility to 2-deoxystreptamine aminoglycosides. To study whether the non-restrictive K42R mutation in RpsL affects 2-deoxystreptamine susceptibility in Mycobacterium smegmatis, we studied the drug susceptibility pattern of various mutants with genetic alterations in the 16S rRNA decoding A-site in the context of wild-type and mutant protein S12. RpsL K42R substitution was found not to affect the drug resistance pattern associated with mutational alterations in 16S rRNA H44.     

RNA Sequence Determinants for Aminoglycoside Binding to an A-site rRNA Model Oligonucleotide

The codon-anticodon interaction on the ribosome occurs in the A site of the 30 S subunit. Aminoglycoside antibiotics, which bind to ribosomal RNA in the A site, cause misreading of the genetic code and inhibit translocation. Biochemical studies and nuclear magnetic resonance spectroscopy were used to characterize the interaction between the aminoglycoside antibiotic paromomycin and a small model oligonucle otide that mimics the A site ofEscherichia coli16 S ribosomal RNA. Upon chemical modification, the RNA oligonucleotide exhibits an accessibility pattern similar to that of 16 S rRNA in the 30 S subunit. In addition, the oligonucleotide binds specifically aminoglycoside antibiotics. The anti biotic binding site forms an asymmetric internal loop, caused by non-canonical base-pairs. Nucleotides that are important for binding of paromomycin were identified by performing quantitative footprinting on oligonucleotide sequence variants and include the C1407·G1494 base-pair, and A·U base-pair at positions 1410/1490, and nucleotides A1408, A1493 and U1495. The asymmetry of the internal loop, which requires the presence of a nucleotide in position 1492, is also crucial for antibiotic binding. Introduction into the oligonucleotide of base changes that are known to confer aminoglycoside resistance in 16 S rRNA result in weaker binding of paromomycin to the oligonucleotide. Oligonucleotides homologous to eukaryotic rRNA sequences show reduced binding of paromomycin, suggesting a physical origin for the species-specific action of aminoglycosides.