Distinct response of yeast ribosomes to a miscoding event during translation

Numerous mechanisms have evolved to control the accuracy of translation, including a recently discovered retrospective quality control mechanism in bacteria. This quality control mechanism is sensitive to perturbations in the codon:anticodon interaction in the P site of the ribosome that trigger a dramatic loss of fidelity in subsequent tRNA and release factor selection events in the A site. These events ultimately lead to premature termination of translation in response to an initial miscoding error. In this work, we extend our investigations of this mechanism to an in vitro reconstituted Saccharomyces cerevisiae translation system. We report that yeast ribosomes do not respond to mismatches in the P site by loss of fidelity in subsequent substrate recognition events. We conclude that retrospective editing, as initially characterized in Escherichia coli, does not occur in S. cerevisiae. These results highlight potential mechanistic differences in the functional core of highly conserved ribosomes.     

Activities of the peptidyl transferase center of ribosomes lacking protein L27

The ribosome is the molecular machine responsible for protein synthesis in all living organisms. Its catalytic core, the peptidyl transferase center (PTC), is built of rRNA, although several proteins reach close to the inner rRNA shell. In the Escherichia coli ribosome, the flexible N-terminal tail of the ribosomal protein L27 contacts the A- and P-site tRNA. Based on computer simulations of the PTC and on previous biochemical evidence, the N-terminal α-amino group of L27 was suggested to take part in the peptidyl-transfer reaction. However, the contribution of this group to catalysis has not been tested experimentally. Here we investigate the role of L27 in peptide-bond formation using fast kinetics approaches. We show that the rate of peptide-bond formation at physiological pH, both with aminoacyl-tRNA or with the substrate analog puromycin, is independent of the presence of L27; furthermore, translation of natural mRNAs is only marginally affected in the absence of L27. The pH dependence of the puromycin reaction is unaltered in the absence of L27, indicating that the N-terminal α-amine is not the ionizing group taking part in catalysis. Likewise, L27 is not required for the peptidyl-tRNA hydrolysis during termination. Thus, apart from the known effect on subunit association, which most likely explains the phenotype of the deletion strains, L27 does not appear to be a key player in the core mechanism of peptide-bond formation on the ribosome.     

Cooperative effects in the peptidyltransferase center of Escherichia coli ribosomes

We have measured the binding isotherms of C--A--C--C--A(3'NH)-[14C]Phe to the 70S ribosomes and 50S subunits of Escherichia coli and proposed a theoretical model for adsorption when cooperative interaction occurs between ligands that are adsorbed on ribosomes. Analysis of the experimental binding isotherms leads to the following conclusions. A ribosome (or subunit) binds two C--A--C--C--A(3'NH)-Phe molecules. The binding of C--A--C--C--A(3'NH)-Phe to a ribosome (or subunit) is a cooperative process, characterized by a cooperativity coefficient tau = 40 +/- 5 or more. The binding of C--A--C--C--A(3'NH)-AcPhe at the donor site of the peptidyltransferase center (association binding constant 1.5 X 10(6) M-1) and the binding of puromycin at the acceptor site also occur cooperatively with a coefficient of 10-25, the association binding constant of puromycin at the acceptor site being (1-2) X 10(4) M-1. The puromycin association binding constant at the donor site multiplied by the cooperativity coefficient of two interacting puromycin molecules absorbed on a ribosome equals 100-200 M-1.     

Composition and structure of the 80S ribosome from the green alga Chlamydomonas reinhardtii: 80S ribosomes are conserved in plants and animals

We have conducted a proteomic analysis of the 80S cytosolic ribosome from the eukaryotic green alga Chlamydomonas reinhardtii, and accompany this with a cryo-electron microscopy structure of the ribosome. Proteins homologous to all but one rat 40S subunit protein, including a homolog of RACK1, and all but three rat 60S subunit proteins were identified as components of the C. reinhardtii ribosome. Expressed Sequence Tag (EST) evidence and annotation of the completed C. reinhardtii genome identified genes for each of the four proteins not identified by proteomic analysis, showing that algae potentially have a complete set of orthologs to mammalian 80S ribosomal proteins. Presented at 25A, the algal 80S ribosome is very similar in structure to the yeast 80S ribosome, with only minor distinguishable differences. These data show that, although separated by billions of years of evolution, cytosolic ribosomes from photosynthetic organisms are highly conserved with their yeast and animal counterparts.     

The role of rRNA bases in the interaction of peptidyltransferase inhibitors with bacterial ribosomes.

Synergism of streptogramins A (virginiamycin M, VM) and B (virginiamycin S, VS), peptidyltransferase inhibitors, was explored in EM4/pLC7-21 (wild type) and EM4/pERY (VS-resistant). These bacterial strains contained multicopy plasmids carrying an rrnH operon with wild type (pLC7-21) or mutated (A2058----U transversion) 23 S rRNA gene. Ribosomes with wild type and mutated rRNA were both present in EM4/pERY. The latter particles did not bind VS; in the presence of VM, however, high affinity VS binding occurred. As shown previously, VS protected against chemical reagents certain bases in domain V rRNA and VM in the stems flanking this loop. Differences between wild type and mutant ribosomes were observed: A2058, A2059, A2062, and G2505, protected by VS and ERY in EM4/pLC7-21, were unshielded in EM4/pERY. A2062 was shielded by VM in EM4/pERY, not in EM4/pLC7-21, and G2505 of mutant ribosomes became protected by VS when VM was simultaneously present. Induction by VM of a high affinity VS binding site in VS-sensitive and -resistant ribosomes indicates A2058 mutation to entail a conformational change of this site, which is counteracted by VM fixation. Accessibility of A2062 to chemical reagents (unlike behavior of EM4/pERY and EM4/pLC7-21 in the presence of VM) implies different conformations for wild type and mutant ribosomes.     

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.     

RNA-protein distance patterns in ribosomes reveal the mechanism of translational attenuation

Elucidating protein translational regulation is crucial for understanding cellular function and drug development. A key molecule in protein translation is ribosome, which is a super-molecular complex extensively studied for more than a half century. The structure and dynamics of ribosome complexes were resolved recently thanks to the development of X-ray crystallography, Cryo-EM, and single molecule biophysics. Current studies of the ribosome have shown multiple functional states, each with a unique conformation. In this study, we analyzed the RNA-protein distances of ribosome (2.5 MDa) complexes and compared these changes among different ribosome complexes. We found that the RNA-protein distance is significantly correlated with the ribosomal functional state. Thus, the analysis of RNA-protein binding distances at important functional sites can distinguish ribosomal functional states and help understand ribosome functions. In particular, the mechanism of translational attenuation by nascent peptides and antibiotics was revealed by the conformational changes of local functional sites.     

Asc1, homolog of human RACK1, prevents frameshifting in yeast by ribosomes stalled at CGA codon repeats

Quality control systems monitor and stop translation at some ribosomal stalls, but it is unknown if halting translation at such stalls actually prevents synthesis of abnormal polypeptides. In yeast, ribosome stalling occurs at Arg CGA codon repeats, with even two consecutive CGA codons able to reduce translation by up to 50%. The conserved eukaryotic Asc1 protein limits translation through internal Arg CGA codon repeats. We show that, in the absence of Asc1 protein, ribosomes continue translating at CGA codons, but undergo substantial frameshifting with dramatically higher levels of frameshifting occurring with additional repeats of CGA codons. Frameshifting depends upon the slow or inefficient decoding of these codons, since frameshifting is suppressed by increased expression of the native tRNA^Arg(ICG) that decodes CGA codons by wobble decoding. Moreover, the extent of frameshifting is modulated by the position of the CGA codon repeat relative to the translation start site. Thus, translation fidelity depends upon Asc1-mediated quality control.     

Multiple effects of S13 in modulating the strength of intersubunit interactions in the ribosome during translation

The ribosomal protein S13 is found in the head region of the small subunit, where it interacts with the central protuberance of the large ribosomal subunit and with the P site-bound tRNA through its extended C terminus. The bridging interactions between the large and small subunits are dynamic, and are thought to be critical in orchestrating the molecular motions of the translation cycle. S13 provides a direct link between the tRNA-binding site and the movements in the head of the small subunit seen during translocation, thereby providing a possible pathway of signal transduction. We have created and characterized an rpsM(S13)-deficient strain of Escherichia coli and have found significant defects in subunit association, initiation and translocation through in vitro assays of S13-deficient ribosomes. Targeted mutagenesis of specific bridge and tRNA contact elements in S13 provides evidence that these two interaction domains play critical roles in maintaining the fidelity of translation. This ribosomal protein thus appears to play a non-essential, yet important role by modulating subunit interactions in multiple steps of the translation cycle.     

Optimization of speed and accuracy of decoding in translation

The speed and accuracy of protein synthesis are fundamental parameters for understanding the fitness of living cells, the quality control of translation, and the evolution of ribosomes. In this study, we analyse the speed and accuracy of the decoding step under conditions reproducing the high speed of translation in vivo. We show that error frequency is close to 10⁻³, consistent with the values measured in vivo. Selectivity is predominantly due to the differences in k_cat values for cognate and near-cognate reactions, whereas the intrinsic affinity differences are not used for tRNA discrimination. Thus, the ribosome seems to be optimized towards high speed of translation at the cost of fidelity. Competition with near- and non-cognate ternary complexes reduces the rate of GTP hydrolysis in the cognate ternary complex, but does not appreciably affect the rate-limiting tRNA accommodation step. The GTP hydrolysis step is crucial for the optimization of both the speed and accuracy, which explains the necessity for the trade-off between the two fundamental parameters of translation.     

The role of SmpB and the ribosomal decoding center in licensing tmRNA entry into stalled ribosomes

In bacteria, stalled ribosomes are recycled by a hybrid transfer-messenger RNA (tmRNA). Like tRNA, tmRNA is aminoacylated with alanine and is delivered to the ribosome by EF-Tu, where it reacts with the growing polypeptide chain. tmRNA entry into stalled ribosomes poses a challenge to our understanding of ribosome function because it occurs in the absence of a codon-anticodon interaction. Instead, tmRNA entry is licensed by the binding of its protein partner, SmpB, to the ribosomal decoding center. We analyzed a series of SmpB mutants and found that its C-terminal tail is essential for tmRNA accommodation but not for EF-Tu activation. We obtained evidence that the tail likely functions as a helix on the ribosome to promote accommodation and identified key residues in the tail essential for this step. In addition, our mutational analysis points to a role for the conserved K(131)GKK tail residues in trans-translation after peptidyl transfer to tmRNA, presumably EF-G-mediated translocation or translation of the tmRNA template. Surprisingly, analysis of A1492, A1493, and G530 mutants reveals that while these ribosomal nucleotides are essential for normal tRNA selection, they play little to no role in peptidyl transfer to tmRNA. These studies clarify how SmpB interacts with the ribosomal decoding center to license tmRNA entry into stalled ribosomes.     

Loss of rRNA modifications in the decoding center of the ribosome impairs translation and strongly delays pre-rRNA processing

The ribosome decoding center is rich in modified rRNA nucleotides and little is known about their effects. Here, we examine the consequences of systematically deleting eight pseudouridine and 2′-O-methylation modifications in the yeast decoding center. Loss of most modifications individually has no apparent effect on cell growth. However, deletions of 2–3 modifications in the A- and P-site regions can cause (1) reduced growth rates (∼15%–50% slower); (2) reduced amino acid incorporation rates (14%–24% slower); and (3) a significant deficiency in free small subunits. Negative and positive interference effects were observed, as well as strong positional influences. Notably, blocking formation of a hypermodified pseudouridine in the P region delays the onset of the final cleavage event in 18S rRNA formation (∼60% slower), suggesting that modification at this site could have an important role in modulating ribosome synthesis.     

Mutations at the accommodation gate of the ribosome impair RF2-dependent translation termination

During protein synthesis, aminoacyl-tRNA (aa-tRNA) and release factors 1 and 2 (RF1 and RF2) have to bind at the catalytic center of the ribosome on the 50S subunit where they take part in peptide bond formation or peptidyl-tRNA hydrolysis, respectively. Computer simulations of aa-tRNA movement into the catalytic site (accommodation) suggested that three nucleotides of 23S rRNA, U2492, C2556, and C2573, form a “gate” at which aa-tRNA movement into the A site is retarded. Here we examined the role of nucleotides C2573 of 23S rRNA, a part of the putative accommodation gate, and of the neighboring A2572 for aa-tRNA binding followed by peptide bond formation and for the RF2-dependent peptide release. Mutations at the two positions did not affect aa-tRNA accommodation, peptide bond formation, or the fidelity of aa-tRNA selection, but impaired RF2-catalyzed peptide release. The data suggest that the ribosome is a robust machine that allows rapid aa-tRNA accommodation despite the defects at the accommodation gate. In comparison, peptide release by RF2 appears more sensitive to these mutations, due to slower accommodation of the factor or effects on RF2 positioning in the A site.     

Peptidyltransferase center of ribosomes: On the mechanism of action of alkaloid lycorine

The molecular mechanism of action of the alkaloid lycorine has been revised. According to our results, lycorine inhibits the binding of CACCA-Leu←Ac to the donor site of the peptidyltranferase center of wheat-germ ribosomes, whereas the transpeptidation reaction in the system with the minimal model donor is not inhibited. The equilibrium constant of CACCA-Leu←Ac to the donor site of 80 S ribosomes is measured.     

rRNA mutants in the yeast peptidyltransferase center reveal allosteric information networks and mechanisms of drug resistance

To ensure accurate and rapid protein synthesis, nearby and distantly located functional regions of the ribosome must dynamically communicate and coordinate with one another through a series of information exchange networks. The ribosome is ~2/3 rRNA and information should pass mostly through this medium. Here, two viable mutants located in the peptidyltransferase center (PTC) of yeast ribosomes were created using a yeast genetic system that enables stable production of ribosomes containing only mutant rRNAs. The specific mutants were C2820U (Escherichia coli C2452) and Ψ2922C (E. coli U2554). Biochemical and genetic analyses of these mutants suggest that they may trap the PTC in the ‘open’ or aa-tRNA bound conformation, decreasing peptidyl-tRNA binding. We suggest that these structural changes are manifested at the biological level by affecting large ribosomal subunit biogenesis, ribosomal subunit joining during initiation, susceptibility/resistance to peptidyltransferase inhibitors, and the ability of ribosomes to properly decode termination codons. These studies also add to our understanding of how information is transmitted both locally and over long distances through allosteric networks of rRNA–rRNA and rRNA–protein interactions.     

Characterization of the termination-reinitiation strategy employed in the expression of influenza B virus BM2 protein

Coupled expression of the M1 and BM2 open-reading frames (ORFs) of influenza B from the dicistronic segment 7 mRNA occurs by a process of termination-dependent reinitiation. The AUG start codon of the BM2 ORF overlaps the stop codon of the upstream M1 ORF in the pentanucleotide UAAUG, and BM2 synthesis is dependent upon translation of the M1 ORF and termination at the stop codon. Here, we have investigated the mRNA sequence requirements for BM2 expression. Termination-reinitiation is dependent upon 45 nucleotide (nt) of RNA immediately upstream of the UAAUG pentanucleotide, which includes an essential stretch complementary to 18S rRNA helix 26. Thus, similar to the caliciviruses, base-pairing between mRNA and rRNA is likely to play a role in tethering the 40S subunit to the mRNA following termination at the M1 stop codon. Consistent with this, repositioning of the M1 stop codon more than 24 nt downstream from the BM2 start codon inhibited BM2 expression. RNA structure probing revealed that the RNA upstream of the UAAUG overlap is not highly structured, but upon encountering the M1 stop codon by the ribosome, a stem-loop may form immediately 5' of the ribosome, with the 18S rRNA complementary region in the apical loop and in close proximity to helix 26. Mutational analysis reveals that the normal requirements for start site selection in BM2 expression are suspended, with little effect of initiation codon context and efficient use of noncanonical initiation codons. This suggests that the full complement of initiation factors is not required for the reinitiation process.     

Characterization of 16S rRNA mutations that decrease the fidelity of translation initiation

In bacteria, initiation of translation is kinetically controlled by factors IF1, IF2, and IF3, which work in conjunction with the 30S subunit to ensure accurate selection of the initiator tRNA (fMet-tRNA(fMet)) and the start codon. Here, we show that mutations G1338A and A790G of 16S rRNA decrease initiation fidelity in vivo and do so in distinct ways. Mutation G1338A increases the affinity of tRNA(fMet) for the 30S subunit, suggesting that G1338 normally forms a suboptimal Type II interaction with fMet-tRNA(fMet). By stabilizing fMet-tRNA(fMet) in the preinitiation complex, G1338A may partially compensate for mismatches in the codon-anti-codon helix and thereby increase spurious initiation. Unlike G1338A, A790G decreases the affinity of IF3 for the 30S subunit. This may indirectly stabilize fMet-tRNA(fMet) in the preinitiation complex and/or promote premature docking of the 50S subunit, resulting in increased levels of spurious initiation.     

A covariant change of the two highly conserved bases in the GTPase-associated center of 28 S rRNA in silkworms and other moths

The GTPase-associated center in 23/28 S rRNA is one of the most conserved functional domains throughout all organisms. We detected a unique sequence of this domain in Bombyx mori species in which the bases at positions 1094 and 1098 (numbering from Escherichia coli 23 S rRNA) are C and G instead of the otherwise universally conserved bases U and A, respectively. These changes were also observed in four other species of moths, but not in organisms other than the moths. Characteristics of the B. mori rRNA domain were investigated by native polyacrylamide gel electrophoresis using RNA fragments containing residues 1030-1128. Although two bands of protein-free RNA appeared on gel, they shifted to a single band when bound to Bombyx ribosomal proteins Bm-L12 and Bm-P complex, equivalent to E. coli L11 and L8, respectively. Bombyx RNA showed lower binding capacity than rat RNA for the ribosomal proteins and anti-28 S autoantibody, specific for a folded structure of the eukaryotic GTPase-associated domain. When the C(1094)/G(1098) bases in Bombyx RNA were replaced by the conserved U/A bases, the protein-free RNA migrated as a single band, and the complex formation with Bm-L12, Bm-P complex, and anti-28 S autoantibody was comparable to that of rat RNA. The results suggest that the GTPase-associated domain of moth-type insects has a labile structural feature that is caused by an unusual covariant change of the U(1094)/A(1098) bases to C/G.     

Iron-mediated degradation of ribosomes under oxidative stress is attenuated by manganese

Protein biosynthesis is fundamental to cellular life and requires the efficient functioning of the translational machinery. At the center of this machinery is the ribosome, a ribonucleoprotein complex that depends heavily on Mg²⁺ for structure. Recent work has indicated that other metal cations can substitute for Mg²⁺, raising questions about the role different metals may play in the maintenance of the ribosome under oxidative stress conditions. Here, we assess ribosomal integrity following oxidative stress both in vitro and in cells to elucidate details of the interactions between Fe²⁺ and the ribosome and identify Mn²⁺ as a factor capable of attenuating oxidant-induced Fe²⁺-mediated degradation of rRNA. We report that Fe²⁺ promotes degradation of all rRNA species of the yeast ribosome and that it is bound directly to RNA molecules. Furthermore, we demonstrate that Mn²⁺ competes with Fe²⁺ for rRNA-binding sites and that protection of ribosomes from Fe²⁺-mediated rRNA hydrolysis correlates with the restoration of cell viability. Our data, therefore, suggest a relationship between these two transition metals in controlling ribosome stability under oxidative stress.