Analysis of recognition of transfer-messenger RNA by the ribosomal decoding center

Bacterial ribosomes stalled on defective mRNAs are rescued by tmRNA that functions as both tRNA and mRNA. The first ribosomal elongation cycle on tmRNA where tmRNA functions as tRNA is highly unusual: occupation of the ribosomal A site by tmRNA occurs without codon:anticodon pairing. Our analysis shows that in this case the role of a codon:anticodon duplex should be accomplished by a single unpaired triplet. In order that tmRNA could participate in the ribosomal elongation cycle, a triplet preceding the mRNA portion of tmRNA (the -1triplet) should be in the A-form and this form should be recognized by the ribosomal decoding center. A rule is derived that determines what triplets cannot be used as the -1triplet. The rule was tested with the -1triplets of all known 414 tmRNA species. All 23 observed -1triplets follow the formulated rule. The rule is also supported by the available data on base substitutions within the -1triplet.     

Energy landscape of the ribosomal decoding center

The ribosome decodes the genetic information that resides in nucleic acids. A key component of the decoding mechanism is a conformational switch in the decoding center of the small ribosomal subunit discovered in high-resolution X-ray crystallography studies. It is known that small subunit nucleotides A1492 and A1493 flip out of helix 44 upon transfer RNA (tRNA) binding; however, the operation principles of this switch remain unknown. Replica molecular dynamics simulations reveal a low free energy barrier between flipped-out and flipped-in states, consistent with a switch that can be controlled by shifting the equilibrium between states. The barrier determined by the simulations is sufficiently small for the binding of ligands, such as tRNAs or aminoglycoside antibiotics, to shift the equilibrium.     

The codon specificity of eubacterial release factors is determined by the sequence and size of the recognition loop

The two codon-specific eubacterial release factors (RF1: UAA/UAG and RF2: UAA/UGA) have specific tripeptide motifs (PXT/SPF) within an exposed recognition loop shown in recent structures to interact with stop codons during protein synthesis termination. The motifs have been inferred to be critical for codon specificity, but this study shows that they are insufficient to determine specificity alone. Swapping the motifs or the entire loop between factors resulted in a loss of codon recognition rather than a switch of codon specificity. From a study of chimeric eubacterial RF1/RF2 recognition loops and an atypical shorter variant in Caenorhabditis elegans mitochondrial RF1 that lacks the classical tripeptide motif PXT, key determinants throughout the whole loop have been defined. It reveals that more than one configuration of the recognition loop based on specific sequence and size can achieve the same desired codon specificity. This study has provided unexpected insight into why a combination of the two factors is necessary in eubacteria to exclude recognition of UGG as stop.     

Stop codon recognition and interactions with peptide release factor RF3 of truncated and chimeric RF1 and RF2 from Escherichia coli

Release factors RF1 and RF2 recognize stop codons present at the A-site of the ribosome and activate hydrolysis of peptidyl-tRNA to release the peptide chain. Interactions with RF3, a ribosome-dependent GTPase, then initiate a series of reactions that accelerate the dissociation of RF1 or RF2 and their recycling between ribosomes. Two regions of Escherichia coli RF1 and RF2 were identified previously as involved in stop codon recognition and peptidyl-tRNA hydrolysis. We show here that removing the N-terminal domain of RF1 or RF2 or exchanging this domain between the two factors does not affect RF specificity but has different effects on the activity of RF1 and RF2: truncated RF1 remains highly active and able to support rapid cell growth, whereas cells with truncated RF2 grow only poorly. Transplanting a loop of 13 amino acid residues from RF2 to RF1 switches the stop codon specificity. The interaction of the truncated factors with RF3 on the ribosome is defective: they fail to stimulate guanine nucleotide exchange on RF3, recycling is not stimulated by RF3, and nucleotide-free RF3 fails to stabilize the binding of RF1 or RF2 to the ribosome. However, the N-terminal domain seems not to be required for the expulsion of RF1 or RF2 by RF3:GTP.     

eIF3j is located in the decoding center of the human 40S ribosomal subunit

Protein synthesis in all cells begins with the ordered binding of the small ribosomal subunit to messenger RNA (mRNA) and transfer RNA (tRNA). In eukaryotes, translation initiation factor 3 (eIF3) is thought to play an essential role in this process by influencing mRNA and tRNA binding through indirect interactions on the backside of the 40S subunit. Here we show by directed hydroxyl radical probing that the human eIF3 subunit eIF3j binds to the aminoacyl (A) site and mRNA entry channel of the 40S subunit, placing eIF3j directly in the ribosomal decoding center. eIF3j also interacts with eIF1A and reduces 40S subunit affinity for mRNA. A high affinity for mRNA is restored upon recruitment of initiator tRNA, even though eIF3j remains in the mRNA-binding cleft in the presence of tRNA. These results suggest that eIF3j functions in part by regulating access of the mRNA-binding cleft in response to initiation factor binding.     

Photolabile anticodon stem-loop analogs of tRNAPhe as probes of ribosomal structure and structural fluctuation at the decoding center

With the recent availability of high-resolution structures of bacterial ribosomes, studies of ribosome-catalyzed protein biosynthesis are now focusing on the nature of conformational changes that occur as the ribosome exerts its complex catalytic function. Photocrosslinking can be relevant for this purpose by providing clues to ribosomal structural fluctuations and dynamics. Here we describe crosslinking experiments on 70S ribosomes using two photolabile anticodon stem-loop derivatives of Escherichia coli tRNAPhe carrying a 4-thiouridine in either position 33 or 37 and denoted Ph-ASLs. One or both of these Ph-ASLs bind to the tRNA A-, P-, and E-sites on the ribosome, with both binding to and photocrosslinking from the E-site showing strong dependence on the presence of a tRNA in the P-site. Both Ph-ASLs crosslink to the extreme 3'-end of 16S rRNA from both the P- and E-sites, providing direct confirmatory evidence in solution for the folding back of the 3'-end toward the decoding region. This suggests that the 3'-end of 16S rRNA may act as a switch in controlling mRNA access to the decoding center, a phenomenon of potential relevance for the translation of leaderless mRNA. E-site bound Ph-ASLs also form photocrosslinks to nucleotides 1395-1398, 1399-1400, and 1491-1494 at the top of helix 44 of 16S rRNA, indicating movement of the decoding center from a position between the A- and P-sites seen in the crystal structure to one neighboring the E-site.     

RF3 induces ribosomal conformational changes responsible for dissociation of class I release factors

During translation termination, class II release factor RF3 binds to the ribosome to promote rapid dissociation of a class I release factor (RF) in a GTP-dependent manner. We present the crystal structure of E. coli RF3*GDP, which has a three-domain architecture strikingly similar to the structure of EF-Tu*GTP. Biochemical data on RF3 mutants show that a surface region involving domains II and III is important for distinct steps in the action cycle of RF3. Furthermore, we present a cryo-electron microscopy (cryo-EM) structure of the posttermination ribosome bound with RF3 in the GTP form. Our data show that RF3*GTP binding induces large conformational changes in the ribosome, which break the interactions of the class I RF with both the decoding center and the GTPase-associated center of the ribosome, apparently leading to the release of the class I RF.     

Genetic code degeneracy is established by the decoding center of the ribosome

The degeneracy of the genetic code confers a wide array of properties to coding sequences. Yet, its origin is still unclear. A structural analysis has shown that the stability of the Watson–Crick base pair at the second position of the anticodon–codon interaction is a critical parameter controlling the extent of non-specific pairings accepted at the third position by the ribosome, a flexibility at the root of degeneracy. Based on recent cryo-EM analyses, the present work shows that residue A1493 of the decoding center provides a significant contribution to the stability of this base pair, revealing that the ribosome is directly involved in the establishment of degeneracy. Building on existing evolutionary models, we show the evidence that the early appearance of A1493 and A1492 established the basis of degeneracy when an elementary kinetic scheme of translation was prevailing. Logical considerations on the expansion of this kinetic scheme indicate that the acquisition of the peptidyl transferase center was the next major evolutionary step, while the induced-fit mechanism, that enables a sharp selection of the tRNAs, necessarily arose later when G530 was acquired by the decoding center.     

Precise alignment of peptidyl tRNA by the decoding center is essential for EF-G-dependent translocation

Translocation is an essential step in the elongation cycle of the protein synthesis that allows for the continual incorporation of new amino acids to the growing polypeptide. Movement of mRNA and tRNAs within the ribosome is catalyzed by EF-G binding and GTP hydrolysis. The 30S subunit decoding center is crucial for the selection of the cognate tRNA. However, it is not clear whether the decoding center participates in translocation. We disrupted the interactions in the decoding center by mutating the universally conserved 16S rRNA bases G530, A1492, and A1493, and the effects of these mutations on translocation were studied. Our results show that point mutation of any of these 16S rRNA bases inhibits EF-G-dependent translocation. Furthermore, the mutant ribosomes showed increased puromycin reactivity in the pretranslocation complexes, indicating that the dynamic equilibrium of the peptidyl tRNA between the classical and hybrid-state configurations is influenced by contacts in the decoding center.     

Fine-tuning of the ribosomal decoding center by conserved methyl-modifications in the Escherichia coli 16S rRNA

In bacterial 16S rRNAs, methylated nucleosides are clustered within the decoding center, and these nucleoside modifications are thought to modulate translational fidelity. The N⁴, 2′-O-dimethylcytidine (m⁴Cm) at position 1402 of the Escherichia coli 16S rRNA directly interacts with the P-site codon of the mRNA. The biogenesis and function of this modification remain unclear. We have identified two previously uncharacterized genes in E. coli that are required for m⁴Cm formation. mraW (renamed rsmH) and yraL (renamed rsmI) encode methyltransferases responsible for the N⁴ and 2′-O-methylations of C1402, respectively. Recombinant RsmH and RsmI proteins employed the 30S subunit (not the 16S rRNA) as a substrate to reconstitute m⁴Cm1402 in the presence of S-adenosylmethionine (Ado-Met) as the methyl donor, suggesting that m⁴Cm1402 is formed at a late step during 30S assembly in the cell. A luciferase reporter assay indicated that the lack of N⁴ methylation of C1402 increased the efficiency of non-AUG initiation and decreased the rate of UGA read-through. These results suggest that m⁴Cm1402 plays a role in fine-tuning the shape and function of the P-site, thus increasing decoding fidelity.     

Class-1 translation termination factors: invariant GGQ minidomain is essential for release activity and ribosome binding but not for stop codon recognition

Previously, we have shown that all class-1 polypeptide release factors (RFs) share a common glycine-glycine-glutamine (GGQ) motif, which is critical for RF activity. Here, we subjected to site-directed mutagenesis two invariant amino acids, Gln185 and Arg189, situated in the GGQ minidomain of human eRF1, followed by determination of RF activity and the ribosome binding capacity for mutant eRF1. We show that replacement of Gln185 with polar amino acid residues causes partial inactivation of RF activity; Gln185Ile, Arg189Ala and Arg189Gln mutants are completely inactive; all mutants that retain partial RF activity respond similarly to three stop codons. We suggest that loss of RF activity for Gln185 and Arg189 mutants is caused by distortion of the conformation of the GGQ minidomain but not by damage of the stop codon recognition site of eRF1. Our data are inconsistent with the model postulating direct involvement of Gln185 side chain in orientation of water molecule toward peptidyl-tRNA ester bond at the ribosomal peptidyl transferase centre. Most of the Gln185 mutants exhibit reduced ability to bind to the ribosome, probably, to rRNA and/or (peptidyl)-tRNA(s). The data suggest that the GGQ motif is implicated both in promoting peptidyl-tRNA hydrolysis and binding to the ribosome.     

A posttermination ribosomal complex is the guanine nucleotide exchange factor for peptide release factor RF3

The mechanism by which peptide release factor RF3 recycles RF1 and RF2 has been clarified and incorporated in a complete scheme for translation termination. Free RF3 is in vivo stably bound to GDP, and ribosomes in complex with RF1 or RF2 act as guanine nucleotide exchange factors (GEF). Hydrolysis of peptidyl-tRNA by RF1 or RF2 allows GTP binding to RF3 on the ribosome. This induces an RF3 conformation with high affinity for ribosomes and leads to rapid dissociation of RF1 or RF2. Dissociation of RF3 from the ribosome requires GTP hydrolysis. Our data suggest that RF3 and its eukaryotic counterpart, eRF3, have mechanistic principles in common.     

Touch induces ATP release in Arabidopsis roots that is modulated by the heterotrimeric G-protein complex

Amongst the many stimuli orienting the growth of plant roots, of critical importance are the touch signals generated as roots explore the mechanically complex soil environment. However, the molecular mechanisms behind these sensory events remain poorly defined. We report an impaired obstacle-avoiding response of roots in Arabidopsis lacking a heterotrimeric G-protein. Obstacle avoidance may utilize a touch-induced release of ATP to the extracellular space. While sequential touch stimulation revealed a strong refractory period for ATP release in response to mechano-stimulation in wild-type plants, the refractory period in mutants was attenuated, resulting in extracellular ATP accumulation. We propose that ATP acts as an extracellular signal released by mechano-stimulation and that the G-protein complex is needed for fine-tuning this response.     

Nucleotide G-1207 of 18S rRNA is an essential component of the human 80S ribosomal decoding center.

mRNA analogues-derivatives of oligoribonucleotides consisting of two different codons and bearing an aryl azide group at the 5'-phosphates-were crosslinked to human 80S ribosomes by UV-irradiation of the various model complexes obtained in the presence of the cognate tRNAs. Three sequences, namely pUUUGUU (coding for Phe and Val), pUUCUAAA (first triplet coding for Phe and second being stop-codon), and pGUGUUU (coding for Val and Phe), have been used. Sequences of 18S rRNA containing nucleotides crosslinked to the mRNA analogues were examined by hydrolysis with RNase H in the presence of various cDNA probes. Crosslinked nucleotides were identified by primer extension. In all cases, only nucleotide G-1207 (equivalent to G-926 in Escherichia coli 16S rRNA) has been detected as crosslinked. Crosslinking of the mRNA analogues to the large ribosomal subunit was negligible.     

Molecular mechanism of stop codon recognition by eRF1: a wobble hypothesis for peptide anticodons

We propose that the amino acid residues 57/58 and 60/61 of eukaryotic release factors (eRF1s) (counted from the N-terminal Met of human eRF1) are responsible for stop codon recognition in protein synthesis. The proposal is based on amino acid exchanges in these positions in the eRF1s of two ciliates that reassigned one or two stop codons to sense codons in evolution and on the crystal structure of human eRF1. The proposed mechanism of stop codon recognition assumes that the amino acid residues 57/58 interact with the second and the residues 60/61 with the third position of a stop codon. The fact that conventional eRF1s recognize all three stop codons but not the codon for tryptophan is attributed to the flexibility of the helix containing these residues. We suggest that the helix is able to assume a partly relaxed or tight conformation depending on the stop codon recognized. The restricted codon recognition observed in organisms with unconventional eRF1s is attributed mainly to the loss of flexibility of the helix due to exchanged amino acids.     

Mutations in conserved regions of ribosomal RNAs decrease the productive association of peptide-chain release factors with the ribosome during translation termination

Early studies provided evidence that peptide-chain release factors (RFs) bind to both ribosomal subunits and trigger translation termination. Although many ribosomal proteins have been implicated in termination, very few data present direct biochemical evidence for the involvement of rRNA. Particularly absent is direct evidence for a role of a large subunit rRNA in RF binding. Previously we demonstrated in vitro that mutations in Escherichia coli rRNAs, known to cause nonsense codon readthrough in vivo, reduce the efficiency of RF2-driven catalysis of peptidyl-tRNA hydrolysis. This reduction was consistent with the idea that in vivo defective termination at the mutant ribosomes contributes to the readthrough. Nevertheless, other explanations were also possible, because still missing was essential biochemical evidence for that idea, namely, decrease in productive association of RFs with the mutant ribosomes. Here we present such evidence using a new realistic in vitro termination assay. This study directly supports in vivo involvement in termination of conserved rRNA regions that also participate in other translational events. Furthermore, this study provides the first strong evidence for involvement of large subunit rRNA in RF binding, indicating that the same rRNA region interacts with factors that determine both elongation and termination of translation.