A stereochemical model of the transpeptidation complex

Molecular models are proposed to describe the relative arrangement of aminoacyl and peptidyl tRNAs when bound to their respective A and P sites on the ribosome. The crystallographically determined structures of tRNAasp and tRNAphe have served as the models for these bound structures, while the imposed steric constraints for the model complexes were based on the results of published experimental data. The constructed models satisfy the stereochemical requirements needed for codon-anticodon interaction and for peptide bond formation. In this paper, the results of the complex containing tRNAphe as the A and P site bound transfer RNAs, is compared to a similarly constructed model which uses tRNAasp as the ribosome-bound transfer RNAs. The models have the following three major features: 1) the aminoacyl and peptidyl transfer RNAs assume an angle of approximately 45 degrees relative to each other; 2) in providing the proper stereochemistry for peptide bond condensation, a significant kink must be present in the messenger RNA between the A site and P site codons; and 3) a comparison of the two model complexes indicates that structural variations between the tRNAs or any allosteric transitions of the transfer RNAs associated with codon-anticodon recognition may be accommodated in the model by way of freedom of rotation about the phosphate backbone bonds in the mRNA between consecutive codons.     

An energy transfer equilibrium between two identical copies of a ribosome-bound fluorescent transfer RNA analogue: implications for the possible structure of codon-anticodon complexes.

When yeast tRNA_Pf^Phe, a derivative of tRNA^Phe in which proflavine replaces the Y base, is bound simultaneously to both the peptidyl and aminoacyl sites of a 70 S Escherichia coli ribosome, there is a rapid mutual energy transfer between the two bound tRNAs. Analysis of this energy transfer yields an upper limit for the proflavine-proflavine distance of 20 Å. It also allows an unequivocal measurement of the emission spectrum of tRNA_Pf^Phe bound at the aminoacyl site. In the presence of message this spectrum is very different from that seen in the peptidyl site, implying that in the two sites the hypermodified bases exist in significantly different environments. The rapid energy transfer leads to some loss of fluorescence anisotropy. This can be analyzed to obtain an estimate of the angle between the two proflavines: 28 ° ± 10 ° or 152 ° ± 10 °. Taken together all of these results place severe constraints on possible models of codon-anticodon complexes. The mutual energy transfer seen and analyzed on the ribosome is a convenient aspect of fluorescence spectroscopy, and it is one that should see broad application where multiple copies of a fluorescent ligand interact on a macromolecular substrate.     

The distance between the anticodon loops of two tRNAs bound to the 70 S Escherichia coli ribosome.

Using singlet-singlet energy transfer, we have measured the distance between the anticodons of two transfer RNAs simultaneously bound to a messengerprogramed Escherichia coli 70 S ribosome. The fluorescent Y base adjacent to the anticodon of yeast tRNA_Y^Phe serves as a donor. A proflavine (Pf) chemically substituted for the Y base in tRNA_Pf^Phe serves as an acceptor. By exploiting the sequential binding properties of 70 S ribosomes for two deacylated tRNAs, we can fill the strong site with either tRNA_Y^Phe or tRNA_Pf^Phe and then the weak site with the other tRNA. In both cases donor quenching and sensitized emission of the acceptor are observed. Analysis of these results leads to an estimate for the Y-proflavine distance of 18 ± 2 Å. This distance is very short and suggests strongly that the two tRNAs are simultaneously in contact with adjacent codons of the message. Separate experiments show that binding of a tRNA to the weak site does not perturb the environment of the hypermodified base of a tRNA bound to the strong site. This supports the assignment of the strong site as the peptidyl site. It also indicates that binding of the second tRNA proceeds without a change in the anticodon structure of a pre-existing tRNA at the peptidyl site.     

Doubly Spin-Labeled RNA as an EPR Reporter for Studying Multicomponent Supramolecular Assemblies

mRNAs are involved in complicated supramolecular complexes with human 40S and 80S ribosomes responsible for the protein synthesis. In this work, a derivative of nonaribonucleotide pUUCGUAAAA with nitroxide spin labels attached to the 5′-phosphate and to the C8 atom of the adenosine in sixth position (mRNA analog) was used for studying such complexes using double electron-electron resonance/pulsed electron-electron double resonance spectroscopy. The complexes were assembled with participation of tRNA^Phe, which targeted triplet UUC of the derivative to the ribosomal peptidyl site and predetermined location of the adjacent GUA triplet coding for Val at the aminoacyl (A) site. The interspin distances were measured between the two labels of mRNA analog attached to the first nucleotide of the peptidyl site bound codon and to the third nucleotide of the A site bound codon, in the absence/presence of second tRNA bound at the A site. The values of the obtained interspin distances agree with those calculated for available near-atomic structures of similar complexes of 40S and 80S ribosomes, showing that neither 60S subunit nor tRNA at the A site have a noticeable effect on arrangement of mRNA at the codon-anticodon interaction area. In addition, the shapes of distance distributions in four studied ribosomal complexes allowed conclusions on conformational flexibility of mRNA in these complexes. Overall, the results of this study are the first, to our knowledge, demonstration of double electron-electron resonance/pulsed electron-electron double resonance application for measurements of intramolecular distances in multicomponent supramolecular complexes involving intricate cellular machineries and for evaluating dynamic properties of ligands bound to these machineries.     

Crystal structure of a transfer‐ribonucleoprotein particle that promotes asparagine formation

Four out of the 22 aminoacyl‐tRNAs (aa‐tRNAs) are systematically or alternatively synthesized by an indirect, two‐step route requiring an initial mischarging of the tRNA followed by tRNA‐dependent conversion of the non‐cognate amino acid. During tRNA‐dependent asparagine formation, tRNA^Asn promotes assembly of a ribonucleoprotein particle called transamidosome that allows channelling of the aa‐tRNA from non‐discriminating aspartyl‐tRNA synthetase active site to the GatCAB amidotransferase site. The crystal structure of the Thermus thermophilus transamidosome determined at 3 Å resolution reveals a particle formed by two GatCABs, two dimeric ND‐AspRSs and four tRNAs^Asn molecules. In the complex, only two tRNAs are bound in a functional state, whereas the two other ones act as an RNA scaffold enabling release of the asparaginyl‐tRNA^Asn without dissociation of the complex. We propose that the crystal structure represents a transient state of the transamidation reaction. The transamidosome constitutes a transfer‐ribonucleoprotein particle in which tRNAs serve the function of both substrate and structural foundation for a large molecular machine.     

Activation of several tRNAs of Escherichia coli by the phenoxyacetyl derivative of N-hydroxysuccinimide

Escherichia coli 15T^? treated with chloramphenicol produces tRNA^phe which is deficient in minor nucleosides. Undermodified tRNA^phe chromatographs as two new peaks from a benzoylated diethylaminoethyl-cellulose column. Chloramphenicol tRNA^phe was purified by phenoxyacetylation of phenylalanyl-tRNA and subsequent chromatography on benzoylated diethylaminoethyl-cellulose. Purified tRNA^phe had an altered Chromatographie profile as a result of the purification procedure. Phenoxyacetylation of an unpurified tRNA preparation, which was either charged with phenylalanine or kept discharged, resulted in a permanent alteration of tRNA^phe which was similar to the alteration of the purified tRNA^phe. The altered tRNAs eluted with higher salt or ethanol concentrations from benzoylated diethylaminoethyl-cellulose. The alteration was also shown for tRNA^phe of phenoxyacetylated tRNA from late log phase E. coli 15T?. tRNA^glu and tRNA^Leu were not changed, but both tRNA^Arg and tRNA^Ile were altered. tRNA₂^Val and tRNA^Met shifted in the elution profile; tRNA₁^Val and tRNA_f^Met were not affected.Comparison of the primary structures of the alterable and nonalterable tRNA's revealed that all alterable tRNA's have the undefined nucleoside X in the extra loop. Phenoxyacetylation of nucleoside X probably was the cause of the altered profiles.tRNA^phe from E. coli 15T^? treated with chloramphenicol was less reactive towards phenoxyacetylation than normal tRNA, possibly because of a different conformation of the modification-deficient molecule relative to the normal tRNA^phe. tRNA^phe from E. coli 15T^?, starved for cysteine and methionine and treated with chloram-phenicol, is more deficient in minor nucleosides and showed even less reactivity.Acceptor capacities of the altered tRNA species were not changed significantly; only the acceptor capacity for tRNA^Ile decreased approximately 25%. The recognition site for the aminoacyl-tRNA synthetases probably is not affected.     

Functional Interaction Between Release Factor One and P-site Peptidyl-tRNA on the Ribosome

Translation termination at UAG is influenced by the nature of the 5′ flanking codon inEscherichia coli. Readthrough of the stop codon is always higher in a strain with mutant (prfA1) as compared to wild-type (prfA⁺) release factor one (RF1). Isocodons, which differ in the last base and are decoded by the same tRNA species, affect termination at UAG differently in strains with mutant or wild-type RF1. No general preference of the last codon base to favour readthrough or termination can be found. The data suggest that RF1 is sensitive to the nature of the wobble base anticodon-codon interaction at the ribosomal peptidyl-tRNA binding site (P-site). For some isoaccepting P-site tRNAs (tRNA₃^ProversustRNA₂^Pro, tRNA₄^ThrversustRNA_1,3^Thr) the effect is different on mutant and wild-type RF1, suggesting an interaction between RF1 at the aminoacyl-tRNA acceptor site (A-site) and the P-site tRNA itself. The glycine codons GGA (tRNA₂^Gly) and GGG (tRNA_2,3^Gly) at the ribosomal P-site are associated with an almost threefold higher readthrough of UAG than any of the other 42 codons tested, including the glycine codons GGU/C, in a strain with wild-type RF1. This differential response to the glycine codons is lost in the strain with the mutant form of RF1 since readthrough is increased to a similar high level for all four glycine codons. High α-helix propensity of the last amino acid residue at the C-terminal end of the nascent peptide is correlated with an increased termination at UAG. The effect is stronger on mutant compared to wild-type RF1. The data suggest that RF1-mediated termination at UAG is sensitive to the nature of the codon-anticodon interaction of the wobble base, the last amino acid residue of the nascent peptide chain, and the tRNA at the ribosomal P-site.     

Elongation factor G stabilizes the hybrid-state conformation of the 70S ribosome

Following peptide bond formation, transfer RNAs (tRNAs) and messenger RNA (mRNA) are translocated through the ribosome, a process catalyzed by elongation factor EF-G. Here, we have used a combination of chemical footprinting, peptidyl transferase activity assays, and mRNA toeprinting to monitor the effects of EF-G on the positions of tRNA and mRNA relative to the A, P, and E sites of the ribosome in the presence of GTP, GDP, GDPNP, and fusidic acid. Chemical footprinting experiments show that binding of EF-G in the presence of the non-hydrolyzable GTP analog GDPNP or GDP.fusidic acid induces movement of a deacylated tRNA from the classical P/P state to the hybrid P/E state. Furthermore, stabilization of the hybrid P/E state by EF-G compromises P-site codon-anticodon interaction, causing frame-shifting. A deacylated tRNA bound to the P site and a peptidyl-tRNA in the A site are completely translocated to the E and P sites, respectively, in the presence of EF-G with GTP or GDPNP but not with EF-G.GDP. Unexpectedly, translocation with EF-G.GTP leads to dissociation of deacylated tRNA from the E site, while tRNA remains bound in the presence of EF-G.GDPNP, suggesting that dissociation of tRNA from the E site is promoted by GTP hydrolysis and/or EF-G release. Our results show that binding of EF-G in the presence of GDPNP or GDP.fusidic acid stabilizes the ribosomal intermediate hybrid state, but that complete translocation is supported only by EF-G.GTP or EF-G.GDPNP.     

In vitro reconstitution of eukaryotic translation reveals cooperativity between release factors eRF1 and eRF3

Eukaryotic translation termination is triggered by peptide release factors eRF1 and eRF3. Whereas eRF1 recognizes all three termination codons and induces hydrolysis of peptidyl tRNA, eRF3's function remains obscure. Here, we reconstituted all steps of eukaryotic translation in vitro using purified ribosomal subunits; initiation, elongation, and termination factors; and aminoacyl tRNAs. This allowed us to investigate termination using pretermination complexes assembled on mRNA encoding a tetrapeptide and to propose a model for translation termination that accounts for the cooperative action of eRF1 and eRF3 in ensuring fast release of nascent polypeptide. In this model, binding of eRF1, eRF3, and GTP to pretermination complexes first induces a structural rearrangement that is manifested as a 2 nucleotide forward shift of the toeprint attributed to pretermination complexes that leads to GTP hydrolysis followed by rapid hydrolysis of peptidyl tRNA. Cooperativity between eRF1 and eRF3 required the eRF3 binding C-terminal domain of eRF1.     

Targeting different RNA motifs by beta carboline alkaloid,harmalol: a comparative photophysical,calorimetric, and molecular docking approach

RNA has attracted recent attention for its key role in gene expression and targeting by small molecules for therapeutic intervention. This work focuses towards understanding interaction of harmalol, a DNA intercalator, with RNAs of different motifs viz. single-stranded A-form poly(A), double-stranded A-form of poly(C)·poly(G), and clover leaf tRNA^phe by different spectroscopic, calorimetric, and molecular modeling techniques. Results of this study converge to suggest that (i) binding constant varied in the order poly(C)·poly(G)?>?tRNA^phe > poly(A), (ii) non-cooperative binding of harmalol to poly(C)·poly(G) and poly(A) and cooperative binding with tRNA^phe, (iii) significant structural changes of poly(C)·poly(G) and tRNA^phe with concomitant induction of optical activity in the bound achiral alkaloid molecules, while with poly(A) no induced Circular dichroism (CD) perturbation was observed, (iv) the binding was predominantly exothermic, enthalpy-driven, entropy-favored with poly(C)·poly(G), while it was entropy driven with tRNA^phe and poly(A), (v) a hydrophobic contribution and comparatively large role of non polyelectrolytic forces to Gibbs energy changes with poly(C)·poly(G) and tRNA^phe and (vi) intercalated state of harmalol inside poly(C)·poly(G) structure as revealed from molecular docking was supported by the viscometric and ferrocyanide quenching data. All these findings unequivocally pointed out that harmalol prefers binding with poly(C)·poly(G), compared to tRNA^phe and poly(A); this results serve as data for the development of RNA-based antiviral drugs.     

Ribosome protection by tRNA derivatives against inactivation by virginiamycin M: evidence for two types of interaction of tRNA with the donor site of peptidyl transferase

Virginiamycin M (VM) was previously shown to interfere with the function of both the A and P sites of ribosomes and to inactivate tRNA-free ribosomes but not particles bearing peptidyl-tRNA. To explain these findings, the shielding ability afforded by tRNA derivatives positioned at the A and P sites against VM-produced inactivation was explored. Unacylated tRNA(Phe) was ineffective, irrespective of its position on the ribosome. Phe-tRNA and Ac-Phe-tRNA provided little protection when bound directly to the P site but were active when present at the A site. Protection by these tRNA derivatives was markedly enhanced by the formation of the first peptide bond and increased further upon elongation of peptide chains. Most of the shielding ability of Ac-Phe-tRNA and Phe-tRNA positioned at the A site was conserved when these tRNAs were translocated to the P site by the action of elongation factor G and GTP. Thus, a 5-10-fold difference in the protection afforded by these tRNAs was observed, depending on their mode of entry to the P site. This indicates the occurrence of two types of interaction of tRNA derivatives with the donor site of peptidyl transferase: one shared by acylated tRNAs directly bound to the ribosomal P site (no protection against VM) and the other characteristic of aminoacyl- or peptidyl-tRNA translocated from the A site (protection of peptidyl transferase against VM). To explain these data and previous observations with other protein synthesis inhibitors, a new model of peptidyl transferase is proposed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Visualization of tRNA movements on the Escherichia coli 70S ribosome during the elongation cycle

Three-dimensional cryomaps have been reconstructed for tRNA-ribosome complexes in pre- and posttranslocational states at 17-A resolution. The positions of tRNAs in the A and P sites in the pretranslocational complexes and in the P and E sites in the posttranslocational complexes have been determined. Of these, the P-site tRNA position is the same as seen earlier in the initiation-like fMet-tRNA(f)(Met)-ribosome complex, where it was visualized with high accuracy. Now, the positions of the A- and E-site tRNAs are determined with similar accuracy. The positions of the CCA end of the tRNAs at the A site are different before and after peptide bond formation. The relative positions of anticodons of P- and E-site tRNAs in the posttranslocational state are such that a codon-anticodon interaction at the E site appears feasible.     

Calculation of the relative geometry of tRNAs in the ribosome from directed hydroxyl-radical probing data

The many interactions of tRNA with the ribosome are fundamental to protein synthesis. During the peptidyl transferase reaction, the acceptor ends of the aminoacyl and peptidyl tRNAs must be in close proximity to allow peptide bond formation, and their respective anticodons must base pair simultaneously with adjacent trinucleotide codons on the mRNA. The two tRNAs in this state can be arranged in two nonequivalent general configurations called the R and S orientations, many versions of which have been proposed for the geometry of tRNAs in the ribosome. Here, we report the combined use of computational analysis and tethered hydroxyl-radical probing to constrain their arrangement. We used Fe(II) tethered to the 5' end of anticodon stem-loop analogs (ASLs) of tRNA and to the 5' end of deacylated tRNA(Phe) to generate hydroxyl radicals that probe proximal positions in the backbone of adjacent tRNAs in the 70S ribosome. We inferred probe-target distances from the resulting RNA strand cleavage intensities and used these to calculate the mutual arrangement of A-site and P-site tRNAs in the ribosome, using three different structure estimation algorithms. The two tRNAs are constrained to the S configuration with an angle of about 45 degrees between the respective planes of the molecules. The terminal phosphates of 3'CCA are separated by 23 A when using the tRNA crystal conformations, and the anticodon arms of the two tRNAs are sufficiently close to interact with adjacent codons in mRNA.     

Modified nucleotides in tRNA(Lys) and tRNA(Val) are important for translocation

Ribosomes translate genetic information encoded by messenger RNAs (mRNAs) into proteins. Accurate decoding by the ribosome depends on the proper interaction between the mRNA codon and the anticodon of transfer RNA (tRNA). tRNAs from all kingdoms of life are enzymatically modified at distinct sites, particularly in and near the anticodon. Yet, the role of these naturally occurring tRNA modifications in translation is not fully understood. Here we show that modified nucleosides at the first, or wobble, position of the anticodon and 3'-adjacent to the anticodon are important for translocation of tRNA from the ribosome's aminoacyl site (A site) to the peptidyl site (P site). Thus, naturally occurring modifications in tRNA contribute functional groups and conformational dynamics that are critical for accurate decoding of mRNA and for translocation to the P site during protein synthesis.     

The interaction between C75 of tRNA and the A loop of the ribosome stimulates peptidyl transferase activity

Ribosomal variants carrying mutations in active site nucleotides are severely compromised in their ability to catalyze peptide bond formation (PT) with minimal aminoacyl tRNA substrates such as puromycin. However, catalysis of PT by these same ribosomes with intact aminoacyl tRNA substrates is uncompromised. These data suggest that these active site nucleotides play an important role in the positioning of minimal aminoacyl tRNA substrates but are not essential for catalysis per se when aminoacyl tRNAs are positioned by more remote interactions with the ribosome. Previously reported biochemical studies and atomic resolution X-ray structures identified a direct Watson-Crick interaction between C75 of the A-site substrate and G2553 of the 23S rRNA. Here we show that the addition of this single cytidine residue (the C75 equivalent) to puromycin is sufficient to suppress the deficiencies of active site ribosomal variants, thus restoring "tRNA-like" behavior to this minimal substrate. Studies of the binding parameters and the pH-dependence of catalysis with this minimal substrate indicate that the interaction between C75 and the ribosomal A loop is an essential feature for robust catalysis and further suggest that the observed effects of C75 on peptidyl transfer activity reflect previously reported conformational rearrangements in this active site.     

Orientations of transfer RNA in the ribosomal A and P sites.

In protein synthesis, peptide bond formation requires that the tRNA carrying the amino acid (A site tRNA) contact the tRNA carrying the growing peptide chain (P site tRNA) at their 3' termini. Two models have been proposed for the orientations of two tRNAs as they would be bound to the mRNA in the ribosome. Viewing the tRNA as an upside down L, anticodon loop pointing down, acceptor stem pointing right, and calling this the front view, the R (Rich) model would have the back of the P site tRNA facing the front of the A site tRNA. In the S (Sundaralingam) model the front of the P site tRNA faces the back of the A site tRNA. Models of two tRNAs bound to mRNA as they would be positioned in the ribosomal A and P sites have been created using MC-SYM, a constraint satisfaction search program designed to build nucleic acid structures. The models incorporate information from fluorescence energy transfer experiments and chemical crosslinks. The models that best answer the constraints are of the S variety, with no R conformations produced consistent with the constraints.     

ArfA recruits release factor 2 to rescue stalled ribosomes by peptidyl‐tRNA hydrolysis in Escherichia coli

The ribosomes stalled at the end of non‐stop mRNAs must be rescued for productive cycles of cellular protein synthesis. Escherichia coli possesses at least three independent mechanisms that resolve non‐productive translation complexes (NTCs). While tmRNA (SsrA) mediates trans‐translation to terminate translation, ArfA (YhdL) and ArfB (YaeJ) induce hydrolysis of ribosome‐tethered peptidyl‐tRNAs. ArfB is a paralogue of the release factors (RFs) and directly catalyses the peptidyl‐tRNA hydrolysis within NTCs. In contrast, the mechanism of the ArfA action had remained obscure beyond its ability to bind to the ribosome. Here, we characterized the ArfA pathway of NTC resolution in vitro and identified RF2 as a factor that cooperates with ArfA to hydrolyse peptidyl‐tRNAs located in the P‐site of the stalled ribosome. This reaction required the GGQ (Gly–Gly–Gln) hydrolysis motif, but not the SPF (Ser–Pro–Phe) codon–recognition sequence, of RF2 and was stimulated by tRNAs. From these results we suggest that ArfA binds to the vacant A‐site of the stalled ribosome with possible aid from association with a tRNA, and then recruits RF2, which hydrolyses peptidyl‐tRNA in a GGQ motif‐dependent but codon‐independent manner. In support of this model, the ArfA‐RF2 pathway did not act on the SecM‐arrested ribosome, which contains an aminoacyl‐tRNA in the A‐site.     

Structural Requirements for a Primitive Adaptor Molecule

Abstract

Adaptor properties of linear hairpin helices have been examined. The analysis suggests that neither right nor left handed hairpin helices can simultaneously read a comma free messenger and align aminoacyl residues for peptide condensation. Comparison of these studies with the model of the present day peptidyl transfer intermediate suggests that the “L” shaped folding of the present day tRNAs may be a prerequisite for adaptor function. Therefore, the three-dimensional organization of the ancestral adaptor molecule must have had structural features similar to its present day counterpart.     

Five transfer RNA genes lacking CCA termini are clustered in the chromosome of Streptomyces rimosus

Summary The nucleotide sequence of a 1105 by Streptomyces rimosus DNA fragment containing five transfer RNA genes was determined. Two tRNA^Gln (CUG) genes, differing by 1 by in the aminoacyl stem, and three identical tRNA^Glu (CUC) genes were identified. The five tRNA genes, arranged in the order: Gln1-Glul-Glu2-Gln2-Glu3, were separated by short, nonhomologous intergenic regions. Surprisingly, none of these tRNA genes encoded the CCA 3 terminus of mature tRNAs. All five encoded tRNAs for the translation of GC rich codons, which are preferentially used in Streptomyces genes (CAG and GAG, respectively). We recently reported nucleotide sequences of two initiator tRNA genes from S. rimosus, which also do not encode the CCA end of mature tRNAs. It is therefore very likely that S. rimosus represents an example of those eubacteria in which the majority of tRNA genes do not encode the 3 terminal CCA end of mature tRNAs. Evolutionary implications of this finding remain to be elucidated.     

The Role of Initiator tRNAi met in Fidelity of Initiation of Protein Synthesis

The proper arrangement of amino acids in a protein determines its proper function, which is vital for the cellular metabolism. This indicates that the process of peptide bond formation requires high fidelity. One of the most important processes for this fidelity is kinetic proofreading. As biochemical experiments suggest that kinetic proofreading plays a major role in ensuring the fidelity of protein synthesis, it is not certain whether or not a misacylated tRNA would be corrected by kinetic proofreading during the peptide bond formation. Using 2-layered ONIOM (QM/MM) computational calculations, we studied the behavior of misacylated tRNAs and compared the results with these for cognate aminoacyl-tRNAs during the process of peptide bond formation to investigate the effect of nonnative amino acids on tRNAs. The difference between the behavior of initiator tRNA_i ^met compared to the one for the elongator tRNAs indicates that only the initiator tRNA_i ^met specifies the amino acid side chain.