Partial spontaneous intersubunit rotations in pretranslocation ribosomes.

The two main steps of translation, peptidyl transfer, and translocation are accompanied by counterclockwise and clockwise rotations of the large and small ribosomal subunits with respect to each other. Upon peptidyl transfer, the small ribosomal subunit rotates counterclockwise relative to the large subunit, placing the ribosome into the rotated conformation. Simultaneously, tRNAs move into the hybrid conformation, and the L1 stalk moves inward toward the P-site tRNA. The conformational dynamics of pretranslocation ribosomes were extensively studied by ensemble and single-molecule methods. Different experimental modalities tracking ribosomal subunits, tRNAs, and the L1 stalk showed that pretranslocation ribosomes undergo spontaneous conformational transitions. Thus, peptidyl transfer unlocks the ribosome and decreases an energy barrier for the reverse ribosome rotation during translocation. However, the tracking of translation with ribosomes labeled at rRNA helices h44 and H101 showed a lack of spontaneous rotations in pretranslocation complexes. Therefore, reverse intersubunit rotations occur during EF-G catalyzed translocation. To reconcile these views, we used high-speed single-molecule microscopy to follow translation in real time. We showed spontaneous rotations in puromycin-released h44-H101 dye-labeled ribosomes. During elongation, the h44-H101 ribosomes undergo partial spontaneous rotations. Spontaneous rotations in h44-H101-labeled ribosomes are restricted prior to aminoacyl-tRNA binding. The pretranslocation h44-H101 ribosomes spontaneously exchanged between three different rotational states. This demonstrates that peptidyl transfer unlocks spontaneous rotations and pretranslocation ribosomes can adopt several thermally accessible conformations, thus supporting the Brownian model of translocation.

Dynamics of release factor recycling during translation termination in bacteria.

In bacteria, release of newly synthesized proteins from ribosomes during translation termination is catalyzed by class-I release factors (RFs) RF1 or RF2, reading UAA and UAG or UAA and UGA codons, respectively. Class-I RFs are recycled from the post-termination ribosome by a class-II RF, the GTPase RF3, which accelerates ribosome intersubunit rotation and class-I RF dissociation. How conformational states of the ribosome are coupled to the binding and dissociation of the RFs remains unclear and the importance of ribosome-catalyzed guanine nucleotide exchange on RF3 for RF3 recycling in vivo has been disputed. Here, we profile these molecular events using a single-molecule fluorescence assay to clarify the timings of RF3 binding and ribosome intersubunit rotation that trigger class-I RF dissociation, GTP hydrolysis, and RF3 dissociation. These findings in conjunction with quantitative modeling of intracellular termination flows reveal rapid ribosome-dependent guanine nucleotide exchange to be crucial for RF3 action in vivo.

Uncovering translation roadblocks during the development of a synthetic tRNA.

Ribosomes are remarkable in their malleability to accept diverse aminoacyl-tRNA substrates from both the same organism and other organisms or domains of life. This is a critical feature of the ribosome that allows the use of orthogonal translation systems for genetic code expansion. Optimization of these orthogonal translation systems generally involves focusing on the compatibility of the tRNA, aminoacyl-tRNA synthetase, and a non-canonical amino acid with each other. As we expand the diversity of tRNAs used to include non-canonical structures, the question arises as to the tRNA suitability on the ribosome. Specifically, we investigated the ribosomal translation of allo-tRNAUTu1, a uniquely shaped (9/3) tRNA exploited for site-specific selenocysteine insertion, using single-molecule fluorescence. With this technique we identified ribosomal disassembly occurring from translocation of allo-tRNAUTu1 from the A to the P site. Using cryo-EM to capture the tRNA on the ribosome, we pinpointed a distinct tertiary interaction preventing fluid translocation. Through a single nucleotide mutation, we disrupted this tertiary interaction and relieved the translation roadblock. With the continued diversification of genetic code expansion, our work highlights a targeted approach to optimize translation by distinct tRNAs as they move through the ribosome.

Continued expansion of the genetic code has required the use of synthetic tRNAs for decoding. Some of these synthetic tRNAs have unique structural features that are not observed in canonical tRNAs. Here, the authors applied single-molecule, biochemical and structural methods to determine whether these distinct features were deleterious for efficient protein translation on the ribosome. With a focus on selenocysteine insertion, the authors explored an allo-tRNA with a 9/3 acceptor domain. They observed a translational roadblock that occurred in A to P site tRNA translocation. This block was mediated by a tertiary interaction across the tRNA core, directing the variable arm position into an unfavorable conformation. A single-nucleotide mutation disrupted this interaction, providing flexibility in the variable arm and promoting efficient protein production.

Mechanisms that ensure speed and fidelity in eukaryotic translation termination.

Translation termination, which liberates a nascent polypeptide from the ribosome specifically at stop codons, must occur accurately and rapidly. We established single-molecule fluorescence assays to track the dynamics of ribosomes and two requisite release factors (eRF1 and eRF3) throughout termination using an in vitro-reconstituted yeast translation system. We found that the two eukaryotic release factors bound together to recognize stop codons rapidly and elicit termination through a tightly regulated, multistep process that resembles transfer RNA selection during translation elongation. Because the release factors are conserved from yeast to humans, the molecular events that underlie yeast translation termination are likely broadly fundamental to eukaryotic protein synthesis.

N 6-Methyladenosines in mRNAs reduce the accuracy of codon reading by transfer RNAs and peptide release factors.

We used quench flow to study how N6-methylated adenosines (m6A) affect the accuracy ratio between kcat/Km (i.e. association rate constant (ka) times probability (Pp) of product formation after enzyme-substrate complex formation) for cognate and near-cognate substrate for mRNA reading by tRNAs and peptide release factors 1 and 2 (RFs) during translation with purified Escherichia coli components. We estimated kcat/Km for Glu-tRNAGlu, EF-Tu and GTP forming ternary complex (T3) reading cognate (GAA and Gm6AA) or near-cognate (GAU and Gm6AU) codons. ka decreased 10-fold by m6A introduction in cognate and near-cognate cases alike, while Pp for peptidyl transfer remained unaltered in cognate but increased 10-fold in near-cognate case leading to 10-fold amino acid substitution error increase. We estimated kcat/Km for ester bond hydrolysis of P-site bound peptidyl-tRNA by RF2 reading cognate (UAA and Um6AA) and near-cognate (UAG and Um6AG) stop codons to decrease 6-fold or 3-fold by m6A introduction, respectively. This 6-fold effect on UAA reading was also observed in a single-molecule termination assay. Thus, m6A reduces both sense and stop codon reading accuracy by decreasing cognate significantly more than near-cognate kcat/Km, in contrast to most error inducing agents and mutations, which increase near-cognate at unaltered cognate kcat/Km.

eIF5B gates the transition from translation initiation to elongation.

Translation initiation determines both the quantity and identity of the protein that is encoded in an mRNA by establishing the reading frame for protein synthesis. In eukaryotic cells, numerous translation initiation factors prepare ribosomes for polypeptide synthesis; however, the underlying dynamics of this process remain unclear1,2. A central question is how eukaryotic ribosomes transition from translation initiation to elongation. Here we use in vitro single-molecule fluorescence microscopy approaches in a purified yeast Saccharomyces cerevisiae translation system to monitor directly, in real time, the pathways of late translation initiation and the transition to elongation. This transition was slower in our eukaryotic system than that reported for Escherichia coli3-5. The slow entry to elongation was defined by a long residence time of eukaryotic initiation factor 5B (eIF5B) on the 80S ribosome after the joining of individual ribosomal subunits-a process that is catalysed by this universally conserved initiation factor. Inhibition of the GTPase activity of eIF5B after the joining of ribosomal subunits prevented the dissociation of eIF5B from the 80S complex, thereby preventing elongation. Our findings illustrate how the dissociation of eIF5B serves as a kinetic checkpoint for the transition from initiation to elongation, and how its release may be governed by a change in the conformation of the ribosome complex that triggers GTP hydrolysis.

Relating Structure and Dynamics in RNA Biology.

Recent advances in structural biology methods have enabled a surge in the number of RNA and RNA-protein assembly structures available at atomic or near-atomic resolution. These complexes are often trapped in discrete conformational states that exist along a mechanistic pathway. Single-molecule fluorescence methods provide temporal resolution to elucidate the dynamic mechanisms of processes involving complex RNA and RNA-protein assemblies, but interpretation of such data often requires previous structural knowledge. Here we highlight how single-molecule tools can directly complement structural approaches for two processes--translation and reverse transcription-to provide a dynamic view of molecular function.

Single-Molecule Fluorescence Applied to Translation.

Single-molecule fluorescence methods have illuminated the dynamics of the translational machinery. Structural and bulk biochemical experiments have provided detailed atomic and global mechanistic views of translation, respectively. Single-molecule studies of translation have bridged these views by temporally connecting the conformational and compositional states defined from structural data within the mechanistic framework of translation produced from biochemical studies. Here, we discuss the context for applying different single-molecule fluorescence experiments, and present recent applications to studying prokaryotic and eukaryotic translation. We underscore the power of observing single translating ribosomes to delineate and sort complex mechanistic pathways during initiation and elongation, and discuss future applications of current and improved technologies.

How Messenger RNA and Nascent Chain Sequences Regulate Translation Elongation.

Translation elongation is a highly coordinated, multistep, multifactor process that ensures accurate and efficient addition of amino acids to a growing nascent-peptide chain encoded in the sequence of translated messenger RNA (mRNA). Although translation elongation is heavily regulated by external factors, there is clear evidence that mRNA and nascent-peptide sequences control elongation dynamics, determining both the sequence and structure of synthesized proteins. Advances in methods have driven experiments that revealed the basic mechanisms of elongation as well as the mechanisms of regulation by mRNA and nascent-peptide sequences. In this review, we highlight how mRNA and nascent-peptide elements manipulate the translation machinery to alter the dynamics and pathway of elongation.

2'-O-methylation in mRNA disrupts tRNA decoding during translation elongation.

Chemical modifications of mRNA may regulate many aspects of mRNA processing and protein synthesis. Recently, 2'-O-methylation of nucleotides was identified as a frequent modification in translated regions of human mRNA, showing enrichment in codons for certain amino acids. Here, using single-molecule, bulk kinetics and structural methods, we show that 2'-O-methylation within coding regions of mRNA disrupts key steps in codon reading during cognate tRNA selection. Our results suggest that 2'-O-methylation sterically perturbs interactions of ribosomal-monitoring bases (G530, A1492 and A1493) with cognate codon-anticodon helices, thereby inhibiting downstream GTP hydrolysis by elongation factor Tu (EF-Tu) and A-site tRNA accommodation, leading to excessive rejection of cognate aminoacylated tRNAs in initial selection and proofreading. Our current and prior findings highlight how chemical modifications of mRNA tune the dynamics of protein synthesis at different steps of translation elongation.

Post-termination Ribosome Intermediate Acts as the Gateway to Ribosome Recycling.

During termination of translation, the nascent peptide is first released from the ribosome, which must be subsequently disassembled into subunits in a process known as ribosome recycling. In bacteria, termination and recycling are mediated by the translation factors RF, RRF, EF-G, and IF3, but their precise roles have remained unclear. Here, we use single-molecule fluorescence to track the conformation and composition of the ribosome in real time during termination and recycling. Our results show that peptide release by RF induces a rotated ribosomal conformation. RRF binds to this rotated intermediate to form the substrate for EF-G that, in turn, catalyzes GTP-dependent subunit disassembly. After the 50S subunit departs, IF3 releases the deacylated tRNA from the 30S subunit, thus preventing reassembly of the 70S ribosome. Our findings reveal the post-termination rotated state as the crucial intermediate in the transition from termination to recycling.

Dynamic basis of fidelity and speed in translation: Coordinated multistep mechanisms of elongation and termination.

As the universal machine that transfers genetic information from RNA to protein, the ribosome synthesizes proteins with remarkably high fidelity and speed. This is a result of the accurate and efficient decoding of mRNA codons via multistep mechanisms during elongation and termination stages of translation. These mechanisms control how the correct sense codon is recognized by a tRNA for peptide elongation, how the next codon is presented to the decoding center without change of frame during translocation, and how the stop codon is discriminated for timely release of the nascent peptide. These processes occur efficiently through coupling of chemical energy expenditure, ligand interactions, and conformational changes. Understanding this coupling in detail required integration of many techniques that were developed in the past two decades. This multidisciplinary approach has revealed the dynamic nature of translational control and uncovered how external cellular factors such as tRNA abundance and mRNA modifications affect the synthesis of the protein product. Insights from these studies will aid synthetic biology and therapeutic approaches to translation.

The molecular choreography of protein synthesis: translational control, regulation, and pathways.

Translation of proteins by the ribosome regulates gene expression, with recent results underscoring the importance of translational control. Misregulation of translation underlies many diseases, including cancer and many genetic diseases. Decades of biochemical and structural studies have delineated many of the mechanistic details in prokaryotic translation, and sketched the outlines of eukaryotic translation. However, translation may not proceed linearly through a single mechanistic pathway, but likely involves multiple pathways and branchpoints. The stochastic nature of biological processes would allow different pathways to occur during translation that are biased by the interaction of the ribosome with other translation factors, with many of the steps kinetically controlled. These multiple pathways and branchpoints are potential regulatory nexus, allowing gene expression to be tuned at the translational level. As research focus shifts toward eukaryotic translation, certain themes will be echoed from studies on prokaryotic translation. This review provides a general overview of the dynamic data related to prokaryotic and eukaryotic translation, in particular recent findings with single-molecule methods, complemented by biochemical, kinetic, and structural findings. We will underscore the importance of viewing the process through the viewpoints of regulation, translational control, and heterogeneous pathways.

N(6)-methyladenosine in mRNA disrupts tRNA selection and translation-elongation dynamics.

N(6)-methylation of adenosine (forming m(6)A) is the most abundant post-transcriptional modification within the coding region of mRNA, but its role during translation remains unknown. Here, we used bulk kinetic and single-molecule methods to probe the effect of m(6)A in mRNA decoding. Although m(6)A base-pairs with uridine during decoding, as shown by X-ray crystallographic analyses of Thermus thermophilus ribosomal complexes, our measurements in an Escherichia coli translation system revealed that m(6)A modification of mRNA acts as a barrier to tRNA accommodation and translation elongation. The interaction between an m(6)A-modified codon and cognate tRNA echoes the interaction between a near-cognate codon and tRNA, because delay in tRNA accommodation depends on the position and context of m(6)A within codons and on the accuracy level of translation. Overall, our results demonstrate that chemical modification of mRNA can change translational dynamics.

A structural role for the PHP domain in E. coli DNA polymerase III.

BACKGROUND: In addition to the core catalytic machinery, bacterial replicative DNA polymerases contain a Polymerase and Histidinol Phosphatase (PHP) domain whose function is not entirely understood. The PHP domains of some bacterial replicases are active metal-dependent nucleases that may play a role in proofreading. In E. coli DNA polymerase III, however, the PHP domain has lost several metal-coordinating residues and is likely to be catalytically inactive. RESULTS: Genomic searches show that the loss of metal-coordinating residues in polymerase PHP domains is likely to have coevolved with the presence of a separate proofreading exonuclease that works with the polymerase. Although the E. coli Pol III PHP domain has lost metal-coordinating residues, the structure of the domain has been conserved to a remarkable degree when compared to that of metal-binding PHP domains. This is demonstrated by our ability to restore metal binding with only three point mutations, as confirmed by the metal-bound crystal structure of this mutant determined at 2.9 Å resolution. We also show that Pol III, a large multi-domain protein, unfolds cooperatively and that mutations in the degenerate metal-binding site of the PHP domain decrease the overall stability of Pol III and reduce its activity. CONCLUSIONS: While the presence of a PHP domain in replicative bacterial polymerases is strictly conserved, its ability to coordinate metals and to perform proofreading exonuclease activity is not, suggesting additional non-enzymatic roles for the domain. Our results show that the PHP domain is a major structural element in Pol III and its integrity modulates both the stability and activity of the polymerase.

Folate binding site of flavin-dependent thymidylate synthase.

The DNA nucleotide thymidylate is synthesized by the enzyme thymidylate synthase, which catalyzes the reductive methylation of deoxyuridylate using the cofactor methylene-tetrahydrofolate (CH(2)H(4)folate). Most organisms, including humans, rely on the thyA- or TYMS-encoded classic thymidylate synthase, whereas, certain microorganisms, including all Rickettsia and other pathogens, use an alternative thyX-encoded flavin-dependent thymidylate synthase (FDTS). Although several crystal structures of FDTSs have been reported, the absence of a structure with folates limits understanding of the molecular mechanism and the scope of drug design for these enzymes. Here we present X-ray crystal structures of FDTS with several folate derivatives, which together with mutagenesis, kinetic analysis, and computer modeling shed light on the cofactor binding and function. The unique structural data will likely facilitate further elucidation of FDTSs' mechanism and the design of structure-based inhibitors as potential leads to new antimicrobial drugs.