tRNA Binding Sites Research Articles

Extraribosomal Functions of Bacterial Ribosomal Proteins-An Update, 2023.

Ribosomal proteins (r-proteins) are abundant, highly conserved, and multifaceted cellular proteins in all domains of life. Most r-proteins have RNA-binding properties and can form protein-protein contacts. Bacterial r-proteins govern the co-transcriptional rRNA folding during ribosome assembly and participate in the formation of the ribosome functional sites, such as the mRNA-binding site, tRNA-binding sites, the peptidyl transferase center, and the protein exit tunnel. In addition to their primary role in a cell as integral components of the protein synthesis machinery, many r-proteins can function beyond the ribosome (the phenomenon known as moonlighting), acting either as individual regulatory proteins or in complexes with various cellular components. The extraribosomal activities of r-proteins have been studied over the decades. In the past decade, our understanding of r-protein functions has advanced significantly due to intensive studies on ribosomes and gene expression mechanisms not only in model bacteria like Escherichia coli or Bacillus subtilis but also in little-explored bacterial species from various phyla. The aim of this review is to update information on the multiple functions of r-proteins in bacteria.

The UFM1 E3 ligase recognizes and releases 60S ribosomes from ER translocons

Stalled ribosomes at the endoplasmic reticulum (ER) are covalently modified with the ubiquitin-like protein UFM1 on the 60S ribosomal subunit protein RPL26 (also known as uL24)1,2. This modification, which is known as UFMylation, is orchestrated by the UFM1 ribosome E3 ligase (UREL) complex, comprising UFL1, UFBP1 and CDK5RAP3 (ref. 3). However, the catalytic mechanism of UREL and the functional consequences of UFMylation are unclear. Here we present cryo-electron microscopy structures of UREL bound to 60S ribosomes, revealing the basis of its substrate specificity. UREL wraps around the 60S subunit to form a C-shaped clamp architecture that blocks the tRNA-binding sites at one end, and the peptide exit tunnel at the other. A UFL1 loop inserts into and remodels the peptidyl transferase centre. These features of UREL suggest a crucial function for UFMylation in the release and recycling of stalled or terminated ribosomes from the ER membrane. In the absence of functional UREL, 60S–SEC61 translocon complexes accumulate at the ER membrane, demonstrating that UFMylation is necessary for releasing SEC61 from 60S subunits. Notably, this release is facilitated by a functional switch of UREL from a ‘writer’ to a ‘reader’ module that recognizes its product—UFMylated 60S ribosomes. Collectively, we identify a fundamental role for UREL in dissociating 60S subunits from the SEC61 translocon and the basis for UFMylation in regulating protein homeostasis at the ER.

Structural and functional insights into tRNA recognition by human tRNA guanine transglycosylase

Eukaryotic tRNA guanine transglycosylase (TGT) is an RNA-modifying enzyme which catalyzes the base exchange of the genetically encoded guanine 34 of tRNAsAsp,Asn,His,Tyr for queuine, a hypermodified 7-deazaguanine derivative. Eukaryotic TGT is a heterodimer comprised of a catalytic and a non-catalytic subunit. While binding of the tRNA anticodon loop to the active site is structurally well understood, the contribution of the non-catalytic subunit to tRNA binding remained enigmatic, as no complex structure with a complete tRNA was available. Here, we report a cryo-EM structure of eukaryotic TGT in complex with a complete tRNA, revealing the crucial role of the non-catalytic subunit in tRNA binding. We decipher the functional significance of these additional tRNA-binding sites, analyze solution state conformation, flexibility, and disorder of apo TGT, and examine conformational transitions upon tRNA binding.

An Atlas of the base inter-RNA stacks involved in bacterial translation

Nucleobase-specific noncovalent interactions play a crucial role in translation. Herein, we provide a comprehensive analysis of the stacks between different RNA components in the crystal structures of the bacterial ribosome caught at different translation stages. Analysis of tRNA||rRNA stacks reveals distinct behaviour; both the A-and E-site tRNAs exhibit unique stacking patterns with 23S rRNA bases, while P-site tRNAs stack with 16S rRNA bases. Furthermore, E-site stacks exhibit diverse face orientations and ring topologies—rare for inter-chain RNA interactions—with higher average interaction energies than A or P-site stacks. This suggests that stacking may be essential for stabilizing tRNA progression through the E-site. Additionally, mRNA||rRNA stacks reveal other geometries, which depend on the tRNA binding site, whereas 16S rRNA||23S rRNA stacks highlight the importance of specific bases in maintaining the integrity of the translational complex by linking the two rRNAs. Furthermore, tRNA||mRNA stacks exhibit distinct geometries and energetics at the E-site, indicating their significance during tRNA translocation and elimination. Overall, both A and E-sites display a more diverse distribution of inter-RNA stacks compared to the P-site. Stacking interactions in the active ribosome are not simply accidental byproducts of biochemistry but are likely invoked to compensate and support the integrity and dynamics of translation.

Dependence of P/E tRNA hybrid formation on subunit rotation in the ribosome.

The ribosome, a massive nucleoprotein assembly that executes protein synthesis in all living cells, undergoes several large-scale functional rearrangements during the protein elongation cycle. Here we explore two such rearrangements in the ribosome bound tRNA molecules during the translocation step in bacteria, namely, P/E tRNA hybrid formation and inter-subunit rotation in the ribosome. We utilize multi-basin all-atom structure-based models and Ribosome Angle Decomposition (RAD) method to study the dependence of P/E hybrid formation on rotation of small subunit body with respect to the large subunit. Structure-based models provide simplified energetics with experimental atomic structures defined to be the potential energy minima. We employ these models to simulate spontaneous transition of the P-tRNA from P to E tRNA binding site on the large subunit for different degrees of small subunit body rotation. RAD method provides a complete coordinate system that describes any orientation of the ribosomal small subunit body and head in terms of rotation, tilt, tilt direction and translation. We identify reference E. coli structures at different degrees of body rotation but with minimal body tilt, head rotation and head tilt from the Ribosome Analysis Database based on RAD. Utilizing these methods, we explore the influence of subunit rotation on the free-energy barrier associated with P/E tRNA hybrid formation and obtain the quantitative dependence of P/E hybrid formation on subunit rotation.

Mutagenesis of Escherichia coli for sm-FRET and FIRMS study of domain IV EF-G studies.

The translation of mRNA into protein is performed by a ribonucleoprotein complex which consists of two subunits which come together to form three tRNA binding sites; the A-, P-, and E-site. The complex is associated with several proteins, one of which is Elongation Factor G (EF-G) which advances the mRNA sequence through the ribosome in a process known as translocation. During translocation tRNAs move from the A- and P-sites to the P- and E-sites, respectively, and the ribosome slides the mRNA exactly three nucleotides. Two key residues (Q508 and H584) in the domain IV region of EF-G form interactions with nucleotides of the A-site tRNA during translocation. The primary goal of this project will be to reveal the function of EF-G mediated translocation via mutagenesis of these two residues. Single molecule fluorescent resonance energy transfer (sm-FRET) will be used to determine the confirmational change of mutant EF-G. Force production of these mutants will also be measured using force-induced remnant magnetization spectroscopy (FIRMS) to determine the overall effect the domain IV mutants have on power-stroke force. We expect that the EF-G mutants will only be able to complete partial translocation and will have a reduction in force exertion, while GTP hydrolysis will not compromised, which will imply an active role in the translocation mechanism by these residues. These findings together will visualize the ribosome translocation mechanism with sub-nucleotide resolution, which only the FIRMS method can do to our best knowledge.

Conformational dynamics of eukaryotic translation.

The ribosome is a molecular machine that adopts multiple conformations during translation. The body of the small ribosomal subunit rotates relatively to the large ribosomal subunit by about 9 degrees. This movement reconfigures tRNA binding sites, which results in a partial movement of tRNAs on the ribosome. At the beginning of the elongation cycle, the ribosome awaits A-site decoding and occupies the non-rotated conformation. Upon A-site tRNA binding, the ribosome rapidly catalyzes peptidyl transfer. It is followed by counterclockwise rotations of the small ribosomal subunit that puts the ribosome into the rotated state. The resultant pre-translocation complex is dynamic and exchanges between rotated and non-rotated conformations. Eukaryotic elongation factor 2 (eEF2) catalyzes translocation, which moves tRNA-mRNA and resets the ribosome into the non-rotated state. Despite extensive biochemical and structural studies, the underlying mechanism of translation elongation remains elusive. We developed a single-molecule fluorescence resonance energy transfer (smFRET) system that allows the observation of intersubunit conformational change of yeast ribosomes in real-time. We followed the ribosome conformational change in pre- and post-translocation ribosome complexes. To interrogate the mechanism of translocation, we applied antifungal sordarin. Sordarin stabilizes eEF2 on the ribosome after GTP hydrolysis and phosphate release. Our results suggest that in the presence of sordarin, the mid and late stages of translocation are thermally driven.

Structural basis for the dynamics of human methionyl-tRNA synthetase in multi-tRNA synthetase complexes.

In mammals, eight aminoacyl-tRNA synthetases (AARSs) and three AARS-interacting multifunctional proteins (AIMPs) form a multi-tRNA synthetase complex (MSC). MSC components possess extension peptides for MSC assembly and specific functions. Human cytosolic methionyl-tRNA synthetase (MRS) has appended peptides at both termini of the catalytic main body. The N-terminal extension includes a glutathione transferase (GST) domain responsible for interacting with AIMP3, and a long linker peptide between the GST and catalytic domains. Herein, we determined crystal structures of the human MRS catalytic main body, and the complex of the GST domain and AIMP3. The structures reveal human-specific structural details of the MRS, and provide a dynamic model for MRS at the level of domain orientation. A movement of zinc knuckles inserted in the catalytic domain is required for MRS catalytic activity. Depending on the position of the GST domain relative to the catalytic main body, MRS can either block or present its tRNA binding site. Since MRS is part of a huge MSC, we propose a dynamic switching between two possible MRS conformations; a closed conformation in which the catalytic domain is compactly attached to the MSC, and an open conformation with a free catalytic domain dissociated from other MSC components.

Inhibitory mechanism of reveromycin A at the tRNA binding site of a class I synthetase

The polyketide natural product reveromycin A (RM-A) exhibits antifungal, anticancer, anti-bone metastasis, anti-periodontitis and anti-osteoporosis activities by selectively inhibiting eukaryotic cytoplasmic isoleucyl-tRNA synthetase (IleRS). Herein, a co-crystal structure suggests that the RM-A molecule occupies the substrate tRNAIle binding site of Saccharomyces cerevisiae IleRS (ScIleRS), by partially mimicking the binding of tRNAIle. RM-A binding is facilitated by the copurified intermediate product isoleucyl-adenylate (Ile-AMP). The binding assays confirm that RM-A competes with tRNAIle while binding synergistically with l-isoleucine or intermediate analogue Ile-AMS to the aminoacylation pocket of ScIleRS. This study highlights that the vast tRNA binding site of the Rossmann-fold catalytic domain of class I aminoacyl-tRNA synthetases could be targeted by a small molecule. This finding will inform future rational drug design.

Differences in structure and hibernation mechanism highlight diversification of the microsporidian ribosome.

Assembling and powering ribosomes are energy-intensive processes requiring fine-tuned cellular control mechanisms. In organisms operating under strict nutrient limitations, such as pathogenic microsporidia, conservation of energy via ribosomal hibernation and recycling is critical. The mechanisms by which hibernation is achieved in microsporidia, however, remain poorly understood. Here, we present the cryo-electron microscopy structure of the ribosome from Paranosema locustae spores, bound by the conserved eukaryotic hibernation and recycling factor Lso2. The microsporidian Lso2 homolog adopts a V-shaped conformation to bridge the mRNA decoding site and the large subunit tRNA binding sites, providing a reversible ribosome inactivation mechanism. Although microsporidian ribosomes are highly compacted, the P. locustae ribosome retains several rRNA segments absent in other microsporidia, and represents an intermediate state of rRNA reduction. In one case, the near complete reduction of an expansion segment has resulted in a single bound nucleotide, which may act as an architectural co-factor to stabilize a protein-protein interface. The presented structure highlights the reductive evolution in these emerging pathogens and sheds light on a conserved mechanism for eukaryotic ribosome hibernation.

Выделение и PBS-дифференциация изолятов Alternaria spp.

Abstract. The purpose of the study. Isolation of Alternaria sp. and their PBS differentiation. The article presents the results of isolation of phytopathogenic fungi of the genus Alternaria spp. from wheat plants and their genetic differentiation using iPBS (Inter Primer Binding Site Polymorphism) analysis. As a result of monitoring studies, it was shown that the fungi Alternaria spp. are the dominant component of the pathocomplex of fungi affecting the embryonic zone of seeds and ears of wheat in the northern regions of Kazakhstan. The pathocomplex of Alternaria is formed by isolates of A. alternata, A. infectoria, and A. tenuissima. Methods. Genetic differentiation of the isolates was performed using iPBS analysis. This method is based on the use of conserved sequences of tRNA binding sites (Primer Binding Sites) as PCR primers. This method is versatile and effective for the direct detection of polymorphism between individuals; therefore, PBS primers can be used in almost any organism, including fungi. Results. Analysis of the PBS primers showed that they all have high resolution in the differentiation of Alternaria spp. The obtained amplification products showed high variability among isolates, both within one species and at the interspecies level. The level of detectable polymorphism varied from 47.43 % to 80.81 %, with an average of 61 %. The size of the amplified PCR fragments ranged from 200 to 3000 bp; on average, amplification was observed from 5 to 15 bands per isolate. Practical significance. This work made it possible to obtain new data on the genetic diversity of Alternaria phytopathogenic fungi for the subsequent development of a strategy for plant protection against Alternaria.

Molecular Phylogenetic Analysis of 16S rRNA Sequences Identified Two Lineages of Helicobacter pylori Strains Detected from Different Regions in Sudan Suggestive of Differential Evolution.

Background Helicobacter pylori (H. pylori) is ubiquitous among humans and one of the best-studied examples of an intimate association between bacteria and humans. Phylogeny and Phylogeography of H. pylori strains are known to mirror human migration patterns and reflect significant demographic events in human prehistory. In this study, we analyzed the molecular evolution of H. pylori strains detected from different tribes and regions of Sudan using 16S rRNA gene and the phylogenetic approach. Materials and methods. A total of 75 gastric biopsies were taken from patients who had been referred for endoscopy from different regions of Sudan. The DNA extraction was performed by using the guanidine chloride method. Two sets of primers (universal and specific for H. pylori) were used to amplify the 16S ribosomal gene. Sanger sequencing was applied, and the resulted sequences were matched with the sequences of the National Center for Biotechnology Information (NCBI) nucleotide database. The evolutionary aspects were analyzed using MEGA7 software. Results Molecular detection of H. pylori has shown that 28 (37.33%) of the patients were positive for H. pylori and no significant differences were found in sociodemographic characteristics, endoscopy series, and H. pylori infection. Nucleotide variations were observed at five nucleotide positions (positions 219, 305, 578, 741, and 763–764), and one insertion mutation (750_InsC_751) was present in sixty-seven percent (7/12) of our strains. These six mutations were detected in regions of the 16S rRNA not closely associated with either tetracycline or tRNA binding sites; 66.67% of them were located in the central domain of 16S rRNA. The phylogenetic analysis of 16S rRNA sequences identified two lineages of H. pylori strains detected from different regions in Sudan. The presence of Sudanese H. pylori strains resembling Hungarian H. pylori strains could reflect the migration of Hungarian people to Sudan or vice versa. Conclusion This finding emphasizes the significance of studying the phylogeny of H. pylori strains as a discriminatory tool to mirror human migration patterns. In addition, the 16S rRNA gene amplification method was found useful for bacterial identification and phylogeny.

Synthetic group A streptogramin antibiotics that overcome Vat resistance.

Natural products serve as chemical blueprints for the majority of antibiotics in our clinical arsenal. The evolutionary process by which these molecules arise is inherently accompanied by the co-evolution of resistance mechanisms that shorten the clinical lifetime of any given class1. Virginiamycin acetyltransferases (Vats) are resistance proteins that provide protection against streptogramins2, potent Gram-positive antibiotics that inhibit the bacterial ribosome3. Due to the challenge of selectively modifying the chemically complex, 23-membered macrocyclic scaffold of group A streptogramins, analogs that overcome Vat resistance have not been previously accessed2. Here we report the design, synthesis, and antibacterial evaluation of group A streptogramin antibiotics with unprecedented structural variability. Using cryo-electron microscopy and forcefield-based refinement, we characterize the binding of eight analogs to the bacterial ribosome at high resolution, revealing new binding interactions that extend into the peptidyl tRNA binding site and towards synergistic binders that occupy the nascent peptide exit tunnel (NPET). One of these analogs has excellent activity against several streptogramin-resistant strains of S. aureus, exhibits decreased acetylation rates in vitro, and is effective at lowering bacterial load in a mouse model of infection. Our results demonstrate that the combination of rational design and modular chemical synthesis can revitalize classes of antibiotics that are limited by naturally arising resistance mechanisms.

Ciliate mitoribosome illuminates evolutionary steps of mitochondrial translation.

To understand the steps involved in the evolution of translation, we used Tetrahymena thermophila, a ciliate with high coding capacity of the mitochondrial genome, as the model organism and characterized its mitochondrial ribosome (mitoribosome) using cryo-EM. The structure of the mitoribosome reveals an assembly of 94-ribosomal proteins and four-rRNAs with an additional protein mass of ~700 kDa on the small subunit, while the large subunit lacks 5S rRNA. The structure also shows that the small subunit head is constrained, tRNA binding sites are formed by mitochondria-specific protein elements, conserved protein bS1 is excluded, and bacterial RNA polymerase binding site is blocked. We provide evidence for anintrinsic protein targeting system through visualization of mitochondria-specific mL105 by the exit tunnel that would facilitate the recruitment of a nascent polypeptide. Functional protein uS3m is encoded by three complementary genes from the nucleus and mitochondrion, establishing a link between genetic drift and mitochondrial translation. Finally, we reannotated nine open reading frames in the mitochondrial genome that code for mitoribosomal proteins.

EEF1A2 mutations in epileptic encephalopathy/intellectual disability: Understanding the potential mechanism of phenotypic variation

EEF1A2 encodes protein elongation factor 1-alpha 2, which is involved in Guanosine triphosphate (GTP)-dependent binding of aminoacyl-transfer RNA (tRNA) to the A-site of ribosomes during protein biosynthesis and is highly expressed in the central nervous system. De novo mutations in EEF1A2 have been identified in patients with extensive neurological deficits, including intractable epilepsy, globe developmental delay, and severe intellectual disability. However, the mechanism underlying phenotype variation is unknown. Using next-generation sequencing, we identified a novel and a recurrent de novo mutation, c.294C>A; p.(Phe98Leu) and c.208G>A; p.(Gly70Ser), in patients with Lennox–Gastaut syndrome. The further systematic analysis revealed that all EEF1A2 mutations were associated with epilepsy and intellectual disability, suggesting its critical role in neurodevelopment. Missense mutations with severe molecular alteration in the t-RNA binding sites or GTP hydrolysis domain were associated with early-onset severe epilepsy, indicating that the clinical expression was potentially determined by the location of mutations and alteration of molecular effects. This study highlights the potential genotype–phenotype relationship in EEF1A2 and facilitates the evaluation of the pathogenicity of EEF1A2 mutations in clinical practice.

АНАЛИЗ ГЕНЕТИЧЕСКОГО МАТЕРИАЛА АБОРИГЕННЫХ СОРТОВ ВИНОГРАДА РОССИЙСКОЙ АМПЕЛОГРАФИЧЕСКОЙ КОЛЛЕКЦИИ

One of the main places in the selection of grapes today belongs to native varieties. Native varieties possess characteristics valuable in economic relations, such as a high-quality berry crop, from which it is subsequently possible to obtain the highest quality wine, as well as resistance to harsh climatic conditions. However, before starting breeding work, it is necessary to conduct various pre-breeding studies, including genetic analysis. This type of analysis is the most informative of all, because it gives the most complete picture of all the genetic predispositions of any organism, as well as what potential the specific varieties possess. The analysis is carried out using a variety of molecular genetic markers, which include IPBS markers. The mechanism of their action is based on the use of primers for PBS retrotransposon sequences (Primer binding site, tRNA binding site). This choice is due to the fact that IPBS markers perform well when detecting polymorphism between samples, which include both varieties and clones of varieties. As a result, the following DNA markers were used: iPBS2074, iPBS2230, iPBS2415, Vine-1, and the following indigenous grape varieties: Kumshatsky, Varyushkin, 9th row of class 19, Chilyaki 1-23 (35-36), Cinnamon white mutation, Slitny, Decent , Augustine, Yay raisin white, Kishmish oval white, (Sultanina), Yay raisin pink, Kishmish red Turkmen (Sultatnina pink), Tsimlyansky black. Based on the results of all the studies, namely polyacrylamide gel electrophoresis (PAGE), interpretation of the obtained photos, and subsequent data processing in Microsoft Excel using the GenAlEx 6.3 macro, analysis in AMOVA, and MEGA7 with UPGMA parameters, the conclusions were made that molecular markers of IPBS were effective in preselection studies of the native grape varieties.

Tryptophanyl-tRNA synthetase mediates high-affinity tryptophan uptake into human cells

The tryptophan (Trp) transport system has a high affinity and selectivity toward Trp, and has been reported to exist in both human and mouse macrophages. Although this system is highly expressed in interferon-γ (IFN-γ)-treated cells and indoleamine 2,3-dioxygenase 1 (IDO1)-expressing cells, its identity remains incompletely understood. Tryptophanyl-tRNA synthetase (TrpRS) is also highly expressed in IFN-γ-treated cells and also has high affinity and selectivity for Trp. Here, we investigated the effects of human TrpRS expression on Trp uptake into IFN-γ-treated human THP-1 monocytes or HeLa cells. Inhibition of human TrpRS expression by TrpRS-specific siRNAs decreased and overexpression of TrpRS increased Trp uptake into the cells. Of note, the TrpRS-mediated uptake system had more than hundred-fold higher affinity for Trp than the known System L amino acid transporter, promoted uptake of low Trp concentrations, and had very high Trp selectivity. Moreover, site-directed mutagenesis experiments indicated that Trp- and ATP-binding sites, but not tRNA-binding sites, in TrpRS are essential for TrpRS-mediated Trp uptake into the human cells. We further demonstrate that the addition of purified TrpRS to cell culture medium increases Trp uptake into cells. Taken together, our results reveal that TrpRS plays an important role in high-affinity Trp uptake into human cells.

Biochemical and Structural Characterization of a Schiff Base in the Radical-Mediated Biosynthesis of 4-Demethylwyosine by TYW1.

TYW1 is a radical S-adenosyl-l-methionine (SAM) enzyme that catalyzes the condensation of pyruvate and N-methylguanosine to form the posttranscriptional modification, 4-demethylwyosine, in situ on transfer RNA (tRNA). Two mechanisms have been proposed for this transformation, with one of the possible mechanisms invoking a Schiff base intermediate formed between a conserved lysine residue and pyruvate. Utilizing a combination of mass spectrometry and X-ray crystallography, we have obtained evidence to support the formation of a Schiff base lysine adduct in TYW1. When 13C labeled pyruvate is used, the mass shift of the adduct matches that of the labeled pyruvate, indicating that pyruvate is the source of the adduct. Furthermore, a crystal structure of TYW1 provides visualization of the Schiff base lysine–pyruvate adduct, which is positioned directly adjacent to the auxiliary [4Fe–4S] cluster. The adduct coordinates the unique iron of the auxiliary cluster through the lysine nitrogen and a carboxylate oxygen, reminiscent of how the radical SAM [4Fe–4S] cluster is coordinated by SAM. The structure provides insight into the binding site for tRNA and further suggests how radical SAM chemistry can be combined with Schiff base chemistry for RNA modification.

Crystal Structure of the Archaeosine Synthase QueF‐Like – Insights into Amidino Transfer and tRNA Recognition by the Tunnel Fold

Crystal Structure of the Archaeosine Synthase QueF‐Like – Insights into Amidino Transfer and tRNA Recognition by the Tunnel Fold

Functional Insights into the Adjacent Stem-Loop in Honey Bee Dicistroviruses That Promotes Internal Ribosome Entry Site-Mediated Translation and Viral Infection

All viruses must successfully harness the host translational apparatus and divert it towards viral protein synthesis. Dicistroviruses use an unusual internal ribosome entry site (IRES) mechanism whereby the IRES adopts a three-pseudoknot structure that accesses the ribosome tRNA binding sites to directly recruit the ribosome and initiate translation from a non-AUG start site. A subset of dicistroviruses, including the honey bee Israeli acute paralysis virus (IAPV), encode an extra stem-loop (SLVI) 5' -adjacent to the IGR IRES. Previously, the function of this additional stem-loop is unknown. Here, we provide mechanistic and functional insights into the role of SLVI in IGR IRES translation and in virus infection. Biochemical analyses of a series of mutant IRESs demonstrated that SLVI does not function in ribosome recruitment but is required for proper ribosome positioning on the IRES to direct translation. Using a chimeric infectious clone derived from the related Cricket paralysis virus, we showed that the integrity of SLVI is important for optimal viral translation and viral yield. Based on structural models of ribosome-IGR IRES complexes, the SLVI is predicted to be in the vicinity of the ribosome E site. We propose that SLVI of IAPV IGR IRES functionally mimics interactions of an E-site tRNA with the ribosome to direct positioning of the tRNA-like domain of the IRES in the A site.IMPORTANCEViral internal ribosome entry sites are RNA elements and structures that allow some positive-sense monopartite RNA viruses to hijack the host ribosome to start viral protein synthesis. We demonstrate that a unique stem-loop structure is essential for optimal viral protein synthesis and for virus infection. Biochemical evidence shows that this viral stem-loop RNA structure impacts a fundamental property of the ribosome to start protein synthesis.