Mechanism of tRNA-mediated +1 ribosomal frameshifting

Accurate translation of the genetic code is critical to ensure expression of proteins with correct amino acid sequences. Certain tRNAs can cause a shift out of frame (i.e., frameshifting) due to imbalances in tRNA concentrations, lack of tRNA modifications or insertions or deletions in tRNAs (called frameshift suppressors). Here, we determined the structural basis for how frameshift-suppressor tRNASufA6 (a derivative of tRNAPro) reprograms the mRNA frame to translate a 4-nt codon when bound to the bacterial ribosome. After decoding at the aminoacyl (A) site, the crystal structure of the anticodon stem-loop of tRNASufA6 bound in the peptidyl (P) site reveals ASL conformational changes that allow for recoding into the +1 mRNA frame. Furthermore, a crystal structure of full-length tRNASufA6 programmed in the P site shows extensive conformational rearrangements of the 30S head and body domains similar to what is observed in a translocation intermediate state containing elongation factor G (EF-G). The 30S movement positions tRNASufA6 toward the 30S exit (E) site disrupting key 16S rRNA–mRNA interactions that typically define the mRNA frame. In summary, this tRNA-induced 30S domain change in the absence of EF-G causes the ribosome to lose its grip on the mRNA and uncouples the canonical forward movement of the tRNAs during elongation.

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EF-G–induced ribosome sliding along the noncoding mRNA

Science Advances ◽

10.1126/sciadv.aaw9049 ◽

2019 ◽

Vol 5 (6) ◽

pp. eaaw9049 ◽

Cited By ~ 5

Author(s):

M. Klimova ◽

T. Senyushkina ◽

E. Samatova ◽

B. Z. Peng ◽

M. Pearson ◽

...

Keyword(s):

Messenger Rna ◽

Elongation Factor ◽

Transfer Rna ◽

Noncoding Region ◽

Forward Movement ◽

Stem Loop ◽

A Site ◽

Hydrolysis Of ◽

Reading Frames ◽

Factor G

Translational bypassing is a recoding event during which ribosomes slide over a noncoding region of the messenger RNA (mRNA) to synthesize one protein from two discontinuous reading frames. Structures in the mRNA orchestrate forward movement of the ribosome, but what causes ribosomes to start sliding remains unclear. Here, we show that elongation factor G (EF-G) triggers ribosome take-off by a pseudotranslocation event using a small mRNA stem-loop as an A-site transfer RNA mimic and requires hydrolysis of about two molecules of guanosine 5′-triphosphate per nucleotide of the noncoding gap. Bypassing ribosomes adopt a hyper-rotated conformation, also observed with ribosomes stalled by the SecM sequence, suggesting common ribosome dynamics during translation stalling. Our results demonstrate a new function of EF-G in promoting ribosome sliding along the mRNA, in contrast to codon-wise ribosome movement during canonical translation, and suggest a mechanism by which ribosomes could traverse untranslated parts of mRNAs.

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Elongation factor 4 remodels the A-site tRNA on the ribosome

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1522932113 ◽

2016 ◽

Vol 113 (18) ◽

pp. 4994-4999 ◽

Cited By ~ 13

Author(s):

Matthieu G. Gagnon ◽

Jinzhong Lin ◽

Thomas A. Steitz

Keyword(s):

Crystal Structure ◽

Conformational Changes ◽

Thermus Thermophilus ◽

Elongation Factor ◽

Peptidyl Transferase ◽

Peptidyl Transferase Center ◽

70S Ribosome ◽

Regulate Protein ◽

A Site ◽

Hydrolysis Of

During translation, a plethora of protein factors bind to the ribosome and regulate protein synthesis. Many of those factors are guanosine triphosphatases (GTPases), proteins that catalyze the hydrolysis of guanosine 5′-triphosphate (GTP) to promote conformational changes. Despite numerous studies, the function of elongation factor 4 (EF-4/LepA), a highly conserved translational GTPase, has remained elusive. Here, we present the crystal structure at 2.6-Å resolution of the Thermus thermophilus 70S ribosome bound to EF-4 with a nonhydrolyzable GTP analog and A-, P-, and E-site tRNAs. The structure reveals the interactions of EF-4 with the A-site tRNA, including contacts between the C-terminal domain (CTD) of EF-4 and the acceptor helical stem of the tRNA. Remarkably, EF-4 induces a distortion of the A-site tRNA, allowing it to interact simultaneously with EF-4 and the decoding center of the ribosome. The structure provides insights into the tRNA-remodeling function of EF-4 on the ribosome and suggests that the displacement of the CCA-end of the A-site tRNA away from the peptidyl transferase center (PTC) is functionally significant.

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Spontaneous ribosomal translocation of mRNA and tRNAs into a chimeric hybrid state

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1901310116 ◽

2019 ◽

Vol 116 (16) ◽

pp. 7813-7818 ◽

Cited By ~ 18

Author(s):

Jie Zhou ◽

Laura Lancaster ◽

John Paul Donohue ◽

Harry F. Noller

Keyword(s):

Crystal Structure ◽

Elongation Factor ◽

Intermediate Complex ◽

Reading Frame ◽

Hybrid State ◽

Head Domain ◽

30S Subunit ◽

A Site ◽

Insight Into ◽

Factor G

The elongation factor G (EF-G)–catalyzed translocation of mRNA and tRNA through the ribosome is essential for vacating the ribosomal A site for the next incoming aminoacyl-tRNA, while precisely maintaining the translational reading frame. Here, the 3.2-Å crystal structure of a ribosome translocation intermediate complex containing mRNA and two tRNAs, formed in the absence of EF-G or GTP, provides insight into the respective roles of EF-G and the ribosome in translocation. Unexpectedly, the head domain of the 30S subunit is rotated by 21°, creating a ribosomal conformation closely resembling the two-tRNA chimeric hybrid state that was previously observed only in the presence of bound EF-G. The two tRNAs have moved spontaneously from their A/A and P/P binding states into ap/P and pe/E states, in which their anticodon loops are bound between the 30S body domain and its rotated head domain, while their acceptor ends have moved fully into the 50S P and E sites, respectively. Remarkably, the A-site tRNA translocates fully into the classical P-site position. Although the mRNA also undergoes movement, codon–anticodon interaction is disrupted in the absence of EF-G, resulting in slippage of the translational reading frame. We conclude that, although movement of both tRNAs and mRNA (along with rotation of the 30S head domain) can occur in the absence of EF-G and GTP, EF-G is essential for enforcing coupled movement of the tRNAs and their mRNA codons to maintain the reading frame.

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Structural basis of the transactivation deficiency of the human PPARγ F360L mutant associated with familial partial lipodystrophy

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004714009638 ◽

2014 ◽

Vol 70 (7) ◽

pp. 1965-1976 ◽

Cited By ~ 9

Author(s):

Clorinda Lori ◽

Alessandra Pasquo ◽

Roberta Montanari ◽

Davide Capelli ◽

Valerio Consalvi ◽

...

Keyword(s):

Crystal Structure ◽

Conformational Changes ◽

Partial Agonist ◽

Salt Bridge ◽

Van Der Waals Interactions ◽

Structural Basis ◽

Wild Type ◽

Partial Lipodystrophy ◽

X Ray ◽

Familial Partial Lipodystrophy

The peroxisome proliferator-activated receptors (PPARs) are transcription factors that regulate glucose and lipid metabolism. The role of PPARs in several chronic diseases such as type 2 diabetes, obesity and atherosclerosis is well known and, for this reason, they are the targets of antidiabetic and hypolipidaemic drugs. In the last decade, some rare mutations in human PPARγ that might be associated with partial lipodystrophy, dyslipidaemia, insulin resistance and colon cancer have emerged. In particular, the F360L mutant of PPARγ (PPARγ2 residue 388), which is associated with familial partial lipodystrophy, significantly decreases basal transcriptional activity and impairs stimulation by synthetic ligands. To date, the structural reason for this defective behaviour is unclear. Therefore, the crystal structure of PPARγ F360L together with the partial agonist LT175 has been solved and the mutant has been characterized by circular-dichroism spectroscopy (CD) in order to compare its thermal stability with that of the wild-type receptor. The X-ray analysis showed that the mutation induces dramatic conformational changes in the C-terminal part of the receptor ligand-binding domain (LBD) owing to the loss of van der Waals interactions made by the Phe360 residue in the wild type and an important salt bridge made by Arg357, with consequent rearrangement of loop 11/12 and the activation function helix 12 (H12). The increased mobility of H12 makes the binding of co-activators in the hydrophobic cleft less efficient, thereby markedly lowering the transactivation activity. The spectroscopic analysis in solution and molecular-dynamics (MD) simulations provided results which were in agreement and consistent with the mutant conformational changes observed by X-ray analysis. Moreover, to evaluate the importance of the salt bridge made by Arg357, the crystal structure of the PPARγ R357A mutant in complex with the agonist rosiglitazone has been solved.

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The expression of antibiotic resistance methyltransferase correlates with mRNA stability independently of ribosome stalling

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01806-16 ◽

2016 ◽

pp. AAC.01806-16 ◽

Cited By ~ 6

Author(s):

Ekaterina Dzyubak ◽

Mee-Ngan F. Yap

Keyword(s):

Leader Peptide ◽

23S Rrna ◽

Cross Resistance ◽

Nonsense Mutations ◽

Stem Loop ◽

Translational Initiation ◽

Ribosome Stalling ◽

Mrna Structure ◽

Bacterial Ribosome ◽

A Site

Members of the Erm methyltransferase family modify 23S rRNA of the bacterial ribosome and render cross-resistance to macrolides and multiple distantly related antibiotics. Previous studies have shown that the expression ofermis activated when a macrolide-bound ribosome stalls the translation of the leader peptide preceding the co-transcribederm. Ribosome stalling is thought to destabilize the inhibitory stem-loop mRNA structure and exposes theermShine-Dalgarno (SD) sequence for translational initiation. Paradoxically, mutations that abolish ribosome stalling are routinely found in hyper-resistant clinical isolates; however, the significance of the stalling-dead leader sequence is largely unknown. Here we show that nonsense mutations in theStaphylococcus aureusErmB leader peptide (ErmBL) lead to high basal and induced expression of downstream ErmB in the absence and presence of macrolide concomitantly with elevated ribosome methylation and resistance. The overexpression of ErmB is associated with the reduced turnover of theermBL-ermBtranscript, and macrolide appears to mitigate mRNA cleavage at a site immediately downstream of theermBLSD sequence. The stabilizing effect of antibiotics on mRNA is not limited toermBL-ermB; cationic antibiotics representing a ribosome stalling inducer and a non-inducer increase the half-life of specific transcripts. These data unveil a new layer ofermBregulation and imply that ErmBL translation or ribosome stalling serves as a “tuner” to suppress aberrant production of ErmB because methylated ribosome may impose a fitness cost on the bacterium as a result of misregulated translation.

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Structural basis of adenylyl cyclase 9 activation

10.1101/2021.08.11.455989 ◽

2021 ◽

Author(s):

Chao Qi ◽

Pia Lavriha ◽

Ved Mehta ◽

Basavraj Khanppnavar ◽

Inayathulla Mohammed ◽

...

Keyword(s):

Secondary Structure ◽

Binding Site ◽

Adenylyl Cyclase ◽

Conformational Changes ◽

Structural Basis ◽

Structural Studies ◽

Nucleotide Analogue ◽

C Terminus ◽

Membrane Bound ◽

A Site

Adenylyl cyclase 9 (AC9) is a membrane-bound enzyme that converts ATP into cAMP. The enzyme is weakly activated by forskolin, fully activated by the G protein Gαs subunit and is autoinhibited by the AC9 C-terminus. Although our recent structural studies of the AC9-Gαs complex provided the framework for understanding AC9 autoinhibition, the conformational changes that AC9 undergoes in response to activator binding remains poorly understood. Here, we present the cryo-EM structures of AC9 in several distinct states: (i) AC9 bound to a nucleotide inhibitor MANT-GTP, (ii) bound to an artificial activator (DARPin C4) and MANT-GTP, (iii) bound to DARPin C4 and a nucleotide analogue ATPαS, (iv) bound to Gαs and MANT-GTP. The artificial activator DARPin C4 partially activates AC9 by binding at a site that overlaps with the Gαs binding site. Together with the previously observed occluded and forskolin-bound conformations, structural comparisons of AC9 in the four new conformations show that secondary structure rearrangements in the region surrounding the forskolin binding site are essential for AC9 activation.

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Structural Basis of APH(3′)-IIIa-Mediated Resistance to N1-Substituted Aminoglycoside Antibiotics

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00062-09 ◽

2009 ◽

Vol 53 (7) ◽

pp. 3049-3055 ◽

Cited By ~ 17

Author(s):

Desiree H. Fong ◽

Albert M. Berghuis

Keyword(s):

Crystal Structure ◽

Bacterial Resistance ◽

Aminoglycoside Antibiotics ◽

Structural Basis ◽

Amino Group ◽

Naturally Occurring ◽

Type Iiia ◽

Bactericidal Properties ◽

A Site ◽

Binding Loop

ABSTRACT Butirosin is unique among the naturally occurring aminoglycosides, having a substituted amino group at position 1 (N1) of the 2-deoxystreptamine ring with an (S)-4-amino-2-hydroxybutyrate (AHB) group. While bacterial resistance to aminoglycosides can be ascribed chiefly to drug inactivation by plasmid-encoded aminoglycoside-modifying enzymes, the presence of an AHB group protects the aminoglycoside from binding to many resistance enzymes, and hence, the antibiotic retains its bactericidal properties. Consequently, several semisynthetic N1-substituted aminoglycosides, such as amikacin, isepamicin, and netilmicin, were developed. Unfortunately, butirosin, amikacin, and isepamicin are not resistant to inactivation by 3′-aminoglycoside O-phosphotransferase type IIIa [APH(3′)-IIIa]. We report here the crystal structure of APH(3′)-IIIa in complex with an ATP analog, AMPPNP [adenosine 5′-(β,γ-imido)triphosphate], and butirosin A to 2.4-Å resolution. The structure shows that butirosin A binds to the enzyme in a manner analogous to other 4,5-disubstituted aminoglycosides, and the flexible antibiotic-binding loop is key to the accommodation of structurally diverse substrates. Based on the crystal structure, we have also constructed a model of APH(3′)-IIIa in complex with amikacin, a commonly used semisynthetic N1-substituted 4,6-disubstituted aminoglycoside. Together, these results suggest a strategy to further derivatize the AHB group in order to generate new aminoglycoside derivatives that can elude inactivation by resistance enzymes while maintaining their ability to bind to the ribosomal A site.

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Crystal structure and functional interpretation of the erythrocyte spectrin tetramerization domain complex

Blood ◽

10.1182/blood-2010-01-261396 ◽

2010 ◽

Vol 115 (23) ◽

pp. 4843-4852 ◽

Cited By ~ 48

Author(s):

Jonathan J. Ipsaro ◽

Sandra L. Harper ◽

Troy E. Messick ◽

Ronen Marmorstein ◽

Alfonso Mondragón ◽

...

Keyword(s):

Crystal Structure ◽

Conformational Changes ◽

Principal Component ◽

Membrane Skeleton ◽

Structural Basis ◽

Functional Interpretation ◽

Erythrocyte Morphology ◽

Tetramerization Domain ◽

Complementarity Analysis ◽

Red Cell Membranes

Abstract As the principal component of the membrane skeleton, spectrin confers integrity and flexibility to red cell membranes. Although this network involves many interactions, the most common hemolytic anemia mutations that disrupt erythrocyte morphology affect the spectrin tetramerization domains. Although much is known clinically about the resulting conditions (hereditary elliptocytosis and pyropoikilocytosis), the detailed structural basis for spectrin tetramerization and its disruption by hereditary anemia mutations remains elusive. Thus, to provide further insights into spectrin assembly and tetramer site mutations, a crystal structure of the spectrin tetramerization domain complex has been determined. Architecturally, this complex shows striking resemblance to multirepeat spectrin fragments, with the interacting tetramer site region forming a central, composite repeat. This structure identifies conformational changes in α-spectrin that occur upon binding to β-spectrin, and it reports the first structure of the β-spectrin tetramerization domain. Analysis of the interaction surfaces indicates an extensive interface dominated by hydrophobic contacts and supplemented by electrostatic complementarity. Analysis of evolutionarily conserved residues suggests additional surfaces that may form important interactions. Finally, mapping of hereditary anemia-related mutations onto the structure demonstrate that most, but not all, local hereditary anemia mutations map to the interacting domains. The potential molecular effects of these mutations are described.

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Structural basis for +1 ribosomal frameshifting during EF-G-catalyzed translocation

Nature Communications ◽

10.1038/s41467-021-24911-1 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Gabriel Demo ◽

Howard B. Gamper ◽

Anna B. Loveland ◽

Isao Masuda ◽

Christine E. Carbone ◽

...

Keyword(s):

16S Rrna ◽

Elongation Factor ◽

Structural Basis ◽

Elongation Factor G ◽

Ribosomal Frameshifting ◽

Elongation Cycle ◽

70S Ribosome ◽

Complex Features ◽

A Site ◽

Factor G

AbstractFrameshifting of mRNA during translation provides a strategy to expand the coding repertoire of cells and viruses. How and where in the elongation cycle +1-frameshifting occurs remains poorly understood. We describe seven ~3.5-Å-resolution cryo-EM structures of 70S ribosome complexes, allowing visualization of elongation and translocation by the GTPase elongation factor G (EF-G). Four structures with a + 1-frameshifting-prone mRNA reveal that frameshifting takes place during translocation of tRNA and mRNA. Prior to EF-G binding, the pre-translocation complex features an in-frame tRNA-mRNA pairing in the A site. In the partially translocated structure with EF-G•GDPCP, the tRNA shifts to the +1-frame near the P site, rendering the freed mRNA base to bulge between the P and E sites and to stack on the 16S rRNA nucleotide G926. The ribosome remains frameshifted in the nearly post-translocation state. Our findings demonstrate that the ribosome and EF-G cooperate to induce +1 frameshifting during tRNA-mRNA translocation.

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Crystal Structure and Cellular Functions of uPAR Dimer

10.21203/rs.3.rs-802899/v1 ◽

2021 ◽

Author(s):

Shujuan Yu ◽

Yaqun Sui ◽

Jiawei Wang ◽

Yongdong Li ◽

Hanlin Li ◽

...

Keyword(s):

Crystal Structure ◽

Conformational Changes ◽

Cell Function ◽

Basal Membrane ◽

Structural Basis ◽

Cellular Functions ◽

Amino Terminal ◽

Type Plasminogen Activator ◽

Dimeric Form ◽

Monomeric Structure

Abstract Receptor dimerization of urokinase-type plasminogen activator receptor (uPAR) was previously identified at protein level and on the cell surface. Recently, a dimeric form of mouse uPAR isoform 2 was proposed, which induced kidney disease. Here, we report the crystal structure of human uPAR dimer at 2.96 Å. The structure reveals enormous conformational changes of the dimer compared to the monomeric structure: D1 of uPAR opens up into a large expanded loop that captures a β-hairpin loop of a neighboring uPAR to form an expanded β-sheet, leading to an elongated, highly intertwined dimeric uPAR. Based on the structure, we identify the E49P mutation promoting dimer formation. The mutation increases receptor binding to amino terminal fragment (ATF) of its primary ligand uPA, induces the receptor to distribute to the basal membrane, promotes cell proliferation, and alters cell morphology via the ERK activation of β1 integrin signaling. These results reveal the structural basis for uPAR dimerization, its effect on cell function, and provide new insight and tools to study this multifunctional receptor.

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