scholarly journals Ribosome dynamics during decoding

2017 ◽  
Vol 372 (1716) ◽  
pp. 20160182 ◽  
Author(s):  
Marina V. Rodnina ◽  
Niels Fischer ◽  
Cristina Maracci ◽  
Holger Stark
Keyword(s):  
Translation Termination ◽  
Elongation Factor ◽  
Translation Elongation ◽  
Gtp Hydrolysis ◽  
Codon Recognition ◽  
Special Cases ◽  
Multistep Process ◽  
Gtpase Activation ◽  
Hydrolysis Of

Elongation factors Tu (EF-Tu) and SelB are translational GTPases that deliver aminoacyl-tRNAs (aa-tRNAs) to the ribosome. In each canonical round of translation elongation, aa-tRNAs, assisted by EF-Tu, decode mRNA codons and insert the respective amino acid into the growing peptide chain. Stop codons usually lead to translation termination; however, in special cases UGA codons are recoded to selenocysteine (Sec) with the help of SelB. Recruitment of EF-Tu and SelB together with their respective aa-tRNAs to the ribosome is a multistep process. In this review, we summarize recent progress in understanding the role of ribosome dynamics in aa-tRNA selection. We describe the path to correct codon recognition by canonical elongator aa-tRNA and Sec-tRNA Sec and discuss the local and global rearrangements of the ribosome in response to correct and incorrect aa-tRNAs. We present the mechanisms of GTPase activation and GTP hydrolysis of EF-Tu and SelB and summarize what is known about the accommodation of aa-tRNA on the ribosome after its release from the elongation factor. We show how ribosome dynamics ensures high selectivity for the cognate aa-tRNA and suggest that conformational fluctuations, induced fit and kinetic discrimination play major roles in maintaining the speed and fidelity of translation. This article is part of the themed issue ‘Perspectives on the ribosome’.

scholarly journals Activation of GTP hydrolysis in mRNA-tRNA translocation by elongation factor G

2015 ◽  
Vol 1 (4) ◽  
pp. e1500169 ◽  
Author(s):  
Wen Li ◽  
Zheng Liu ◽  
Ravi Kiran Koripella ◽  
Robert Langlois ◽  
Suparna Sanyal ◽  
...  
Keyword(s):  
Elongation Factor ◽  
Elongation Factor G ◽  
Direct Role ◽  
Gtp Hydrolysis ◽  
Ribosomal Subunits ◽  
Trna Translocation ◽  
Gtpase Activation ◽  
Guanosine Triphosphatase ◽  
Factor G

During protein synthesis, elongation of the polypeptide chain by each amino acid is followed by a translocation step in which mRNA and transfer RNA (tRNA) are advanced by one codon. This crucial step is catalyzed by elongation factor G (EF-G), a guanosine triphosphatase (GTPase), and accompanied by a rotation between the two ribosomal subunits. A mutant of EF-G, H91A, renders the factor impaired in guanosine triphosphate (GTP) hydrolysis and thereby stabilizes it on the ribosome. We use cryogenic electron microscopy (cryo-EM) at near-atomic resolution to investigate two complexes formed by EF-G H91A in its GTP state with the ribosome, distinguished by the presence or absence of the intersubunit rotation. Comparison of these two structures argues in favor of a direct role of the conserved histidine in the switch II loop of EF-G in GTPase activation, and explains why GTP hydrolysis cannot proceed with EF-G bound to the unrotated form of the ribosome.

scholarly journals Role of a ribosomal RNA phosphate oxygen during the EF-G–triggered GTP hydrolysis

2015 ◽  
Vol 112 (20) ◽  
pp. E2561-E2568 ◽  
Author(s):  
Miriam Koch ◽  
Sara Flür ◽  
Christoph Kreutz ◽  
Eric Ennifar ◽  
Ronald Micura ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Electrostatic Interactions ◽  
Elongation Factor ◽  
Direct Interaction ◽  
Phosphate Group ◽  
Gtp Hydrolysis ◽  
Elongation Cycle ◽  
Close Proximity ◽  
Catalytically Active ◽  
Gtpase Activation

Elongation factor-catalyzed GTP hydrolysis is a key reaction during the ribosomal elongation cycle. Recent crystal structures of G proteins, such as elongation factor G (EF-G) bound to the ribosome, as well as many biochemical studies, provide evidence that the direct interaction of translational GTPases (trGTPases) with the sarcin-ricin loop (SRL) of ribosomal RNA (rRNA) is pivotal for hydrolysis. However, the precise mechanism remains elusive and is intensively debated. Based on the close proximity of the phosphate oxygen of A2662 of the SRL to the supposedly catalytic histidine of EF-G (His87), we probed this interaction by an atomic mutagenesis approach. We individually replaced either of the two nonbridging phosphate oxygens at A2662 with a methyl group by the introduction of a methylphosphonate instead of the natural phosphate in fully functional, reconstituted bacterial ribosomes. Our major finding was that only one of the two resulting diastereomers, the SP methylphosphonate, was compatible with efficient GTPase activation on EF-G. The same trend was observed for a second trGTPase, namely EF4 (LepA). In addition, we provide evidence that the negative charge of the A2662 phosphate group must be retained for uncompromised activity in GTP hydrolysis. In summary, our data strongly corroborate that the nonbridging proSP phosphate oxygen at the A2662 of the SRL is critically involved in the activation of GTP hydrolysis. A mechanistic scenario is supported in which positioning of the catalytically active, protonated His87 through electrostatic interactions with the A2662 phosphate group and H-bond networks are key features of ribosome-triggered activation of trGTPases.

Selenocysteine Inserting RNA Elements Modulate GTP Hydrolysis of Elongation Factor SelB†

Biochemistry ◽  
1998 ◽  
Vol 37 (3) ◽  
pp. 885-890 ◽  
Author(s):  
Alexander Hüttenhofer ◽  
August Böck
Keyword(s):  
Elongation Factor ◽  
Gtp Hydrolysis ◽  
Rna Elements ◽  
Hydrolysis Of

scholarly journals Bacterial dynamin-like protein DynA mediates lipid and content mixing and shows phospholipid specificity

2018 ◽  
Author(s):  
Lijun Guo ◽  
Marc Bramkamp
Keyword(s):  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Membrane Stress ◽  
Gtp Hydrolysis ◽  
Cellular Processes ◽  
Organelle Division ◽  
Membrane Tethering ◽  
Hydrolysis Of

ABSTRACTThe dynamins family of GTPases is involved in key cellular processes in eukaryotes, including vesicle trafficking and organelle division. The GTP hydrolysis cycle of dynamin translates to a conformational change in the protein structure, which forces the underlying lipid layer into an energetically unstable conformation that promotes membrane rearrangements. Many bacterial genomes encode dynamin-like proteins, but the biological function of these proteins has remained largely enigmatic. In recent years, our group has reported that the dynamin-like protein DynA from Bacillus subtilis mediates nucleotide-independent membrane tethering in vitro and contributes to the innate immunity of bacteria against membrane stress and phage infection. However, so far the mechanism of membrane stress response and the role of GTP hydrolysis remain unclear. Here, we employed content mixing and lipid mixing assays in reconstituted systems to study if the dynamin-like protein DynA from B. subtilis induces membrane full fusion, and further test the possibility that GTP hydrolysis of DynA may act on the fusion-through-hemifusion pathway. Our results based on fluorescence resonance energy transfer (FRET) indicated that DynA could induce aqueous content mixing even in absence of GTP. Moreover, DynA-induced membrane fusion in vitro is a thermo-promoted slow response. Surprisingly, digestion of protein mediated an instantl rise of content exchange, supporting the assumption that disassembly of DynA is the fundamental power for fusion-through-hemifusion.

scholarly journals A dynamic RNA loop in an IRES affects multiple steps of elongation factor-mediated translation initiation

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Marisa D Ruehle ◽  
Haibo Zhang ◽  
Ryan M Sheridan ◽  
Somdeb Mitra ◽  
Yuanwei Chen ◽  
...  
Keyword(s):  
Elongation Factor ◽  
Model Systems ◽  
Initiation Mechanism ◽  
Translation Elongation ◽  
80S Ribosome ◽  
Internal Ribosome Entry ◽  
Elongation Cycle ◽  
Different Types ◽  
Rna Pseudoknot

Internal ribosome entry sites (IRESs) are powerful model systems to understand how the translation machinery can be manipulated by structured RNAs and for exploring inherent features of ribosome function. The intergenic region (IGR) IRESs from the Dicistroviridae family of viruses are structured RNAs that bind directly to the ribosome and initiate translation by co-opting the translation elongation cycle. These IRESs require an RNA pseudoknot that mimics a codon-anticodon interaction and contains a conformationally dynamic loop. We explored the role of this loop and found that both the length and sequence are essential for translation in different types of IGR IRESs and from diverse viruses. We found that loop 3 affects two discrete elongation factor-dependent steps in the IRES initiation mechanism. Our results show how the IRES directs multiple steps after 80S ribosome placement and highlights the often underappreciated significance of discrete conformationally dynamic elements within the context of structured RNAs.

scholarly journals Switch of the interactions between the ribosomal stalk and EF1A in the GTP- and GDP-bound conformations

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Kei Maruyama ◽  
Hirotatsu Imai ◽  
Momoko Kawamura ◽  
Sonoko Ishino ◽  
Yoshizumi Ishino ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Large Subunit ◽  
Elongation Factor ◽  
Binding Mode ◽  
Translation Elongation ◽  
Gtp Hydrolysis ◽  
Before And After ◽  
Ribosomal Stalk ◽  
Biochemical Analyses ◽  
Similar Affinity

Abstract Translation elongation factor EF1A delivers aminoacyl-tRNA to the ribosome in a GTP-bound form, and is released from the ribosome in a GDP-bound form. This association/dissociation cycle proceeds efficiently via a marked conformational change in EF1A. EF1A function is dependent on the ribosomal “stalk” protein of the ribosomal large subunit, although the precise mechanism of action of the stalk on EF1A remains unclear. Here, we clarify the binding mode of archaeal stalk aP1 to GTP-bound aEF1A associated with aPelota. Intriguingly, the C-terminal domain (CTD) of aP1 binds to aEF1A•GTP with a similar affinity to aEF1A•GDP. We have also determined the crystal structure of the aP1-CTD•aEF1A•GTP•aPelota complex at 3.0 Å resolution. The structure shows that aP1-CTD binds to a space between domains 1 and 3 of aEF1A. Biochemical analyses show that this binding is crucial for protein synthesis. Comparison of the structures of aP1-CTD•aEF1A•GTP and aP1-CTD•aEF1A•GDP demonstrates that the binding mode of aP1 changes markedly upon a conformational switch between the GTP- and GDP-bound forms of aEF1A. Taking into account biochemical data, we infer that aP1 employs its structural flexibility to bind to aEF1A before and after GTP hydrolysis for efficient protein synthesis.

scholarly journals The role of translation elongation factor eEF1 subunits in neurodevelopmental disorders

2018 ◽  
Vol 40 (2) ◽  
pp. 131-141 ◽  
Author(s):  
Fiona McLachlan ◽  
Anna Martinez Sires ◽  
Catherine M. Abbott
Keyword(s):  
Neurodevelopmental Disorders ◽  
Elongation Factor ◽  
Translation Elongation Factor ◽  
Translation Elongation

scholarly journals Three tRNAs on the ribosome slow translation elongation

2017 ◽  
Vol 114 (52) ◽  
pp. 13691-13696 ◽  
Author(s):  
Junhong Choi ◽  
Joseph D. Puglisi
Keyword(s):  
Protein Synthesis ◽  
Ionic Strength ◽  
Binding Sites ◽  
Elongation Factor ◽  
Transfer Rna ◽  
Dissociation Kinetics ◽  
Translation Elongation ◽  
Trna Species ◽  
Trna Binding

During protein synthesis, the ribosome simultaneously binds up to three different transfer RNA (tRNA) molecules. Among the three tRNA binding sites, the regulatory role of the exit (E) site, where deacylated tRNA spontaneously dissociates from the translational complex, has remained elusive. Here we use two donor–quencher pairs to observe and correlate both the conformation of ribosomes and tRNAs as well as tRNA occupancy. Our results reveal a partially rotated state of the ribosome wherein all three tRNA sites are occupied during translation elongation. The appearance and lifetime of this state depend on the E-site tRNA dissociation kinetics, which may vary among tRNA species and depends on temperature and ionic strength. The 3-tRNA partially rotated state is not a proper substrate for elongation factor G (EF-G), thus inhibiting translocation until the E-site tRNA dissociates. Our result presents two parallel kinetic pathways during translation elongation, underscoring the ability of E-site codons to modulate the dynamics of protein synthesis.

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