Ribosome-associated GTPases: The role of RNA for GTPase activation

Clementi, Nina; Polacek, Norbert

doi:10.4161/rna.7.5.12467

Cited by 33 publications

(28 citation statements)

References 80 publications

(118 reference statements)

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“…Many of those factors are guanosine triphosphatases (GTPases), proteins that use energy from guanosine 5′-triphosphate (GTP) to promote conformational changes that lead to transitions between ribosome functional states (1,2). In bacteria, for instance, initiation of protein synthesis is largely regulated by initiation factor 2 (IF-2), a GTPase that stabilizes the initiator tRNA in the P site of the ribosome (3).…”

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confidence: 99%

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

Gagnon

Lin

Steitz

2016

Proc. Natl. Acad. Sci. U.S.A.

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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.T ranslation of the genetic information requires protein factors that interact with the ribosome sequentially, regulate its activity, and guide it through the protein synthesis cycle in a concerted manner. Many of those factors are guanosine triphosphatases (GTPases), proteins that use energy from guanosine 5′-triphosphate (GTP) to promote conformational changes that lead to transitions between ribosome functional states (1, 2). In bacteria, for instance, initiation of protein synthesis is largely regulated by initiation factor 2 (IF-2), a GTPase that stabilizes the initiator tRNA in the P site of the ribosome (3). Subsequently, the elongation step is catalyzed by two universally conserved GTPases, elongation factor Tu (EF-Tu) and elongation factor G (EF-G). The ternary complex, consisting of EF-Tu, GTP, and the aminoacyl-tRNA, interacts with the ribosome to decode the codon in the A site of the ribosome. Following accommodation of the aminoacyl-tRNA in the A site of the ribosome and subsequent peptide bond formation, the tRNA-mRNA duplex is translocated by one codon-a process catalyzed by EF-G and GTP (4-6). Termination of protein synthesis is triggered when a stop codon is reached, upon which the newly synthesized protein is released with the help of release factor 3 (RF-3), yet another GTPase (7).Elongation factor 4 (EF-4/LepA) is a highly conserved protein structurally similar to EF-G (8) and has a ribosome-dependent GTPase activity (9-13). However, despite numerous studies, its function has remained elusive (9-20). Fast kinetic studies showed that EF-4 competes with EF-G during elongation for binding to the pretranslocation (PRE) ribosome, with tRNAs in the A and P sites (17). Despite this, EF-4 was also shown to increase the rate of protein synthesis at high intracellular ionic strength (16), without any effect on translational accuracy (16, 18). Conversely, EF-4 was also reported...

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confidence: 99%

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

Gagnon

Lin

Steitz

2016

Proc. Natl. Acad. Sci. U.S.A.

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“…G proteins can be regarded as molecular switches that are driven by GTP hydrolysis and cycle between the active GTP-bound state and the inactive GDP-bound form. Although many G proteins require an external GTPase activating protein (GAP) to trigger efficient GTP hydrolysis (4,5), trGTPases that interact with the ribosome appear to be activated by RNA rather than by a classical GAP (6). In bacteria, ribosome-activated GTPases include the universally present elongation factors (EF)-Tu, EF-G and EF-4 (LepA) and the initiation factor IF2, as well as the less frequently found, yet highly conserved, release factor RF3, tetracycline resistance proteins (TetO), or the selenocystein incorporation factor SelB (7).…”

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confidence: 99%

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

Koch

Flür

Kreutz

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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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.

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“…[29,30] A2660 resides in the so-called sarcin-ricin loop (SRL) which has been shown previously by biochemical and structural studies (reviewed in ref. [30]) to be in immediate proximity to the GTP bound in the active site of translational GTPases such as EF-G or EF-Tu. Our atomic mutagenesis studies highlighted the C6 exocyclic amino group at A2660 as a potential trigger of EF-G GTPase activity (Fig.…”

Section: Making Proteins: the Ribosomal Elongation Cyclementioning

confidence: 81%

“…Stop codons are usually not recognized by tRNA anticodons but by specific peptide motifs of class I release factors (RF1 and RF2 in bacteria). Binding of a class I RF to the A-site of the ribosome results in the hydrolysis of the tional GTPases, [29,30] and tRNA translocation. [31] One of the most surprising results of our recent research was the finding that a single rRNA backbone group, namely the ribose 2'-OH of the inner core PTC residue A2451, rather than any of the universally conserved nucleobases is crucial for peptide bond synthesis (Fig.…”

Section: Making Proteins: the Ribosomal Elongation Cyclementioning

confidence: 99%

“…[30] Structural data suggest an alternate or additional ribosomal trigger to fire GTPase activity on ribosome-bound elongation factors, namely a non-bridging oxygen at the phosphordiester bond between SRL residues G2661 and A2662. [38] Equipped with the atomic mutagenesis approach this model can now be tested experimentally since it enables us to individually manipulate the suspected rRNA backbone groups in that region of the SRL.…”

Section: Making Proteins: the Ribosomal Elongation Cyclementioning

confidence: 99%

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Atomic Mutagenesis of the Ribosome: Towards a Molecular Understanding of Translation

Polacek

2013

Chimia

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The multifaceted repertoire of non-protein-coding RNAs (ncRNAs) in organisms of all three domains of life emphasizes their fundamental role in biology. Research in my lab focuses on revealing the regulatory and catalytic function of small and large ncRNAs in different model organisms. In particular we are interested in understanding ncRNA/protein complexes such as the vault complex or the ribosome. The ribosome, the central enzyme of protein biosynthesis, is a multifunctional ribonucleoprotein particle composed of two unequal subunits that translates the genome's message into all proteins needed for life. The crucial role the translation machinery plays in gene expression is also mirrored by the fact that the ribosome represents the main target for antibiotics. Decades of genetic, biochemical and recent crystallographic studies revealed the ribosome as an RNA-enzyme with roots in the 'RNA world'. Despite these experimental insights, the catalytic and regulatory mechanisms of the ribosomal RNA are still not fully understood at the molecular level. To unravel the detailed contributions of rRNA nucleotides for protein synthesis we have developed and applied an 'atomic mutagenesis' approach. This tool allows the role of specific 23S rRNA functional groups and even individual atoms to be studied during various stages of the ribosomal elongation cycle with thus far unequalled precision. This experimental approach bridges the disciplines of biochemistry and organic chemistry and has recently revealed specific functional 23S rRNA groups involved in peptide bond synthesis, peptidyl-tRNA hydrolysis, GTPase activation, and tRNA translocation.

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Ribosome-associated GTPases: The role of RNA for GTPase activation

Cited by 33 publications

References 80 publications

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

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

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

Atomic Mutagenesis of the Ribosome: Towards a Molecular Understanding of Translation

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