Active role of elongation factor G in maintaining the mRNA reading frame during translation

Peng, Bee-Zen; Bock, Lars V.; Belardinelli, Riccardo; Peske, Frank; Grubmüller, Helmut; Rodnina, Marina V.

doi:10.1126/sciadv.aax8030

Cited by 43 publications

(93 citation statements)

References 40 publications

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“…Interestingly, all three suppressor mutants had mutations affecting the essential translation elongation factor G, in two strains (GP3101 and GP3103) the highly conserved Ala-579 was replaced by a Val (see S9 Fig ). Ala-579 is located in immediate vicinity of a loop (Loop II) that is required for tRNA translocation in the ribosome [ 76 , 77 ], suggesting that the mutation interferes with translation. In the third strain (GP3102) we found an A21T substitution.…”

Section: Resultsmentioning

confidence: 99%

Essentiality of c-di-AMP in Bacillus subtilis: Bypassing mutations converge in potassium and glutamate homeostasis

et al. 2021

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In order to adjust to changing environmental conditions, bacteria use nucleotide second messengers to transduce external signals and translate them into a specific cellular response. Cyclic di-adenosine monophosphate (c-di-AMP) is the only known essential nucleotide second messenger. In addition to the well-established role of this second messenger in the control of potassium homeostasis, we observed that glutamate is as toxic as potassium for a c-di-AMP-free strain of the Gram-positive model bacterium Bacillus subtilis. In this work, we isolated suppressor mutants that allow growth of a c-di-AMP-free strain under these toxic conditions. Characterization of glutamate resistant suppressors revealed that they contain pairs of mutations, in most cases affecting glutamate and potassium homeostasis. Among these mutations, several independent mutations affected a novel glutamate transporter, AimA (Amino acid importer A, formerly YbeC). This protein is the major transporter for glutamate and serine in B. subtilis. Unexpectedly, some of the isolated suppressor mutants could suppress glutamate toxicity by a combination of mutations that affect phospholipid biosynthesis and a specific gain-of-function mutation of a mechanosensitive channel of small conductance (YfkC) resulting in the acquisition of a device for glutamate export. Cultivation of the c-di-AMP-free strain on complex medium was an even greater challenge because the amounts of potassium, glutamate, and other osmolytes are substantially higher than in minimal medium. Suppressor mutants viable on complex medium could only be isolated under anaerobic conditions if one of the two c-di-AMP receptor proteins, DarA or DarB, was absent. Also on complex medium, potassium and osmolyte toxicity are the major bottlenecks for the growth of B. subtilis in the absence of c-di-AMP. Our results indicate that the essentiality of c-di-AMP in B. subtilis is caused by the global impact of the second messenger nucleotide on different aspects of cellular physiology.

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Section: Resultsmentioning

confidence: 99%

Essentiality of c-di-AMP in Bacillus subtilis: Bypassing mutations converge in potassium and glutamate homeostasis

et al. 2021

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“…During translocation, A and P site tRNAs move together with their respective mRNA codons. It has been shown that bacterial EFG is crucial to maintain the mRNA‐tRNA interaction during tRNA movement, as the absence or mutation of EFG leads to increased frameshifting and decreased translocation efficiency (Martemyanov et al , ; Savelsbergh et al , 2000a; Holtkamp et al , ; Peng et al , ; Zhou et al , ). An important parameter to maintain the mRNA reading frame is the interaction of mtEFG1 domain IV with the tRNA‐mRNA module via two apical loops that engage with the minor groove of the codon–anticodon base pairs and with the backbone of the peptidyl‐tRNA (Gao et al , ).…”

Section: Resultsmentioning

confidence: 99%

“…This interaction is conserved in mitochondrial translocation as mtEFG1 retained a critical di‐glycine motif (G544/G545) at the tip of loop1 that enables the loop to sterically fit into the minor groove of the mRNA‐tRNA module (Figs C and EV3C). Loops 1 and 2 in addition contain conserved residues Q542 and H617 (Q500 and H573 in T. thermophilus ) that contact the tRNA backbone and prevent tRNA slippage (Figs C and EV3C) (Gao et al , ; Ramrath et al , ; Zhou et al , ; Peng et al , ).…”

Section: Resultsmentioning

confidence: 99%

Structural insights into mammalian mitochondrial translation elongation catalyzed by mt EFG 1

Kummer

Ban

2020

The EMBO Journal

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Mitochondria are eukaryotic organelles of bacterial origin where respiration takes place to produce cellular chemical energy. These reactions are catalyzed by the respiratory chain complexes located in the inner mitochondrial membrane. Notably, key components of the respiratory chain complexes are encoded on the mitochondrial chromosome and their expression relies on a dedicated mitochondrial translation machinery. Defects in the mitochondrial gene expression machinery lead to a variety of diseases in humans mostly affecting tissues with high energy demand such as the nervous system, the heart, or the muscles. The mitochondrial translation system has substantially diverged from its bacterial ancestor, including alterations in the mitoribosomal architecture, multiple changes to the set of translation factors and striking reductions in otherwise conserved tRNA elements. Although a number of structures of mitochondrial ribosomes from different species have been determined, our mechanistic understanding of the mitochondrial translation cycle remains largely unexplored. Here, we present two cryo‐EM reconstructions of human mitochondrial elongation factor G1 bound to the mammalian mitochondrial ribosome at two different steps of the tRNA translocation reaction during translation elongation. Our structures explain the mechanism of tRNA and mRNA translocation on the mitoribosome, the regulation of mtEFG1 activity by the ribosomal GTPase‐associated center, and the basis of decreased susceptibility of mtEFG1 to the commonly used antibiotic fusidic acid.

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“…EF-G residues located in the tip of domain IV interact with the anticodon stem-loop of the A-site tRNA during translocation to the P site ( Zhou et al, 2013 ) and mutations in this domain slow the rate of translocation ( Rodnina et al, 1997 ; Savelsbergh et al, 2000 ). Recent studies using slippery codon sequences where spontaneous frameshifting occurs, EF-G can restrict frameshifting and helps to maintain the mRNA frame ( Peng et al, 2019 ). It is therefore an enticing hypothesis that EF-G may have a role in mRNA frame maintenance in addition to its established function in translocation.…”

Section: Discussionmentioning

confidence: 99%

Structural insights into mRNA reading frame regulation by tRNA modification and slippery codon–anticodon pairing

Hoffer

Hong

Sunita

et al. 2020

eLife

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Modifications in the tRNA anticodon loop, adjacent to the three-nucleotide anticodon, influence translation fidelity by stabilizing the tRNA to allow for accurate reading of the mRNA genetic code. One example is the N1-methylguaonosine modification at guanine nucleotide 37 (m1G37) located in the anticodon loop, immediately adjacent to the anticodon nucleotides 34-36. The absence of m1G37 in tRNAPro causes +1 frameshifting on polynucleotide, slippery codons. Here, we report structures of the bacterial ribosome containing tRNAPro bound to either cognate or slippery codons to determine how the m1G37 modification prevents mRNA frameshifting. The structures reveal that certain codon-anticodon contexts and m1G37 destabilize interactions of tRNAPro with the peptidyl site, causing large conformational changes typically only seen during EF-G mediated translocation of the mRNA-tRNA pairs. These studies provide molecular insights into how m1G37 stabilizes the interactions of tRNAPro with the ribosome and the influence of slippery codons on the mRNA reading frame.

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Active role of elongation factor G in maintaining the mRNA reading frame during translation

Cited by 43 publications

References 40 publications

Essentiality of c-di-AMP in Bacillus subtilis: Bypassing mutations converge in potassium and glutamate homeostasis

Essentiality of c-di-AMP in Bacillus subtilis: Bypassing mutations converge in potassium and glutamate homeostasis

Structural insights into mammalian mitochondrial translation elongation catalyzed by mt EFG 1

Structural insights into mRNA reading frame regulation by tRNA modification and slippery codon–anticodon pairing

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