Insights into the ribosome function from the structures of non-arrested ribosome–nascent chain complexes

Syroegin, Egor A.; Aleksandrova, Elena V.; Polikanov, Yury S.

doi:10.1038/s41557-022-01073-1

Cited by 25 publications

(47 citation statements)

References 77 publications

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“…The end result is a conformation in which the α-amine of the A-site acyl-tRNA is positioned optimally for amide bond formation. 4,5,16,28,29 As observed in all three structures presented here, the aromatic rings of the monomers pack tightly against the backbone of nucleotide U2506 in 23S rRNA, part of the inner ring of nucleotides in the PTC (Figure 4, Supplementary Figure S13). This packing prevents U2506 from adopting the position seen with natural acyl-tRNA substrates and thus fails to position the attacking nucleophile with respect to the carbonyl carbon of the P-site monomer.…”

Section: Discussionmentioning

confidence: 54%

“…The distance between the α-amine and reactive carbonyl for a properly positioned α-amino acid is 2.9 - 3.3 Å with the expected Bürgi-Dunitz angle of 90-110° (Figure 3A). 5,16,28 Comparison of the three cryo-EM models with aminobenzoic acid monomers in the A site revealed that, while the conserved base pair between C75 in A-site tRNA and G2553 in 23S rRNA is retained (Figure 2), the positioning of the aromatic ring in the A-site cleft formed by A2451 and C2452 shifts the position of the monomer within the PTC, especially the nucleophilic amine, relative to that of an l -α-amino acid (Supplementary Figure S12). In the case of the m ABZ monomer, the aromatic ring could be modeled in two distinct conformations that differ by a 180° rotation about the aryl-carbonyl bond (Figure 1C).…”

Section: Resultsmentioning

confidence: 99%

“…Notably, in all three experimental structures (Apy, o ABZ, and m ABZ), the P-site carbonyl oxygen is rotated towards the A-site monomer and lies almost in the same plane as the A-site nucleophile (Figure 3). 5,16,28 . Although these distances and orientations differ from those seen for properly positioned α-amino acids, 5,16,23 the observation of in vitro reactivity suggests that, at least for Apy and o ABZ, the exocyclic amino group is positioned just close enough to allow amide bond formation under physiological conditions.…”

Section: Resultsmentioning

confidence: 99%

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Aminobenzoic acid derivatives obstruct induced fit in the catalytic center of the ribosome

Majumdar

Walker

Francis

et al. 2023

Preprint

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TheEscherichia coliribosome can incorporate a variety of non-L-α-amino acid monomers into polypeptide chains, but with poor efficiency. Although these monomers span a diverse set of compounds, there exists no high-resolution structural information regarding their positioning within the catalytic center of the ribosome, the peptidyl transferase center (PTC). Thus, details regarding the mechanism of amide bond formation and the structural basis for differences and defects in incorporation efficiency remain unknown. Within a set of three aminobenzoic acid derivatives—3-aminopyridine-4-carboxylic acid (Apy),ortho-aminobenzoic acid (oABZ), andmeta-aminobenzoic acid (mABZ)—the ribosome incorporates Apy into polypeptide chains with the highest efficiency, followed byoABZ and thenmABZ, a trend that does not track with the nucleophilicity of the reactive amines. Here, we report high resolution cryo-EM structures of the ribosome with these three aminobenzoic acid derivatives charged on tRNA bound in the aminoacyl-tRNA site (A site). These structures reveal how the aromatic ring of each monomer sterically blocks positioning of nucleotide U2506, thereby preventing rearrangement of nucleotide U2585 and the resulting induced fit in the PTC required for efficient amide bond formation. They also reveal disruptions to the "proton wire" responsible for facilitating formation and breakdown of the tetrahedral intermediate. Together, the cryo-EM structures reported here provide a clear rationale for differences in reactivity of aminobenzoic acid derivatives relative to L-α-amino acids and each other, and point to stereochemical constraints on the size and geometry of non-proteinogenic monomers that can be accepted efficiently by wild-type ribosomes.

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Section: Discussionmentioning

confidence: 54%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Aminobenzoic acid derivatives obstruct induced fit in the catalytic center of the ribosome

Majumdar

Walker

Francis

et al. 2023

Preprint

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“…Collection and processing of the X-ray diffraction data, model building, and structure refinement were performed as described in our previous reports ( 22 , 23 , 25 , 26 , 28 , 29 ). Diffraction data were collected at beamlines 24ID-C and 24ID-E at the Advanced Photon Source (Argonne National Laboratory).…”

Section: Materials and Methodsmentioning

confidence: 99%

Insights into the molecular mechanism of translation inhibition by the ribosome-targeting antibiotic thermorubin

Paranjpe

Marina

Grachev

et al. 2022

Nucleic Acids Research

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Thermorubin (THR) is an aromatic anthracenopyranone antibiotic active against both Gram-positive and Gram-negative bacteria. It is known to bind to the 70S ribosome at the intersubunit bridge B2a and was thought to inhibit factor-dependent initiation of translation and obstruct the accommodation of tRNAs into the A site. Here, we show that thermorubin causes ribosomes to stall in vivo and in vitro at internal and termination codons, thereby allowing the ribosome to initiate protein synthesis and translate at least a few codons before stalling. Our biochemical data show that THR affects multiple steps of translation elongation with a significant impact on the binding stability of the tRNA in the A site, explaining premature cessation of translation. Our high-resolution crystal and cryo-EM structures of the 70S-THR complex show that THR can co-exist with P- and A-site tRNAs, explaining how ribosomes can elongate in the presence of the drug. Remarkable is the ability of THR to arrest ribosomes at the stop codons. Our data suggest that by causing structural re-arrangements in the decoding center, THR interferes with the accommodation of tRNAs or release factors into the ribosomal A site.

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“…The distance between the α-amine and reactive carbonyl for a properly positioned reaction pair is 2.9−3.3 Å with the expected Burgi-Dunitz angle of 90−110°(Figure 3A). 5,20,39 Comparison of the three cryo-EM models with aminobenzoic acid monomers in the A-site revealed that while the conserved base pair between C75 in A-site tRNA and G2553 in 23S rRNA is retained (Figure 2), the positioning of the aromatic ring in the A-site cleft formed by A2451 and C2452 shifts the position of the monomer within the PTC, especially the nucleophilic amine, relative to that of an L-α-amino acid (Supplementary Figure S12). In the case of the mABZ monomer, the aromatic ring could be modeled in two distinct conformations that differ in a 180°rotation about the aryl-carbonyl bond (Figure 1D).…”

Section: Structures Of Oabz Mabz and Apy Within The Ptc Of The Wild-t...mentioning

confidence: 99%

Aminobenzoic Acid Derivatives Obstruct Induced Fit in the Catalytic Center of the Ribosome

et al. 2023

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The Escherichia coli (E. coli) ribosome can incorporate a variety of non-l-α-amino acid monomers into polypeptide chains in vitro but with poor efficiency. Although these monomers span a diverse set of compounds, there exists no high-resolution structural information regarding their positioning within the catalytic center of the ribosome, the peptidyl transferase center (PTC). Thus, details regarding the mechanism of amide bond formation and the structural basis for differences and defects in incorporation efficiency remain unknown. Within a set of three aminobenzoic acid derivatives3-aminopyridine-4-carboxylic acid (Apy), ortho-aminobenzoic acid (oABZ), and meta-aminobenzoic acid (mABZ)the ribosome incorporates Apy into polypeptide chains with the highest efficiency, followed by oABZ and then mABZ, a trend that does not track with the nucleophilicity of the reactive amines. Here, we report high-resolution cryo-EM structures of the ribosome with each of these three aminobenzoic acid derivatives charged on tRNA bound in the aminoacyl-tRNA site (A-site). The structures reveal how the aromatic ring of each monomer sterically blocks the positioning of nucleotide U2506, thereby preventing rearrangement of nucleotide U2585 and the resulting induced fit in the PTC required for efficient amide bond formation. They also reveal disruptions to the bound water network that is believed to facilitate formation and breakdown of the tetrahedral intermediate. Together, the cryo-EM structures reported here provide a mechanistic rationale for differences in reactivity of aminobenzoic acid derivatives relative to l-α-amino acids and each other and identify stereochemical constraints on the size and geometry of non-monomers that can be accepted efficiently by wild-type ribosomes.

show abstract

Insights into the ribosome function from the structures of non-arrested ribosome–nascent chain complexes

Cited by 25 publications

References 77 publications

Aminobenzoic acid derivatives obstruct induced fit in the catalytic center of the ribosome

Aminobenzoic acid derivatives obstruct induced fit in the catalytic center of the ribosome

Insights into the molecular mechanism of translation inhibition by the ribosome-targeting antibiotic thermorubin

Aminobenzoic Acid Derivatives Obstruct Induced Fit in the Catalytic Center of the Ribosome

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