Functional elucidation of a key contact between tRNA and the large ribosomal subunit rRNA during decoding

Ortiz‐Meoz, Rodrigo F.; Green, Rachel

doi:10.1261/rna.2232710

Cited by 16 publications

(21 citation statements)

References 36 publications

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“…In our previous work, we found that tRNA Trp -miscoding tRNAs exploit an interaction with residue A1913 of H69 to effectively accommodate on certain near-cognate codon ribosome complexes (21). These results are extended in Fig.…”

Section: Transient Kinetic Analysis Of Effects Of H69 Mutations On Sesupporting

confidence: 65%

“…Although there are numerous examples in the literature where both GTPase activation and accommodation are similarly affected by a miscoding tRNA (11,12), we have recently shown that a mutation in the elbow region of tRNA Trp-G59A stimulates accommodation, but not GTPase activation, on a near-cognate codon (21). Interestingly, this variant tRNA also depends on H69 residue A1913 to stimulate this accommodation step.…”

Section: Discussionmentioning

confidence: 71%

“…Kinetic Analysis-Accommodation and GTPase activation measurements were carried out essentially as described previously (11,12,21). Peptide release experiments were carried out as described previously (19), where 0.25 m initiation complexes were typically mixed with 5 M purified release factor protein in a rapid quench apparatus.…”

Section: Methodsmentioning

confidence: 99%

“…Peptide release experiments were carried out as described previously (19), where 0.25 m initiation complexes were typically mixed with 5 M purified release factor protein in a rapid quench apparatus. To account for any subunit association defects of variant ribosomes, all initiation complexes were supplemented with purified WT 30 S ribosomal subunits (21).…”

Section: Methodsmentioning

confidence: 99%

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Helix 69 Is Key for Uniformity during Substrate Selection on the Ribosome

Ortiz‐Meoz

Green

2011

Journal of Biological Chemistry

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Structural studies of ribosome complexes with bound tRNAs and release factors show considerable contacts between these factors and helix 69 (H69) of 23 S rRNA. Although biochemical and genetic studies have provided some general insights into the role of H69 in tRNA and RF selection, a detailed understanding of these contributions remains elusive. Here, we present a presteady-state kinetic analysis establishing that two distinct regions of H69 make critical contributions to substrate selection. The loop of H69 (A1913) forms contacts necessary for the efficient accommodation of a subset of natural tRNA species, whereas the base of the stem (G1922) is specifically critical for UGA codon recognition by the class 1 release factor RF2. These data define a broad and critical role for this centrally located intersubunit helix (H69) in accurate and efficient substrate recognition by the ribosome.The ribosome is the ribonucleoprotein machine responsible for the faithful translation of the genetic material into the encoded polypeptide. The core RNA components of the ribosome (the 16 S and 23 S rRNA) that reflect its earliest evolution play a key role in interactions that specify the selection of tRNAs and release factors on recognition of sense and stop codons, respectively. During translation elongation, the selection of a cognate ternary complex (composed of aa-tRNA, EFTu, and GTP) from solution is specified primarily in the small ribosomal subunit where three nucleotides in 16 S rRNA directly evaluate the geometry of the codon-anticodon interaction (1). Though different in molecular detail, the selection of class 1 release factors takes place in the same decoding center of the small ribosomal subunit where conserved protein features of the RF engage the stop codon. Thus, for both decoding events, molecular interactions in the small subunit "decoding center" are early and critical contributors to the selection of cognate substrates during the translational cycle.In addition to these interactions in the decoding center, it is believed that other molecular interactions between the ribosome and these substrates contribute to binding and specificity. Structural studies of tRNA (2-5), release factors (6 -8), and ribosome recycling factor (9, 10) bound to the ribosome reveal that H69 2 of the large subunit rRNA makes extensive contacts with all of these factors. This universally conserved helix forms an intersubunit bridge that is proximal to the substrate binding cleft and directly contacts the functionally critical h44 of 16 S rRNA which contains two of the three nucleotides known to directly interact with the decoding helix (codon-anticodon) during tRNA selection (A1492 and A1493).What is the specific nature of these molecular interactions, and what do they suggest about the role of H69 in ribosome function? H69 contacts incoming tRNA in both the pre-and postaccommodation steps near positions 25/26 of the D stem and position 38, located near the anticodon stem; tRNA mutations at these positions are linked to miscoding pheno...

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Section: Transient Kinetic Analysis Of Effects Of H69 Mutations On Sesupporting

confidence: 65%

Section: Discussionmentioning

confidence: 71%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Helix 69 Is Key for Uniformity during Substrate Selection on the Ribosome

Ortiz‐Meoz

Green

2011

Journal of Biological Chemistry

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“…8 H69 contains three modified nucleotides, two pseudouridines (Ψ) 7b and one 3-methylpseudouridine (m 3 Ψ) 7a (Figure 2), which are important for ribosome function 8a, 8b, 9 and modulation of the loop structure. 9c, 9d, 10 H69 has also been implicated in a number of key ribosome functions such as ribosome recycling, 11 tRNA selection, 12 translational fidelity, 13 and subunit association, 8a, 9a making it an attractive target for antibiotic drug design.…”

Section: Introductionmentioning

confidence: 99%

The development of peptide ligands that target helix 69 rRNA of bacterial ribosomes

Dremann

Chow

2016

Bioorganic & Medicinal Chemistry

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Antibiotic resistance prevents successful treatment of common bacterial infections, making it clear that new target locations and drugs are required to resolve this ongoing challenge. The bacterial ribosome is a common target for antibacterials due to its essential contribution to cell viability. The focus of this work is a region of the ribosome called helix 69 (H69), which was recently identified as a secondary target site for aminoglycoside antibiotics. H69 has key roles in essential ribosomal processes such as subunit association, ribosome recycling, and tRNA selection. Conserved across phylogeny, bacterial H69 also contains two pseudouridines and one 3-methylpseudouridine. Phage display revealed a heptameric peptide sequence that targeted H69. Using solid-phase synthesis, peptide variants with higher affinity and improved selectivity to modified H69 were generated. Electrospray ionization mass spectrometry was used to determine relative apparent dissociation constants of the RNA-peptide complexes.

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Helix 69: A Multitasking RNA Motif as a Novel Drug Target

Jiang

Sakakibara²,

Chow

2013

Israel Journal of Chemistry

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High‐resolution structures have shown that helix 69 (H69) of the large ribosomal subunit can assume variable conformational states during translation. Solution studies on small model RNAs, isolated subunits, and complete ribosomes also revealed a variety of H69 conformations. Specific nucleotides of H69 that undergo changes in their relative orientations within the ribosome structure play important roles in both translation and ribosome rescue. Furthermore, the presence of multiple pseudouridines influences the global conformational states of H69, and these highly conserved modifications play a role in regulating the positioning of key functional residues. Helix 69 has recently been identified as a novel antibiotic target site. Small molecules such as aminoglycosides bind to specific conformational states of H69 in bacterial ribosomes and affect the translation process. A variety of techniques have been employed to study H69, ranging from chemical synthesis to X‐ray crystallography, highlighting the importance of a detailed evaluation of the underlying principles of RNA conformational dynamics and drug targeting.

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Functional elucidation of a key contact between tRNA and the large ribosomal subunit rRNA during decoding

Cited by 16 publications

References 36 publications

Helix 69 Is Key for Uniformity during Substrate Selection on the Ribosome

Helix 69 Is Key for Uniformity during Substrate Selection on the Ribosome

The development of peptide ligands that target helix 69 rRNA of bacterial ribosomes

Helix 69: A Multitasking RNA Motif as a Novel Drug Target

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