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| 2. |
- Choi, Junhong, et al.
(författare)
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N-6-methyladenosine in mRNA disrupts tRNA selection and translation-elongation dynamics
- 2016
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Ingår i: Nature Structural & Molecular Biology. - : Springer Science and Business Media LLC. - 1545-9993 .- 1545-9985. ; 23:2, s. 110-
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Tidskriftsartikel (refereegranskat)abstract
- N-6-methylation of adenosine (forming m(6)A) is the most abundant post-transcriptional modification within the coding region of mRNA, but its role during translation remains unknown. Here, we used bulk kinetic and single-molecule methods to probe the effect of m(6)A in mRNA decoding. Although m(6)A base-pairs with uridine during decoding, as shown by X-ray crystallographic analyses of Thermus thermophilus ribosomal complexes, our measurements in an Escherichia coli translation system revealed that m(6)A modification of mRNA acts as a barrier to tRNA accommodation and translation elongation. The interaction between an m(6)A-modified codon and cognate tRNA echoes the interaction between a near-cognate codon and tRNA, because delay in tRNA accommodation depends on the position and context of m(6)A within codons and on the accuracy level of translation. Overall, our results demonstrate that chemical modification of mRNA can change translational dynamics.
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| 3. |
- Ieong, Ka-Weng, 1985-, et al.
(författare)
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N-6-Methyladenosines in mRNAs reduce the accuracy of codon reading by transfer RNAs and peptide release factors
- 2021
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Ingår i: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 49:5, s. 2684-2699
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Tidskriftsartikel (refereegranskat)abstract
- We used quench flow to study how N-6-methylated adenosines (m(6)A) affect the accuracy ratio between k(cat)/K-m (i.e. association rate constant (k(a)) times probability (P-p) of product formation after enzyme-substrate complex formation) for cognate and near-cognate substrate for mRNA reading by tRNAs and peptide release factors 1 and 2 (RFs) during translation with purified Escherichia coli components. We estimated k(cat)/K-m for Glu-tRNA(Glu), EF-Tu and GTP forming ternary complex (T-3) reading cognate (GAA and Gm(6)AA) or near-cognate (GAU and Gm(6)AU) codons. k(a) decreased 10-fold by m(6)A introduction in cognate and near-cognate cases alike, while P-p for peptidyl transfer remained unaltered in cognate but increased 10-fold in near-cognate case leading to 10-fold amino acid substitution error increase. We estimated k(cat)/K-m for ester bond hydrolysis of P-site bound peptidyl-tRNA by RF2 reading cognate (UAA and Um(6)AA) and near-cognate (UAG and Um(6)AG) stop codons to decrease 6-fold or 3-fold by m(6)A introduction, respectively. This 6-fold effect on UAA reading was also observed in a single-molecule termination assay. Thus, m(6)A reduces both sense and stop codon reading accuracy by decreasing cognate significantly more than near-cognate k(cat)/K-m, in contrast to most error inducing agents and mutations, which increase near-cognate at unaltered cognate k(cat)/K-m.
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| 4. |
- Prabhakar, Arjun, et al.
(författare)
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Dynamics of release factor recycling during translation termination in bacteria
- 2023
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Ingår i: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 51:11, s. 5774-5790
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Tidskriftsartikel (refereegranskat)abstract
- In bacteria, release of newly synthesized proteins from ribosomes during translation termination is catalyzed by class-I release factors (RFs) RF1 or RF2, reading UAA and UAG or UAA and UGA codons, respectively. Class-I RFs are recycled from the post-termination ribosome by a class-II RF, the GTPase RF3, which accelerates ribosome intersubunit rotation and class-I RF dissociation. How conformational states of the ribosome are coupled to the binding and dissociation of the RFs remains unclear and the importance of ribosome-catalyzed guanine nucleotide exchange on RF3 for RF3 recycling in vivo has been disputed. Here, we profile these molecular events using a single-molecule fluorescence assay to clarify the timings of RF3 binding and ribosome intersubunit rotation that trigger class-I RF dissociation, GTP hydrolysis, and RF3 dissociation. These findings in conjunction with quantitative modeling of intracellular termination flows reveal rapid ribosome-dependent guanine nucleotide exchange to be crucial for RF3 action in vivo.
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| 5. |
- Prabhakar, Arjun, et al.
(författare)
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Uncovering translation roadblocks during the development of a synthetic tRNA
- 2022
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Ingår i: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 50:18, s. 10201-10211
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Tidskriftsartikel (refereegranskat)abstract
- Ribosomes are remarkable in their malleability to accept diverse aminoacyl-tRNA substrates from both the same organism and other organisms or domains of life. This is a critical feature of the ribosome that allows the use of orthogonal translation systems for genetic code expansion. Optimization of these orthogonal translation systems generally involves focusing on the compatibility of the tRNA, aminoacyl-tRNA synthetase, and a non-canonical amino acid with each other. As we expand the diversity of tRNAs used to include non-canonical structures, the question arises as to the tRNA suitability on the ribosome. Specifically, we investigated the ribosomal translation of allo-tRNAUTu1, a uniquely shaped (9/3) tRNA exploited for site-specific selenocysteine insertion, using single-molecule fluorescence. With this technique we identified ribosomal disassembly occurring from translocation of allo-tRNAUTu1 from the A to the P site. Using cryo-EM to capture the tRNA on the ribosome, we pinpointed a distinct tertiary interaction preventing fluid translocation. Through a single nucleotide mutation, we disrupted this tertiary interaction and relieved the translation roadblock. With the continued diversification of genetic code expansion, our work highlights a targeted approach to optimize translation by distinct tRNAs as they move through the ribosome.
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