Functionalized polystyrene supports for solid-phase synthesis of glycyl-, alanyl-, and isoleucyl-RNA conjugates as hydrolysis-resistant mimics of peptidyl-tRNAs

Steger, Jessica; Micura, Ronald

doi:10.1016/j.bmc.2011.07.018

Cited by 18 publications

(16 citation statements)

References 41 publications

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“…The nh-Ala-microhelix Pro containing a hydrolysis-resistant 3′-amide-linked Ala moiety (Fig. 2) was synthesized as described previously (45) using a solid-phase synthesis approach with 3′-aminoacylamino-3′-deoxyadenosine-modified functionalized solid sup-ports (46) and 2′-O-TOM nucleoside phosphoramidites (47). Before the experiments, RNAs were refolded by heating at 80°C for 2 min then 60°C for 2 min, followed by the addition of MgCl 2 to 10 mM and then slow cooling at room temperature and equilibration with either NMR or AUC buffer using SEC, as described above for protein preparation.…”

Section: Methodsmentioning

confidence: 99%

Conformational and chemical selection by a trans -acting editing domain

Danhart

Bakhtina

Cantara

et al. 2017

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

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Molecular sieves ensure proper pairing of tRNAs and amino acids during aminoacyl-tRNA biosynthesis, thereby avoiding detrimental effects of mistranslation on cell growth and viability. Mischarging errors are often corrected through the activity of specialized editing domains present in some aminoacyl-tRNA synthetases or via single-domain -editing proteins. ProXp-ala is a ubiquitous-editing enzyme that edits Ala-tRNA, the product of Ala mischarging by prolyl-tRNA synthetase, although the structural basis for discrimination between correctly charged Pro-tRNA and mischarged Ala-tRNA is unclear. Deacylation assays using substrate analogs reveal that size discrimination is only one component of selectivity. We used NMR spectroscopy and sequence conservation to guide extensive site-directed mutagenesis of ProXp-ala, along with binding and deacylation assays to map specificity determinants. Chemical shift perturbations induced by an uncharged tRNA acceptor stem mimic, microhelix, or a nonhydrolyzable mischarged Ala-microhelix substrate analog identified residues important for binding and deacylation. Backbone N NMR relaxation experiments revealed dynamics for a helix flanking the substrate binding site in free ProXp-ala, likely reflecting sampling of open and closed conformations. Dynamics persist on binding to the uncharged microhelix, but are attenuated when the stably mischarged analog is bound. Computational docking and molecular dynamics simulations provide structural context for these findings and predict a role for the substrate primary α-amine group in substrate recognition. Overall, our results illuminate strategies used by a-editing domain to ensure acceptance of only mischarged Ala-tRNA, including conformational selection by a dynamic helix, size-based exclusion, and optimal positioning of substrate chemical groups.

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Section: Methodsmentioning

confidence: 99%

Conformational and chemical selection by a trans -acting editing domain

Danhart

Bakhtina

Cantara

et al. 2017

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

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“…The ACC-puromycin conjugate was produced as previously described [32]. The ACCA-Leu-Phe conjugate was produced according to the following references [33,34]. The ACCA-Pro-Pro and ACCA-Pro conjugates were produced as outlined in Fig EV1 and Fmoc)), 4.23 (m, 4H, HC(4 0 ) and HC(a, Pro) and OCH 2 (Fmoc)), 3.75 (s, 6H, 2xOCH 3 (DMT)), 3.70 (m, 2H, N (CH 2 CH 2 CH 2 CH 3 ) 2 ), 3.52-3.36 (m, 6H, H 2 C(5 0 ) and CH 2 (Pro) and N (CH 2 CH 2 CH 2 CH 3 ) 2 ), 2.51 and 2.36 (s, 2H, OOCH 2 CH 2 CH 2 CH 2 COO), 2.02-1.80 (m, 4H, 2xCH 2 (Pro)), 1.62 (m, 8H, OOCH 2 CH 2 CH 2 CH 2 COO and N(CH 2 CH 2 CH 2 CH 3 ) 2 ), 1.35 (m, 4H, N(CH 2 CH 2 CH 2 CH 3 ) 2 ), 0.93 (m, 6H, N(CH 2 CH 2 CH 2 CH 3 ) 2 ).…”

Section: Synthesis Of Hydrolysis-resistant Aminoacyl-and Peptidyl-trnmentioning

confidence: 99%

Molecular insights into protein synthesis with proline residues

et al. 2016

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Proline is an amino acid with a unique cyclic structure that facilitates the folding of many proteins, but also impedes the rate of peptide bond formation by the ribosome. As a ribosome substrate, proline reacts markedly slower when compared with other amino acids both as a donor and as an acceptor of the nascent peptide. Furthermore, synthesis of peptides with consecutive proline residues triggers ribosome stalling. Here, we report crystal structures of the eukaryotic ribosome bound to analogs of mono-and diprolyl-tRNAs. These structures provide a high-resolution insight into unique properties of proline as a ribosome substrate. They show that the cyclic structure of proline residue prevents proline positioning in the amino acid binding pocket and affects the nascent peptide chain position in the ribosomal peptide exit tunnel. These observations extend current knowledge of the protein synthesis mechanism. They also revise an old dogma that amino acids bind the ribosomal active site in a uniform way by showing that proline has a binding mode distinct from other amino acids.

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“…In principle, solid supports 8 and 10 can be utilized for elongation of a peptide by automated solid-phase peptide synthesis based on N -(9-fluorenyl)methoxycarbonyl (Fmoc) protected amino acids, as we have shown previously on the analogous 3′-aminoacylamino-3′-deoxyadenosine-functionalized solid support series. 22,26,36 Here, the support 8 was manually coupled with the corresponding Fmoc-protected amino acid using the tetramethyl- O -(1 H -benzotriazol-1-yl)uronium (HBTU) reagent. Subsequently, solid supports 8 and 10 were directly supplied to solid phase RNA synthesis.…”

Section: Resultsmentioning

confidence: 99%