ORCID Profile
0000-0002-5844-6916
Current Organisations
University of Adelaide
,
Uppsala Universitet
,
Harvard Medical School
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Publisher: Wiley
Date: 03-2020
DOI: 10.1002/ENG2.12126
Publisher: American Chemical Society (ACS)
Date: 16-01-2019
DOI: 10.1021/ACSCHEMBIO.8B00952
Abstract: Despite the stereospecificity of translation for l-amino acids (l-AAs) in vivo, synthetic biologists have enabled ribosomal incorporation of d-AAs in vitro toward encoding polypeptides with pharmacologically desirable properties. However, the steps in translation limiting d-AA incorporation need clarification. In this work, we compared d- and l-Phe incorporation in translation by quench-flow kinetics, measuring 250-fold slower incorporation into the dipeptide for the d isomer from a tRNA
Publisher: Elsevier BV
Date: 12-2020
Publisher: Wiley
Date: 03-2020
DOI: 10.1002/ENG2.12163
Publisher: Cold Spring Harbor Laboratory
Date: 08-03-2022
Abstract: Escherichia coli rRNAs are post-transcriptionally modified at 36 positions but their modification enzymes are dispensable in idually for growth, bringing into question their significance. However, a major growth defect was reported for deletion of the RlmE enzyme, which abolished a 2′ O methylation near the peptidyl transferase center (PTC) of the 23S rRNA. Additionally, an adjacent 80-nt “critical region” around the PTC had to be modified to yield significant peptidyl transferase activity in vitro. Surprisingly, we discovered that an absence of just two rRNA modification enzymes is conditionally lethal (at 20°C): RlmE and RluC. At a permissive temperature (37°C), this double knockout was shown to abolish four modifications and be defective in ribosome assembly, though not more so than the RlmE single knockout. However, the double knockout exhibited an even lower rate of tripeptide synthesis than did the single knockout, suggesting an even more defective ribosomal translocation. A combination knockout of the five critical-region-modifying enzymes RluC, RlmKL, RlmN, RlmM, and RluE (not RlmE), which synthesize five of the seven critical-region modifications and 14 rRNA and tRNA modifications altogether, was viable (minor growth defect at 37°C, major at 20°C). This was surprising based on prior in vitro studies. This five-knockout combination had minimal effects on ribosome assembly and frameshifting at 37°C, but greater effects on ribosome assembly and in vitro peptidyl transferase activity at cooler temperatures. These results establish the conditional essentiality of bacterial rRNA modification enzymes and also reveal unexpected plasticity of modification of the PTC region in vivo.
Publisher: Springer Science and Business Media LLC
Date: 27-07-2017
DOI: 10.1038/S41598-017-06991-6
Abstract: Aminoacyl-tRNAs containing a deoxy substitution in the penultimate nucleotide (C75 2′OH → 2′H) have been widely used in translation for incorporation of unnatural amino acids (AAs). However, this supposedly innocuous modification surprisingly increased peptidyl-tRNA Ala ugc drop off in biochemical assays of successive incorporations. Here we predict the function of this tRNA 2′OH in the ribosomal A, P and E sites using recent co-crystal structures of ribosomes and tRNA substrates and test these structure-function models by systematic kinetics analyses. Unexpectedly, the C75 2′H did not affect A- to P-site translocation nor peptidyl donor activity of tRNA Ala ugc . Rather, the peptidyl acceptor activity of the A-site Ala-tRNA Ala ugc and the translocation of the P-site deacylated tRNA Ala ugc to the E site were impeded. Delivery by EF-Tu was not significantly affected. This broadens our view of the roles of 2′OH groups in tRNAs in translation.
Publisher: American Chemical Society (ACS)
Date: 07-04-2022
Publisher: Elsevier BV
Date: 10-2018
DOI: 10.1016/J.CBPA.2018.07.009
Abstract: Technologies for genetically programming ribosomal incorporation of unnatural amino acids are expanding and have created many exciting applications. However, these applications are generally limited by low efficiencies of the unnatural incorporations. Here we review our current mechanistic understanding of these limitations delineated from in vitro fast kinetics. Rate limitation occurs by different mechanisms, depending on the classes of the unnatural amino acids and the tRNA adaptors. This new understanding has led to several ways of improving the incorporation efficiencies, as well as challenges of dogma on rate-limiting steps in protein synthesis in natural cells.
No related grants have been discovered for Anthony Forster.