Professional Education


  • Master of Science, Uppsala Universitet (2012)
  • Doctor of Philosophy, Uppsala Universitet (2016)
  • Bachelor of Science, University of Science and Technology Beijing (2010)

Stanford Advisors


All Publications


  • Dynamic basis of fidelity and speed in translation: Coordinated multistep mechanisms of elongation and termination. Protein science Prabhakar, A., Choi, J., Wang, J., Petrov, A., Puglisi, J. D. 2017

    Abstract

    As the universal machine that transfers genetic information from RNA to protein, the ribosome synthesizes proteins with remarkably high fidelity and speed. This is a result of the accurate and efficient decoding of mRNA codons via multistep mechanisms during elongation and termination stages of translation. These mechanisms control how the correct sense codon is recognized by a tRNA for peptide elongation, how the next codon is presented to the decoding center without change of frame during translocation, and how the stop codon is discriminated for timely release of the nascent peptide. These processes occur efficiently through coupling of chemical energy expenditure, ligand interactions, and conformational changes. Understanding this coupling in detail required integration of many techniques that were developed in the past two decades. This multidisciplinary approach has revealed the dynamic nature of translational control and uncovered how external cellular factors such as tRNA abundance and mRNA modifications affect the synthesis of the protein product. Insights from these studies will aid synthetic biology and therapeutic approaches to translation.

    View details for DOI 10.1002/pro.3190

    View details for PubMedID 28480640

  • Translational roles of the C75 2'OH in an in vitro tRNA transcript at the ribosomal A, P and E sites. Scientific reports Wang, J., Forster, A. C. 2017; 7 (1): 6709

    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.

    View details for DOI 10.1038/s41598-017-06991-6

    View details for PubMedID 28751745

    View details for PubMedCentralID PMC5532260

  • Kinetics of tRNA(Pyl)-mediated amber suppression in Escherichia coli translation reveals unexpected limiting steps and competing reactions BIOTECHNOLOGY AND BIOENGINEERING Wang, J., Kwiatkowski, M., Forster, A. C. 2016; 113 (7): 1552-1559

    Abstract

    The utility of ribosomal incorporation of unnatural amino acids (AAs) in vivo is generally restricted by low efficiencies, even with the most widely used suppressor tRNA(Pyl) . Because of the difficulties of studying incorporation in vivo, almost nothing is known about the limiting steps after tRNA charging. Here, we measured the kinetics of all subsequent steps using a purified Escherichia coli translation system. Dipeptide formation from initiator fMet-tRNA(fMet) and tRNA(Pyl) charged with allylglycine or methylserine displayed unexpectedly sluggish biphasic kinetics, ∼30-fold slower than for native substrates. The amplitude of the fast phases increased with increasing EF-Tu concentration, allowing measurement of Kd values of EF-Tu binding, both of which were ∼25-fold weaker than normal. However, binding could be increased ∼30-fold by lowering temperature. The fast phase rates were limited by the surprisingly ∼10-fold less efficient binding of EF-Tu:GTP:AA-tRNA(Pyl) ternary complex to the ribosomes, not GTP hydrolysis or peptide bond formation. Furthermore, processivity was unexpectedly impaired as ∼40% of the dipeptidyl-tRNA(Pyl) could not be elongated to tripeptide. Dipeptide formation was slow enough that termination due to misreading the UAG codon by non-cognate RF2 became very significant. This new understanding provides a framework for improving unnatural AA incorporation by amber suppression. Biotechnol. Bioeng. 2016;113: 1552-1559. © 2015 Wiley Periodicals, Inc.

    View details for DOI 10.1002/bit.25917

    View details for Web of Science ID 000377527900017

    View details for PubMedID 26705134

  • Ribosomal Peptide Syntheses from Activated Substrates Reveal Rate Limitation by an Unexpected Step at the Peptidyl Site. Journal of the American Chemical Society Wang, J., Kwiatkowski, M., Forster, A. C. 2016; 138 (48): 15587–95

    Abstract

    Protein synthesis (translation) is central to cellular function and antibiotic development. Interestingly, the key chemical step of translation, peptide bond formation, is among the slower enzymatic reactions. The reason for this remains controversial because of reliance on studies using highly modified, severely minimized, or unreactive substrate analogues. Here, we investigated this problem by fast kinetics using full-length aminoacyl-tRNA substrates with atomic substitutions that activated the ester electrophile. While trifluoro substitution of hydrogens in nonconserved positions of the peptidyl-site substrate dramatically increased the ester reactivity in solution assays, a large hastening of the combined rates of ribosomal accommodation and peptidyl transfer was observed only with a slowly reacting aminoacyl-site nucleophile, proline. With a fast-reacting A-site nucleophile, phenylalanine, effects did not correlate at all with electrophilicities. As effects were observed using the same, natural, aminoacyl-tRNA at the A site and all rates of accommodation/peptidyl transfer were pH dependent, we concluded that rate limitation was not by A-site accommodation but rather by peptidyl transfer and a hitherto unexpected step at the P site. This new slow step, which we term P-site accommodation, has implications for the activation or inhibition of ribosome function in vitro and in vivo.

    View details for DOI 10.1021/jacs.6b06936

    View details for PubMedID 27934010

  • Kinetics of Ribosome-Catalyzed Polymerization Using Artificial Aminoacyl-tRNA Substrates Clarifies Inefficiencies and Improvements ACS CHEMICAL BIOLOGY Wang, J., Kwiatkowski, M., Forster, A. C. 2015; 10 (10): 2187-2192

    Abstract

    Ribosomal synthesis of polymers of unnatural amino acids (AAs) is limited by low incorporation efficiencies using the artificial AA-tRNAs, but the kinetics have yet to be studied. Here, kinetics were performed on five consecutive incorporations using various artificial AA-tRNAs with all intermediate products being analyzed. Yields within a few seconds displayed similar trends to our prior yields after 30 min without preincubation, demonstrating the relevance of fast kinetics to traditional long-incubation translations. Interestingly, the two anticodon swaps were much less inhibitory in the present optimized system, which should allow more flexibility in the engineering of artificial AA-tRNAs. The biggest kinetic defect was caused by the penultimate dC introduced from the standard, chemoenzymatic, charging method. This prompted peptidyl-tRNA drop-off, decreasing processivities during five consecutive AA incorporations. Indeed, two tRNA charging methods that circumvented the dC dramatically improved efficiencies of ribosomal, consecutive, unnatural AA incorporations to give near wild-type kinetics.

    View details for DOI 10.1021/acschembio.5b00335

    View details for Web of Science ID 000363225100002

    View details for PubMedID 26191973

  • Facile Synthesis of N-Acyl-aminoacyl-pCpA for Preparation of Mischarged Fully Ribo tRNA BIOCONJUGATE CHEMISTRY Kwiatkowski, M., Wang, J., Forster, A. C. 2014; 25 (11): 2086-2091

    Abstract

    Chemical synthesis of N-acyl-aminoacyl-pdCpA and its ligation to tRNA(minus CA) is widely used for the preparation of unnatural aminoacyl-tRNA substrates for ribosomal translation. However, the presence of the unnatural deoxyribose can decrease incorporation yield in translation and there is no straightforward method for chemical synthesis of the natural ribo version. Here, we show that pCpA is surprisingly stable to treatment with strong organic bases provided that anhydrous conditions are used. This allowed development of a facile method for chemical aminoacylation of pCpA. Preparative synthesis of pCpA was also simplified by using t-butyl-dithiomethyl protecting group methodology, and a more reliable pCpA postpurification treatment method was developed. Such aminoacyl-pCpA analogues ligated to tRNA(minus CA) transcripts are highly active in a purified translation system, demonstrating utility of our synthetic method.

    View details for DOI 10.1021/bc500441b

    View details for Web of Science ID 000345309000020

    View details for PubMedID 25338217

  • Peptide Formation by N-Methyl Amino Acids in Translation Is Hastened by Higher pH and tRNA(Pro) ACS CHEMICAL BIOLOGY Wang, J., Kwiatkowski, M., Pavlov, M. Y., Ehrenberg, M., Forster, A. C. 2014; 9 (6): 1303-1311

    Abstract

    Applications of N-methyl amino acids (NMAAs) in drug discovery are limited by their low efficiencies of ribosomal incorporation, and little is known mechanistically about the steps leading to incorporation. Here, we demonstrate that a synthetic tRNA body based on a natural N-alkyl amino acid carrier, tRNA(Pro), increases translation incorporation rates of all three studied NMAAs compared with tRNA(Phe)- and tRNA(Ala)-based bodies. We also investigate the pH dependence of the incorporation rates and find that the rates increase dramatically in the range of pH 7 to 8.5 with the titration of a single proton. Results support a rate-limiting peptidyl transfer step dependent on deprotonation of the N-nucleophile of the NMAA. Competition experiments demonstrate that several futile cycles of delivery and rejection of A-site NMAA-tRNA are required per peptide bond formed and that increasing magnesium ion concentration increases incorporation yield. Data clarify the mechanism of ribosomal NMAA incorporation and provide three generalizable ways to improve incorporation of NMAAs in translation.

    View details for DOI 10.1021/cb500036a

    View details for Web of Science ID 000337870500012

    View details for PubMedID 24673854