The ribosome can discriminate the chirality of amino acids within its peptidyl-transferase center.
The cellular translational machinery (TM) synthesizes proteins using exclusively L- or achiral aminoacyl-tRNAs (aa-tRNAs), despite the presence of D-amino acids in nature and their ability to be aminoacylated onto tRNAs by aa-tRNA synthetases. The ubiquity of L-amino acids in proteins has led to the hypothesis that D-amino acids are not substrates for the TM. Supporting this view, protein engineering efforts to incorporate D-amino acids into proteins using the TM have thus far been unsuccessful. Nonetheless, a mechanistic understanding of why D-aa-tRNAs are poor substrates for the TM is lacking. To address this deficiency, we have systematically tested the translation activity of D-aa-tRNAs using a series of biochemical assays. We find that the TM can effectively, albeit slowly, accept D-aa-tRNAs into the ribosomal aa-tRNA binding (A) site, use the A-site D-aa-tRNA as a peptidyl-transfer acceptor, and translocate the resulting peptidyl-D-aa-tRNA into the ribosomal peptidyl-tRNA binding (P) site. During the next round of continuous translation, however, we find that ribosomes carrying a P-site peptidyl-D-aa-tRNA partition into subpopulations that are either translationally arrested or that can continue translating. Consistent with its ability to arrest translation, chemical protection experiments and molecular dynamics simulations show that P site-bound peptidyl-D-aa-tRNA can trap the ribosomal peptidyl-transferase center in a conformation in which peptidyl transfer is impaired. Our results reveal a novel mechanism through which D-aa-tRNAs interfere with translation, provide insight into how the TM might be engineered to use D-aa-tRNAs, and increase our understanding of the physiological role of a widely distributed enzyme that clears D-aa-tRNAs from cells.
Integrated Program in Cellular, Molecular and Biomedical Sciences, Department of Chemistry, and.
Michael T
MT
Englander
Department of Chemistry, Columbia University, New York, NY 10027;
Joshua L
JL
Avins
Department of Chemistry, Columbia University, New York, NY 10027;
Rachel C
RC
Fleisher
Center for Biophysics and Computational Biology.
Bo
B
Liu
Integrated Program in Cellular, Molecular and Biomedical Sciences, Department of Chemistry, and.
Philip R
PR
Effraim
Department of Chemistry, Columbia University, New York, NY 10027;
Jiangning
J
Wang
Center for Biophysics and Computational Biology, Department of Physics, and Beckman Institute, University of Illinois at Urbana-Champaign, Urbana, IL 61801; and.
Klaus
K
Schulten
Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461.
Thomas S
TS
Leyh
Department of Chemistry, Columbia University, New York, NY 10027; rlg2118@columbia.edu vc114@columbia.edu.
Ruben L
RL
Gonzalez
Department of Chemistry, Columbia University, New York, NY 10027; rlg2118@columbia.edu vc114@columbia.edu.
Virginia W
VW
Cornish
D000596:Amino Acids / Q000737:chemistry*; D001665:Binding Sites; D002855:Chromatography, Thin Layer; D004926:Escherichia coli / Q000201:enzymology; D056004:Molecular Dynamics Simulation; D010455:Peptides / Q000737:chemistry; D010458:Peptidyl Transferases / Q000737:chemistry*; D010652:Phenylalanine-tRNA Ligase / Q000737:chemistry; D011485:Protein Binding; D014176:Protein Biosynthesis; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D012343:RNA, Transfer / Q000737:chemistry*; D012346:RNA, Transfer, Amino Acyl / Q000737:chemistry; D012270:Ribosomes / Q000737:chemistry*; D013237:Stereoisomerism; D013379:Substrate Specificity
D000596:Amino Acids; D010455:Peptides; D012346:RNA, Transfer, Amino Acyl; C019037:tRNA, peptidyl-; D012343:RNA, Transfer; D010458:Peptidyl Transferases; D010652:Phenylalanine-tRNA Ligase
D-amino acids; D-aminoacyl-tRNA deacylase; ribosome; translation arrest