The puromycin reaction and its relation to protein synthesis

Traut, Robert R.; Monro, R.E.

doi:10.1016/s0022-2836(64)80028-0

Cited by 367 publications

(99 citation statements)

References 17 publications

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“…To test this possibility, we reacted ECs carrying fMet-D-Phe-tRNA Phe at the P site with the antibiotic puromycin (Pmn). Pmn is an aminoacyl-mononucleotide mimic of the 3′ terminus of aa-tRNA that has been extensively used to monitor the reactivity of P site-bound peptidyl-tRNAs as peptidyl-transfer donors (20). In striking agreement with the yield of the fMet-D-Phe-Lys tripeptide synthesis reaction ( Fig.…”

mentioning

confidence: 66%

The ribosome can discriminate the chirality of amino acids within its peptidyl-transferase center

Englander¹,

Avins

Fleisher

et al. 2015

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

136

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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-aatRNA 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.A lthough the ribosome catalyzes protein synthesis using more than 20 chemically diverse natural amino acids, these natural amino acids are all of L-or achiral configurations. As a consequence, the ability of the ribosome to incorporate L-aminoacyltRNAs (L-aa-tRNAs) with both a high degree of speed and accuracy has been the focus of decades of intense mechanistic and structural investigations (1-3). In contrast, the response of the ribosome to D-aa-tRNAs has not been as well characterized. Nonetheless, a comprehensive mechanistic understanding of how the translational machinery (TM) responds to D-aa-tRNAs is of interest for several reasons. First, improved incorporation of D-amino acids by the TM would be useful for protein engineering applications that seek to use the synthetic power of the ribosome to create novel polymers (4) as well as for mechanistic applications that seek to probe protein structure and folding with unnatural amino acids (5). Second, there is growing evidence that ribosomes may have to contend with D-aa-tRNAs in vivo: D-amino acids are synthesized by racemase enzymes (6) and can be found at high concentrations in cells (7), and a growing number of aa-tRNA synthetase (aaRS) enzymes exhibit the ability to misacylate tRNAs with D-amino acids (8...

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mentioning

confidence: 66%

The ribosome can discriminate the chirality of amino acids within its peptidyl-transferase center

Englander¹,

Avins

Fleisher

et al. 2015

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

136

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“…The peptidyltransferase activity of surfaceimmobilized ribosomes was assayed with the antibiotic puromycin, an analogue of the 3Ј terminal residue of aa-tRNA that acts as an acceptor in the peptidyltransferase reaction when the A site is vacant (46). A single-molecule version of the puromycin assay was developed by using ribosomes initiated with Cy3-MettRNA fMet in the P site, dye-labeled with Cy3-NHS dye at the ␣-amino group of the amino acid.…”

Section: Surface-immobilized Ribosomes Are Highly Active In Peptide Bondmentioning

confidence: 99%

tRNA dynamics on the ribosome during translation

Blanchard

Kim

Gonzalez

et al. 2004

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

454

575

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Using single-molecule fluorescence spectroscopy, time-resolved conformational changes between fluorescently labeled tRNA have been characterized within surface-immobilized ribosomes proceeding through a complete cycle of translation elongation. Fluorescence resonance energy transfer was used to observe aminoacyl-tRNA (aa-tRNA) stably accommodating into the aminoacyl site (A site) of the ribosome via a multistep, elongation factor-Tu dependent process. Subsequently, tRNA molecules, bound at the peptidyl site and A site, fluctuate between two configurations assigned as classical and hybrid states. The lifetime of classical and hybrid states, measured for complexes carrying aa-tRNA and peptidyl-tRNA at the A site, shows that peptide bond formation decreases the lifetime of the classical-state tRNA configuration by Ϸ6-fold. These data suggest that the growing peptide chain plays a role in modulating fluctuations between hybrid and classical states. Single-molecule fluorescence resonance energy transfer was also used to observe aa-tRNA accommodation coupled with elongation factor G-mediated translocation. Dynamic rearrangements in tRNA configuration are also observed subsequent to the translocation reaction. This work underscores the importance of dynamics in ribosome function and demonstrates single-particle enzymology in a system of more than two components. P rotein synthesis, catalyzed by the ribosome, is rapid, processive, and highly regulated. In bacteria, two RNA-protein subunits consisting of a small 30S subunit and a large 50S subunit assemble around a mRNA template into a roughly spherical 70S particle that translates the nucleotide sequence into a polypeptide chain through repetitive, codon-dependent binding of aminoacylated tRNA.The landmark structures of the 30S (1, 2), 50S (3, 4), and 70S (5) ribosomal particles provide a molecular basis for understanding ribosome function. tRNA molecules bind to the ribosome in a solvent-accessible channel at the subunit interface. Three binding sites for tRNA, called the aminoacyl site (A site), peptidyl site (P site), and exit site (E site), have been identified on both the large and small subunit (Fig. 1). Each tRNA is separated from its neighbor at the elbow region (the site of Ϸ90°b ending) by 25-45 Å (5, 6). The anticodon stem loops of A-and P-site tRNA form Watson-Crick base pairs with adjacent mRNA codons on the 30S subunit (5, 7), whereas the 3Ј CCA terminal residues of A-and P-site tRNAs base-pair with conserved ribosomal RNA (rRNA) loops (8-10) within the 50S subunit peptidyltransferase center, the site of peptide bond formation (11). Additional interactions with rRNA and ribosomal proteins position tRNA molecules on the ribosome (5).The essential components and principal steps of translation have been delineated through genetic, biochemical, and kinetic methods (12). The process of polypeptide elongation has been most extensively characterized (13). Binding of aminoacyl-tRNA (aa-tRNA) to the A site occurs as a ''ternary complex'' with the GTPase elongation ...

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“…Formyl-methionyltRNA Met and peptidyl-tRNA's are known to react with puromycin only when bound at the P site of the ribosome [ 13]. Our results indicate that the formylated mischarged tRNAf Met species react with puromycin as well as formyl-methionyl-tRNAf Met does.…”

Section: Puromycin Reactionsmentioning

confidence: 62%

“…For the puromycin reaction 100 tzl of a solution of the antibiotic (puromycin dihydrochloride, 0.7 mg/ml; Tris-HC1 pH 7.5 50 mM; NH4C1, 80 mM; magnesium acetate, 5 mM;/3-mercaptoethanol, 7 mM) was added to each sample after 20 min-incubation in the same conditions as in the binding determination. Incubation was continued for 5 min at 37°C and then the sample treated as alr6ady published [ 13,14] and counted in the same scintillation counter. Results are expressed in picomoles of (formyl)-aminoacylMet tRNAf bound to ribosomes or in picomoles of (formyl)-aminoacyl-puromyein.…”

Section: Methodsmentioning

confidence: 99%