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

2015 ◽  
Vol 112 (19) ◽  
pp. 6038-6043 ◽  
Author(s):  
Michael T. Englander ◽  
Joshua L. Avins ◽  
Rachel C. Fleisher ◽  
Bo Liu ◽  
Philip R. Effraim ◽  
...  
Keyword(s):  
Amino Acids ◽  
Physiological Role ◽  
Peptidyl Transferase ◽  
Translational Machinery ◽  
Peptidyl Transferase Center ◽  
Peptidyl Transfer ◽  
Site Use ◽  
Dynamics Simulations ◽  
A Site ◽  
Trna Binding

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.

scholarly journals Induced-fit of the peptidyl-transferase center of the ribosome and conformational freedom of the esterified amino acids

2015 ◽  
Author(s):  
Jean Lehmann
Keyword(s):  
Amino Acids ◽  
Similar Rate ◽  
Catalytic Site ◽  
Side Chain ◽  
Peptidyl Transferase ◽  
Induced Fit ◽  
Independent Manner ◽  
Peptidyl Transferase Center ◽  
Conformational Freedom ◽  
A Site

The catalytic site of most enzymes can essentially deal with only one substrate. In contrast, the ribosome is capable of polymerizing at a similar rate at least 20 different kinds of amino acids from aminoacyl-tRNA carriers while using just one catalytic site, the peptidyl-transferase center (PTC). An induced-fit mechanism has been uncovered in the PTC, but a possible connection between this mechanism and the uniform handling of the substrates has not been investigated. We present an analysis of published ribosome structures supporting the hypothesis that the induced-fit eliminates unreactive rotamers predominantly populated for some A-site aminoacyl esters before induction. We show that this hypothesis is fully consistent with the wealth of kinetic data obtained with these substrates. Our analysis reveals that induction constrains the amino acids into a reactive conformation in a side-chain independent manner. It allows us to highlight the rationale of the PTC structural organization, which confers to the ribosome the very unusual ability to handle large as well as small substrates.

scholarly journals Mechanism of ribosome rescue by ArfA and RF2

2016 ◽  
Author(s):  
Gabriel Demo ◽  
Egor Svidritskiy ◽  
Rohini Madireddy ◽  
Ruben Diaz-Avalos ◽  
Timothy Grant ◽  
...  
Keyword(s):  
Structural Dynamics ◽  
Stop Codon ◽  
Release Factor ◽  
Peptidyl Transferase ◽  
Peptidyl Transferase Center ◽  
C Terminus ◽  
Peptide Release ◽  
N Terminus ◽  
70S Ribosome ◽  
A Site

AbstractArfA rescues ribosomes stalled on truncated mRNAs by recruiting the release factor RF2, which normally binds stop codons to catalyze peptide release. We report two 3.2-Å resolution cryo-EM structures – determined from a single sample – of the 70S ribosome with ArfA∙RF2 in the A site. In both states, the ArfA C-terminus occupies the mRNA tunnel downstream of the A site. One state contains a compact inactive RF2 conformation, hitherto unobserved in 70S termination complexes. Ordering of the ArfA N-terminus in the second state rearranges RF2 into an extended conformation that docks the catalytic GGQ motif into the peptidyl-transferase center. Our work thus reveals the structural dynamics of ribosome rescue. The structures demonstrate how ArfA “senses” the vacant mRNA tunnel and activates RF2 to mediate peptide release without a stop codon, allowing stalled ribosomes to be recycled.

scholarly journals Elongation factor 4 remodels the A-site tRNA on the ribosome

2016 ◽  
Vol 113 (18) ◽  
pp. 4994-4999 ◽  
Author(s):  
Matthieu G. Gagnon ◽  
Jinzhong Lin ◽  
Thomas A. Steitz
Keyword(s):  
Crystal Structure ◽  
Conformational Changes ◽  
Thermus Thermophilus ◽  
Elongation Factor ◽  
Peptidyl Transferase ◽  
Peptidyl Transferase Center ◽  
70S Ribosome ◽  
Regulate Protein ◽  
A Site ◽  
Hydrolysis Of

During translation, a plethora of protein factors bind to the ribosome and regulate protein synthesis. Many of those factors are guanosine triphosphatases (GTPases), proteins that catalyze the hydrolysis of guanosine 5′-triphosphate (GTP) to promote conformational changes. Despite numerous studies, the function of elongation factor 4 (EF-4/LepA), a highly conserved translational GTPase, has remained elusive. Here, we present the crystal structure at 2.6-Å resolution of the Thermus thermophilus 70S ribosome bound to EF-4 with a nonhydrolyzable GTP analog and A-, P-, and E-site tRNAs. The structure reveals the interactions of EF-4 with the A-site tRNA, including contacts between the C-terminal domain (CTD) of EF-4 and the acceptor helical stem of the tRNA. Remarkably, EF-4 induces a distortion of the A-site tRNA, allowing it to interact simultaneously with EF-4 and the decoding center of the ribosome. The structure provides insights into the tRNA-remodeling function of EF-4 on the ribosome and suggests that the displacement of the CCA-end of the A-site tRNA away from the peptidyl transferase center (PTC) is functionally significant.

scholarly journals Essential Mechanisms in the Catalysis of Peptide Bond Formation on the Ribosome

2005 ◽  
Vol 280 (43) ◽  
pp. 36065-36072 ◽  
Author(s):  
Malte Beringer ◽  
Christian Bruell ◽  
Liqun Xiong ◽  
Peter Pfister ◽  
Peter Bieling ◽  
...  
Keyword(s):  
Ph Dependence ◽  
Bond Formation ◽  
Peptide Bond ◽  
Activation Entropy ◽  
Peptide Bond Formation ◽  
Peptidyl Transferase ◽  
Peptidyl Transferase Center ◽  
Chemical Catalysis ◽  
Catalytic Function ◽  
Peptidyl Transfer

Peptide bond formation is the main catalytic function of the ribo-some. The mechanism of catalysis is presumed to be highly conserved in all organisms. We tested the conservation by comparing mechanistic features of the peptidyl transfer reaction on ribosomes from Escherichia coli and the Gram-positive bacterium Mycobacterium smegmatis. In both cases, the major contribution to catalysis was the lowering of the activation entropy. The rate of peptide bond formation was pH independent with the natural substrate, amino-acyl-tRNA, but was slowed down 200-fold with decreasing pH when puromycin was used as a substrate analog. Mutation of the conserved base A2451 of 23 S rRNA to U did not abolish the pH dependence of the reaction with puromycin in M. smegmatis, suggesting that A2451 did not confer the pH dependence. However, the A2451U mutation alters the structure of the peptidyl transferase center and changes the pattern of pH-dependent rearrangements, as probed by chemical modification of 23 S rRNA. A2451 seems to function as a pivot point in ordering the structure of the peptidyl transferase center rather than taking part in chemical catalysis.

scholarly journals Peptidyl transferase center decompaction and structural constraints during early protein elongation on the ribosome

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Bin Jia ◽  
Tianlong Wang ◽  
Jean Lehmann
Keyword(s):  
Peptide Bond ◽  
Nucleophilic Attack ◽  
Structural Constraints ◽  
Peptide Bond Formation ◽  
Peptidyl Transferase ◽  
Peptidyl Transferase Center ◽  
Early Protein ◽  
Physico Chemical ◽  
Exit Tunnel ◽  
A Site

AbstractPeptide bond formation on the ribosome requires that aminoacyl-tRNAs and peptidyl-tRNAs are properly positioned on the A site and the P site of the peptidyl transferase center (PTC) so that nucleophilic attack can occur. Here we analyse some constraints associated with the induced-fit mechanism of the PTC, that promotes this positioning through a compaction around the aminoacyl ester orchestrated by U2506. The physical basis of PTC decompaction, that allows the elongated peptidyl-tRNA to free itself from that state and move to the P site of the PTC, is still unclear. From thermodynamics considerations and an analysis of published ribosome structures, the present work highlights the rational of this mechanism, in which the free-energy released by the new peptide bond is used to kick U2506 away from the reaction center. Furthermore, we show the evidence that decompaction is impaired when the nascent peptide is not yet anchored inside the exit tunnel, which may contribute to explain why the first rounds of elongation are inefficient, an issue that has attracted much interest for about two decades. Results in this field are examined in the light of the present analysis and a physico-chemical correlation in the genetic code, which suggest that elementary constraints associated with the size of the side-chain of the amino acids penalize early elongation events.

scholarly journals Nascent Peptide in the Ribosome Exit Tunnel Affects Functional Properties of the A-Site of the Peptidyl Transferase Center

2011 ◽  
Vol 41 (3) ◽  
pp. 321-330 ◽  
Author(s):  
Haripriya Ramu ◽  
Nora Vázquez-Laslop ◽  
Dorota Klepacki ◽  
Qing Dai ◽  
Joseph Piccirilli ◽  
...  
Keyword(s):  
Functional Properties ◽  
Peptidyl Transferase ◽  
Peptidyl Transferase Center ◽  
Nascent Peptide ◽  
Exit Tunnel ◽  
Ribosome Exit Tunnel ◽  
A Site

scholarly journals NAC covers ribosome-associated nascent chains thereby forming a protective environment for regions of nascent chains just emerging from the peptidyl transferase center.

1995 ◽  
Vol 130 (3) ◽  
pp. 519-528 ◽  
Author(s):  
S Wang ◽  
H Sakai ◽  
M Wiedmann
Keyword(s):  
Amino Acids ◽  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Peptidyl Transferase ◽  
Nascent Polypeptide ◽  
Peptidyl Transferase Center ◽  
Protease Resistance ◽  
Protective Environment ◽  
Cytosolic Factors

We demonstrate that nascent polypeptide-associated complex (NAC) is one of the first cytosolic factors that newly synthesized nascent chains encounter. When NAC is present, nascent chains are segregated from the cytosol until approximately 30 amino acids in length, a finding consistent with the well-documented protease resistance of short ribosome-associated nascent chains. When NAC is removed, the normally protected nascent chains are susceptible to proteolysis. Therefore NAC, by covering COOH-terminal segments of nascent chains on the ribosome, perhaps together with ribosomal proteins, forms a protective environment for regions of nascent chains just emerging from the peptidyl transferase center. Since NAC is not a core ribosomal protein, the emergence of nascent chains from the ribosome may be more dynamic than previously thought.

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