Ancient machinery embedded in the contemporary ribosome

2010 ◽  
Vol 38 (2) ◽  
pp. 422-427 ◽  
Author(s):  
Matthew J. Belousoff ◽  
Chen Davidovich ◽  
Ella Zimmerman ◽  
Yaron Caspi ◽  
Itai Wekselman ◽  
...  
Keyword(s):  
Gene Fusion ◽  
Bond Formation ◽  
Ribosomal Subunit ◽  
Peptide Bond ◽  
Peptide Bond Formation ◽  
Bacterial Organism ◽  
Or Gene ◽  
Definition Of ◽  
Folded Rna ◽  
Structural Definition

Structural analysis, supported by biochemical, mutagenesis and computational evidence, indicates that the peptidyltransferase centre of the contemporary ribosome is a universal symmetrical pocket composed solely of rRNA. This pocket seems to be a relic of the proto-ribosome, an ancient ribozyme, which was a dimeric RNA assembly formed from self-folded RNA chains of identical, similar or different sequences. This could have occurred spontaneously by gene duplication or gene fusion. This pocket-like entity was capable of autonomously catalysing various reactions, including peptide bond formation and non-coded or semi-coded amino acid polymerization. Efforts toward the structural definition of the early entity capable of genetic decoding involve the crystallization of the small ribosomal subunit of a bacterial organism harbouring a single functional rRNA operon.

scholarly journals Structural determinants for peptide-bond formation by asparaginyl ligases

2019 ◽  
pp. 201818568 ◽  
Author(s):  
Xinya Hemu ◽  
Abbas El Sahili ◽  
Side Hu ◽  
Kaho Wong ◽  
Yu Chen ◽  
...  
Keyword(s):  
Cyclic Peptides ◽  
Cyclic Peptide ◽  
Bond Formation ◽  
Peptide Bond ◽  
Cysteine Proteases ◽  
Peptide Bond Formation ◽  
Structural Determinants ◽  
Functional Studies ◽  
Definition Of ◽  
Neutral Conditions

Asparaginyl endopeptidases (AEPs) are cysteine proteases which break Asx (Asn/Asp)–Xaa bonds in acidic conditions. Despite sharing a conserved overall structure with AEPs, certain plant enzymes such as butelase 1 act as a peptide asparaginyl ligase (PAL) and catalyze Asx–Xaa bond formation in near-neutral conditions. PALs also serve as macrocyclases in the biosynthesis of cyclic peptides. Here, we address the question of how a PAL can function as a ligase rather than a protease. Based on sequence homology of butelase 1, we identified AEPs and PALs from the cyclic peptide-producing plants Viola yedoensis (Vy) and Viola canadensis (Vc) of the Violaceae family. Using a crystal structure of a PAL obtained at 2.4-Å resolution coupled to mutagenesis studies, we discovered ligase-activity determinants flanking the S1 site, namely LAD1 and LAD2 located around the S2 and S1′ sites, respectively, which modulate ligase activity by controlling the accessibility of water or amine nucleophile to the S-ester intermediate. Recombinantly expressed VyPAL1–3, predicted to be PALs, were confirmed to be ligases by functional studies. In addition, mutagenesis studies on VyPAL1–3, VyAEP1, and VcAEP supported our prediction that LAD1 and LAD2 are important for ligase activity. In particular, mutagenesis targeting LAD2 selectively enhanced the ligase activity of VyPAL3 and converted the protease VcAEP into a ligase. The definition of structural determinants required for ligation activity of the asparaginyl ligases presented here will facilitate genomic identification of PALs and engineering of AEPs into PALs.

scholarly journals Elongation factor-Tu can repetitively engage aminoacyl-tRNA within the ribosome during the proofreading stage of tRNA selection

2020 ◽  
Vol 117 (7) ◽  
pp. 3610-3620 ◽  
Author(s):  
Justin C. Morse ◽  
Dylan Girodat ◽  
Benjamin J. Burnett ◽  
Mikael Holm ◽  
Roger B. Altman ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Ternary Complex ◽  
Bond Formation ◽  
Critical Role ◽  
Ribosomal Subunit ◽  
Elongation Factor ◽  
Peptide Bond ◽  
Elongation Factor Tu ◽  
Peptide Bond Formation ◽  
Gtp Hydrolysis

The substrate for ribosomes actively engaged in protein synthesis is a ternary complex of elongation factor Tu (EF-Tu), aminoacyl-tRNA (aa-tRNA), and GTP. EF-Tu plays a critical role in mRNA decoding by increasing the rate and fidelity of aa-tRNA selection at each mRNA codon. Here, using three-color single-molecule fluorescence resonance energy transfer imaging and molecular dynamics simulations, we examine the timing and role of conformational events that mediate the release of aa-tRNA from EF-Tu and EF-Tu from the ribosome after GTP hydrolysis. Our investigations reveal that conformational changes in EF-Tu coordinate the rate-limiting passage of aa-tRNA through the accommodation corridor en route to the peptidyl transferase center of the large ribosomal subunit. Experiments using distinct inhibitors of the accommodation process further show that aa-tRNA must at least partially transit the accommodation corridor for EF-Tu⋅GDP to release. aa-tRNAs failing to undergo peptide bond formation at the end of accommodation corridor passage after EF-Tu release can be reengaged by EF-Tu⋅GTP from solution, coupled to GTP hydrolysis. These observations suggest that additional rounds of ternary complex formation can occur on the ribosome during proofreading, particularly when peptide bond formation is slow, which may serve to increase both the rate and fidelity of protein synthesis at the expense of GTP hydrolysis.

Antibiotic inhibition of the movement of tRNA substrates through a peptidyl transferase cavity

1995 ◽  
Vol 73 (11-12) ◽  
pp. 877-885 ◽  
Author(s):  
Bo T. Porse ◽  
Cristina Rodriguez-Fonseca ◽  
Ilia Leviev ◽  
Roger A. Garrett
Keyword(s):  
Bond Formation ◽  
Ribosomal Subunit ◽  
Peptide Bond ◽  
Inhibitory Mechanism ◽  
Large Ribosomal Subunit ◽  
Peptide Bond Formation ◽  
Peptidyl Transferase ◽  
Subunit Interactions ◽  
Antibiotic Inhibition ◽  
Peptidyl Transferase Centre

The present review attempts to deal with movement of tRNA substrates through the peptidyl transferase centre on the large ribosomal subunit and to explain how this movement is interrupted by antibiotics. It builds on the concept of hybrid tRNA states forming on ribosomes and on the observed movement of the 5′ end of P-site-bound tRNA relative to the ribosome that occurs on peptide bond formation. The 3′ ends of the tRNAs enter, and move through, a catalytic cavity where antibiotics are considered to act by at least three primary mechanisms: (i) they interfere with the entry of the aminoacyl moiety into the catalytic cavity before peptide bond formation; (ii) they inhibit movement of the nascent peptide along the peptide channel, a process that may generally involve destabilization of the peptidyl tRNA, and (iii) they prevent movement of the newly deacylated tRNA between the P/P and hybrid P/E sites on peptide bond formation.Key words: peptidyl transferase cavity, transient tRNA states, antibiotics, inhibitory mechanism, subunit–subunit interactions.

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