An intact ribose moiety at A2602 of 23S rRNA is key to trigger peptidyl-tRNA hydrolysis during translation termination

Peptide bond formation and peptidyl-tRNA hydrolysis are the two elementary chemical reactions of protein synthesis catalyzed by the ribosomal peptidyl transferase ribozyme. Due to the combined effort of structural and biochemical studies, details of the peptidyl transfer reaction have become increasingly clearer. However, significantly less is known about the molecular events that lead to peptidyl-tRNA hydrolysis at the termination phase of translation. Here we have applied a recently introduced experimental system, which allows the ribosomal peptidyl transferase center (PTC) to be chemically engineered by the introduction of non-natural nucleoside analogs. By this approach single functional group modifications are incorporated, thus allowing their functional contributions in the PTC to be unravelled with improved precision. We show that an intact ribose sugar at the 23S rRNA residue A2602 is crucial for efficient peptidyl-tRNA hydrolysis, while having no apparent functional relevance for transpeptidation. Despite the fact that all investigated active site residues are universally conserved, the removal of the complete nucleobase or the ribose 2′-hydroxyl at A2602, U2585, U2506, A2451 or C2063 has no or only marginal inhibitory effects on the overall rate of peptidyl-tRNA hydrolysis. These findings underscore the exceptional functional importance of the ribose moiety at A2602 for triggering peptide release

Chemical engineering of the peptidyl transferase center reveals an important role of the 2′-hydroxyl group of A2451

The main enzymatic reaction of the large ribosomal subunit is peptide bond formation. Ribosome crystallography showed that A2451 of 23S rRNA makes the closest approach to the attacking amino group of aminoacyl-tRNA. Mutations of A2451 had relatively small effects on transpeptidation and failed to unequivocally identify the crucial functional group(s). Here, we employed an in vitro reconstitution system for chemical engineering the peptidyl transferase center by introducing non-natural nucleosides at position A2451. This allowed us to investigate the peptidyl transfer reaction performed by a ribosome that contained a modified nucleoside at the active site. The main finding is that ribosomes carrying a 2′-deoxyribose at A2451 showed a compromised peptidyl transferase activity. In variance, adenine base modifications and even the removal of the entire nucleobase at A2451 had only little impact on peptide bond formation, as long as the 2′-hydroxyl was present. This implicates a functional or structural role of the 2′-hydroxyl group at A2451 for transpeptidation

The role of the universally conserved A2450–C2063 base pair in the ribosomal peptidyl transferase center

Despite the fact that all 23S rRNA nucleotides that build the ribosomal peptidyl transferase ribozyme are universally conserved, standard and atomic mutagenesis studies revealed the nucleobase identities being non-critical for catalysis. This indicates that these active site residues are highly conserved for functions distinct from catalysis. To gain insight into potential contributions, we have manipulated the nucleobases via an atomic mutagenesis approach and have utilized these chemically engineered ribosomes for in vitro translation reactions. We show that most of the active site nucleobases could be removed without significant effects on polypeptide production. Our data however highlight the functional importance of the universally conserved non-Watson-Crick base pair at position A2450–C2063. Modifications that disrupt this base pair markedly impair translation activities, while having little effects on peptide bond formation, tRNA drop-off and ribosome-dependent EF-G GTPase activity. Thus it seems that disruption of the A2450–C2063 pair inhibits a reaction following transpeptidation and EF-G action during the elongation cycle. Cumulatively our data are compatible with the hypothesis that the integrity of this A-C wobble base pair is essential for effective tRNA translocation through the peptidyl transferase center during protein synthesis

Aptazyme-Mediated Regulation of 16S Ribosomal RNA

Developing artificial genetic switches in order to control gene expression via an external stimulus is an important aim in chemical and synthetic biology. Here, we expand the application range of RNA switches to the regulation of 16S rRNA function in Escherichia coli. For this purpose, we incorporated hammerhead ribozymes at several positions into orthogonalized 16S rRNA. We observed that ribosomal function is remarkably tolerant toward the incorporation of large additional RNA fragments at certain sites of the 16S rRNA. However, ribozyme-mediated cleavage results in severe reduction of 16S rRNA stability. We carried out an in vivo screen for the identification of sequences acting as ligand-responsive RNA switches, enabling thiamine-dependent switching of 16S rRNA function. In addition to expanding the regulatory toolbox, the presented artificial riboswitches should prove valuable to study aspects of rRNA folding and stability in bacteria

An intact ribose moiety at A2602 of 23S rRNA is

key to trigger peptidyl-tRNA hydrolysis during translation terminatio

Peptidyl transferase activities of gapped-cp-reconstituted subunits containing 2′-deoxyribose modifications at A2451 using CC-puromycin as acceptor substrate

<p><b>Copyright information:</b></p><p>Taken from "Chemical engineering of the peptidyl transferase center reveals an important role of the 2′-hydroxyl group of A2451"</p><p>Nucleic Acids Research 2005;33(5):1618-1627.</p><p>Published online 14 Mar 2005</p><p>PMCID:PMC1065261.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> () The reaction between -acetyl-Phe-tRNA and [P]CC-puromycin was carried out for 120 min, a time point that corresponded to the endpoint of the reaction catalyzed by reconstituted wild-type large subunits. The product -acetyl-Phe-CC-puromycin (CC-Pmn-AcPhe) was resolved from CC-puromycin (CC-Pmn) by gel electrophoresis (). The control reaction (ctrl) contained the whole reaction mixture except ribosomal particles. The relative yields of product formation by gapped-cp-reconstituted subunits containing adenosine (wt), 2′-deoxyadenosine (dA), or the deoxy-abasic analog (d-aba) at 2451 are shown below the gel. () The initial rates of peptide bond formation catalyzed by the wt or the deoxy-A2451-modified large ribosomal subunit were determined from experimental points within the first 45 min of incubation. During this incubation time, no product formation with ribosomal particles carrying the deoxy-abasic site analog at position 2451 could be measured (n.d.). The rates were normalized to the rate of reconstituted subunits containing the synthetic wild-type RNA fragment (wt)