[Show abstract][Hide abstract] ABSTRACT: During the elongation cycle of protein biosynthesis, tRNAs traverse through the ribosome by consecutive binding to the 3 ribosomal binding sites (A-, P-, and E- sites). While the ribosomal A- and P-sites have been functionally well characterized in the past, the contribution of the E-site to protein biosynthesis is still poorly understood in molecular terms. Previous studies suggested an important functional interaction of the terminal residue A76 of E-tRNA with the nucleobase of the universally conserved 23S rRNA residue C2394. Using an atomic mutagenesis approach to introduce non-natural nucleoside analogs into the 23S rRNA, we could show that removal of the nucleobase or the ribose 2'-OH at C2394 had no effect on protein synthesis. On the other hand, our data disclose the importance of the highly conserved E-site base pair G2421-C2395 for effective translation. Ribosomes with a disrupted G2421-C2395 base pair are defective in tRNA binding to the E-site. This results in an impaired translation of genuine mRNAs, while homo-polymeric templates are not affected. Cumulatively our data emphasize the importance of E-site tRNA occupancy and in particular the intactness of the 23S rRNA base pair G2421-C2395 for productive protein biosynthesis.
[Show abstract][Hide abstract] ABSTRACT: The ribosome is a huge ribonucleoprotein complex in charge of protein synthesis in every living cell. The catalytic center of this dynamic molecular machine is entirely built up of 23S ribosomal RNA and therefore the ribosome can be referred to as the largest natural ribozyme known so far. The in vitro reconstitution approach of large ribosomal subunits described herein allows nucleotide analog interference studies to be performed. The approach is based on the site-specific introduction of nonnatural nucleotide analogs into the peptidyl transferase center, the active site located on the interface side of the large ribosomal subunit. This method combined with standard tests of ribosomal functions broadens the biochemical repertoire to investigate the mechanism of diverse aspects of translation considerably and adds another layer of molecular information on top of structural and mutational studies of the ribosome.
[Show abstract][Hide abstract] ABSTRACT: The protocol describes the site-specific chemical modification of 23S rRNA of Thermus aquaticus ribosomes. The centerpiece of this 'atomic mutagenesis' approach is the site-specific incorporation of non-natural nucleoside analogs into 23S rRNA in the context of the entire 70S ribosome. This technique exhaustively makes use of the available crystallographic structures of the ribosome for designing detailed biochemical experiments aiming at unraveling molecular insights of ribosomal functions. The generation of chemically engineered ribosomes carrying a particular non-natural 23S rRNA residue at the site of interest, a procedure that typically takes less than 2 d, allows the study of translation at the molecular level and goes far beyond the limits of standard mutagenesis approaches. This methodology, in combination with the presented tests for ribosomal functions adapted to chemically engineered ribosomes, allows unprecedented molecular insight into the mechanisms of protein biosynthesis.
[Show abstract][Hide abstract] ABSTRACT: 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.
Nucleic Acids Research 04/2010; 38(14):4844-55. DOI:10.1093/nar/gkq213 · 9.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Chemically modified RNA nucleotides have been introduced in the past into various ribozymes in order to understand RNA folding and the mechanism of RNA catalysis. Recently the ribosome, the largest natural ribozyme known to date, has been added to the list of enzymes amenable to synthetic biology. The chemically engineered ribosomes were active in various functional assays including single-turnover peptidyl transfer reaction as well as in vitro translation assays. Solid-phase synthesis of several non-natural nucleotide analogs and their subsequent introduction into the catalytic center of the ribosome, revealed the ribose 2'-OH at position A2451 of 23S ribosomal RNA as key functional group for amide bond synthesis. By altering the chemical characteristics of the ribose at A2451 by replacing its 2'-OH with selected functional groups demonstrated that hydrogen donor capability is essential for efficient transpeptidation. These findings in combination with data that accumulated over the past years allowed to propose a comprehensive model for peptide bond synthesis in which the A2451 2'-OH directly assists in positioning one of the tRNA substrates via hydrogen-bond formation and thus supports amide bond synthesis via a proton shuttle mechanism. It is conceivable that cell-free translation systems employing rationally designed chemically engineered ribosomes can be established in the near future to produce peptides and proteins harboring unnatural amino acids.
Current Organic Chemistry 12/2009; 14(2):148-161. DOI:10.2174/138527210790069848 · 2.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Role of RNA backbone groups for ribosomal catalysis S u m m a r y The ribosomal peptidyl transferase ribozyme resides in the large ribosomal subunit and catalyzes the two principal chemical reactions of protein synthesis, peptide bond formation and peptidyl-tRNA hydrolysis. With the presentations of atomic structures of the large ribosomal subunit, the questions how an RNA active site can catalyze these chemical reactions gained a new level of molecular significance. The peptidyl transferase center represents the most intense accu-mulation of universally conserved ribosomal RNA nucleotides in the entire ribo-some. Thus, it came as a surprise that recent findings revealed that the nucleobase identities of active site residues are actually not critical for catalysis. Instead RNA backbone groups have been identified as key players in transpeptidation and peptide release. While the ribose 2'-OH of the 23S rRNA residue A2451 plays an important role in peptidyl transfer, its contribution to peptidyl-tRNA hydrolysis is only minor. On the other hand, the ribose 2'-OH of the terminal adenosine of P-site bound tRNA seems to play equally crucial roles in peptide bond formation and tRNA hydrolysis. While it seems that details of ri-bosome-catalyzed peptidyl-tRNA hydrolysis are just emerging, our molecular in-sights into transpeptidation are already very advanced. It has been realized that an intricate interaction between the ribose 2'-OH groups of 23S rRNA residue A2451 and tRNA nucleotide A76 is crucial for proton shuttling that is required for efficient amide bond synthesis.
[Show abstract][Hide abstract] ABSTRACT: Peptide bond formation is a fundamental reaction in biology, catalyzed by the ribosomal peptidyl-transferase ribozyme. Although all active-site 23S ribosomal RNA nucleotides are universally conserved, atomic mutagenesis suggests that these nucleobases do not carry functional groups directly involved in peptide bond formation. Instead, a single ribose 2'-hydroxyl group at A2451 was identified to be of pivotal importance. Here, we altered the chemical characteristics by replacing its 2'-hydroxyl with selected functional groups and demonstrate that hydrogen donor capability is essential for transpeptidation. We propose that the A2451-2'-hydroxyl directly hydrogen bonds to the P-site tRNA-A76 ribose. This promotes an effective A76 ribose C2'-endo conformation to support amide synthesis via a proton shuttle mechanism. Simultaneously, the direct interaction of A2451 with A76 renders the intramolecular transesterification of the peptide from the 3'- to 2'-oxygen unfeasible, thus promoting effective peptide bond synthesis.
[Show abstract][Hide abstract] ABSTRACT: Over time the mechanistic concepts to describe the two principal chemical reactions that are catalyzed by the ribosome, peptide bond formation and peptidyl-tRNA hydrolysis, have undergone dramatic changes. While the initial models were based on a ribosomal protein-based mechanism, evidence for a direct functional contribution of ribosomal RNA for catalysis has accumulated over the past years. The presentation of high resolution crystallographic structures of the large ribosomal subunit at the beginning of the new millennium dramatically increased our molecular insight into the organization of the active center and finally placed the ribosome amongst the list of RNA enzymes. Combined with elaborate biochemical and biophysical approaches the translation field has made significant progress in understanding mechanistic details of ribosomal catalysis. While it seems that the mechanism of ribosome-catalyzed peptidyl-tRNA hydrolysis is just emerging, the knowledge on transpeptidation is already very advanced. It has been realized that intricate interactions between ribosomal RNA and the transfer RNA substrate are crucial for proton shuttling that is required for efficient amide bond formation.
[Show abstract][Hide abstract] ABSTRACT: 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.
Nucleic Acids Research 02/2007; 35(15):5130-40. DOI:10.1093/nar/gkm539 · 9.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The ribosomal peptidyl transferase center is a ribozyme catalyzing peptide bond synthesis in all organisms. We applied a novel modified nucleoside interference approach to identify functional groups at 9 universally conserved active site residues. Owing to their immediate proximity to the chemical center, the 23S rRNA nucleosides A2451, U2506 and U2585 were of particular interest. Our study ruled out U2506 and U2585 as contributors of vital chemical groups for transpeptidation. In contrast the ribose 2'-OH of A2451 was identified as the prime ribosomal group with potential functional importance. This 2'-OH renders almost full catalytic power to the ribosome even when embedded into an active site of six neighboring 2'-deoxyribose nucleosides. These data highlight the unique functional role of the A2451 2'-OH for peptide bond synthesis among all other functional groups at the ribosomal peptidyl transferase active site.
Journal of the American Chemical Society 05/2006; 128(13):4453-9. DOI:10.1021/ja0588454 · 12.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Nucleic Acids Research 02/2005; 33(5):1618-27. DOI:10.1093/nar/gki308 · 9.11 Impact Factor