Efficient 50S ribosome-catalyzed peptide bond synthesis with an aminoacyl minihelix.

RNA minihelices that recreate the amino acid acceptor domain of the two-domain L-shaped tRNA molecule are substrates for specific aminoacylation by tRNA synthetases. Some lines of evidence suggest that this domain arose independently of and predated the second, anticodon-containing domain. With puromycin and a minihelix charged with alanine, we show here efficient 50S ribosome catalyzed peptide synthesis. The aminoacyl minihelix is as active as aminoacyl tRNA in the synthetic reaction. The high efficiency of the charged minihelix is due to a relatively strong interaction with the 50S particle. In contrast, an aminoacyl RNA fragment that recreates the 3'-side of the tRNA acceptor stem has a much weaker interaction with the 50S particle. These results are consistent with the minihelix domain being the major loci for tRNA interactions with the 50S ribosome. They may also have implications for the historical development of RNA-based systems of peptide synthesis.     

Oxazolidinones mechanism of action: inhibition of the first peptide bond formation

Oxazolidinones are potent inhibitors of bacterial protein biosynthesis. Previous studies have demonstrated that this new class of antimicrobial agent blocks translation by inhibiting initiation complex formation, while post-initiation translation by polysomes and poly(U)-dependent translation is not a target for these compounds. We found that oxazolidinones inhibit translation of natural mRNA templates but have no significant effect on poly(A)-dependent translation. Here we show that various oxazolidinones inhibit ribosomal peptidyltransferase activity in the simple reaction of 70 S ribosomes using initiator-tRNA or N-protected CCA-Phe as a P-site substrate and puromycin as an A-site substrate. Steady-state kinetic analysis shows that oxazolidinones display a competitive inhibition pattern with respect to both the P-site and A-site substrates. This is consistent with a rapid equilibrium, ordered mechanism of the peptidyltransferase reaction, wherein binding of the A-site substrate can occur only after complex formation between peptidyltransferase and the P-site substrate. We propose that oxazolidinones inhibit bacterial protein biosynthesis by interfering with the binding of initiator fMet-tRNA(i)(Met) to the ribosomal peptidyltransferase P-site, which is vacant only prior to the formation of the first peptide bond.     

Mechanism of peptide bond formation on the ribosome--controversions

During past five years there have been published many experimental data concerning structure and function of the ribosome. With the presentation of atomic structures we obtained a new data about composition of peptidyl transferase center. It is now obvious that PTC is composed entirely of rRNA. It is also known that the proper substrate alignment is the major factor for ribosome's catalytic activity. However, more detailed mechanism of peptide bond formation on the ribosome still remains unclear. Several issues remain unsolved. For example, are there any chemical components coming from ribosome itself, that enhance the rate of the reaction? Do intact ribosomes perform peptidyltransfer in the same way as the isolated ribosomal subunits that have been the source of most of the data? In this article we present different opinions and controversions around peptide bond formation on the ribosome.     

Mechanism of peptide bond formation on the ribosome

Peptide bond formation is the fundamental reaction of ribosomal protein synthesis. The ribosome's active site--the peptidyl transferase center--is composed of rRNA, and thus the ribosome is the largest known RNA catalyst. The ribosome accelerates peptide bond formation by 10(7)-fold relative to the uncatalyzed reaction. Recent progress of structural, biochemical and computational approaches has provided a fairly detailed picture of the catalytic mechanisms employed by the ribosome. Energetically, catalysis is entirely entropic, indicating an important role of solvent reorganization, substrate positioning, and/or orientation of the reacting groups within the active site. The ribosome provides a pre-organized network of electrostatic interactions that stabilize the transition state and facilitate proton shuttling involving ribose hydroxyl groups of tRNA. The catalytic mechanism employed by the ribosome suggests how ancient RNA-world enzymes may have functioned.     

Optimization of enzyme-mediated peptide bond formation

Enzyme-catalyzed peptide bond formation requires thorough examination and optimization of each coupling step. In order to identify factors influencing the selectivity between aminolysis and hydrolysis, a systematic study was carried out for the kinetically controlled peptide synthesis. The reaction temperature, the type of C-terminal protecting group, and different organic cosolvents showed little influence on the selectivity. The enzyme, excess nucleophile, pH, N-terminal protecting group, and ionic strength of the solution were identified as major factors controlling the selectivity and, therefore, the yield of the dipeptide synthesis. Under optimized conditions, the selectivity of the chymotrypsin-catalyzed synthesis of PheSer could be increased from 35 to 100%.     

Ribosomal tolerance and peptide bond formation

In the ribosome, the decoding and peptide bond formation sites are composed entirely of ribosomal RNA, thus confirming that the ribosome is a ribozyme. Precise alignment of the aminoacylated and peptidyl tRNA 3'-ends, which is the major enzymatic contribution of the ribosome, is dominated by remote interactions of the tRNA double helical acceptor stem with the distant rims of the peptidyl transferase center. An elaborate architecture and a sizable symmetry-related region within the otherwise asymmetric ribosome guide the A --> P passage of the tRNA 3'-end by a spiral rotatory motion, and ensures its outcome: stereochemistry suitable for peptide bond formation and geometry facilitating the entrance of newly formed proteins into their exit tunnel.     

Substrate-assisted catalysis of peptide bond formation by the ribosome

The ribosome accelerates the rate of peptide bond formation by at least 10(7)-fold, but the catalytic mechanism remains controversial. Here we report evidence that a functional group on one of the tRNA substrates plays an essential catalytic role in the reaction. Substitution of the P-site tRNA A76 2' OH with 2' H or 2' F results in at least a 10(6)-fold reduction in the rate of peptide bond formation, but does not affect binding of the modified substrates. Such substrate-assisted catalysis is relatively uncommon among modern protein enzymes, but it is a property predicted to be essential for the evolution of enzymatic function. These results suggest that substrate assistance has been retained as a catalytic strategy during the evolution of the prebiotic peptidyl transferase center into the modern ribosome.     

Investigations on the mechanism of the salt-induced peptide formation

The applicability of the salt-induced peptide formation in aqueous solution — the simplest model so far for peptide synthesis under primitive earth conditions — is demonstrated for valine as another amino acid, and the formation of mixed peptides in systems containing glycine, alanine and valine is investigated. The dominant dipeptides formed are Gly-Gly, Gly-Ala and Gly-Val, at longer reaction times sequence inversion produces Ala-Gly and, considerably slower, Val-Gly. Ala-Ala is also produced and the relative amounts of the diastereomers prove the high conservation of optical purity of the original amino acids over a considerable time. The results lead to some further conclusions about the reaction mechanism and the possible dominance of peptide sequences in primordial dipeptides.     

Essential mechanisms in the catalysis of peptide bond formation on the ribosome

Peptide bond formation is the main catalytic function of the ribosome. 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.     

Movement of tRNA but not the nascent peptide during peptide bond formation on ribosomes.

The results from experiments involving nonradiative energy transfer indicate that a fluorescent probe on the 5'-end of tRNA(Phe) moves more than 20 A towards probes on ribosomal protein L1 as a peptide bond is formed during the peptidyl transferase reaction on Escherichia coli ribosomes. The peptide itself moves no more than a few angstroms during peptide bond formation, as judged by the movement of fluorescent probes attached to the phenylalanine amino group of phenylalanyl-tRNA. Other results demonstrate that an analogue of peptidyl-tRNA, deacylated tRNA, and puromycin can be bound simultaneously to the same ribosome, indicating that there are three physically distinct sites to which tRNA is bound during the reaction steps by which peptides are elongated. The results appear to be consistent with the displacement model of peptide elongation.     

The effect of smectite composition on the catalysis of peptide bond formation

Clay-catalyzed glycine and diglycine oligomerizations were performed as drying/wetting cycles at 80°C. Two trioctahedral smectites (hectorite and saponite), three pure montmorillonites, a ferruginous smectite, an Fe(II)-rich smectite, and three smectites containing goethite admixture were used as catalysts. Highest peptide bond formation was found with trioctahedral smectites. About 7% of glycine was converted to diglycine and diketopiperazine on hectorite after 7 days. In the case of dioctahedral smectites, highest yields were achieved using clays with a negative-layer charge localized in the octahedral sheets (up to 2% of converted glycine after 7 days). The presence of Fe(II) in clay is reflected in a higher efficiency in catalyzing amino acid dimerization (about 3.5% of converted glycine after 7 days). The possible significance of the results for prebiotic chemistry is discussed. Received: 20 February 1996 / Accepted: 26 April 1996     

Energetics of peptide bond formation at elevated temperatures

Summary The free energies of formation of the peptide bond between carbobenzoxy-glycine and L-phenylalanine amide in aqueous solution at temperatures up to 60°C were calculated from experimentally determined equilibrium constants. The reaction was catalyzed by a thermophylic enzyme. The thermodynamic energy barrier to peptide bond formation was found to decrease with increasing temperature: the standard free energy of peptide bond formation did appear to become negative in the region of 60°C. The possible significance of these results for peptide bond formation under prebiotic conditions is discussed.     

Peptide bond formation on the ribosome: structure and mechanism

The peptidyl transferase reaction on the ribosome is catalyzed by RNA. Pre-steady-state kinetic studies using Escherichia coli ribosomes suggest that catalysis (>10(5)-fold overall acceleration) is, to a large part, a result of substrate positioning, in agreement with crystal structures of large ribosomal subunits with bound substrate or product analogs. The rate of peptide bond formation is inhibited approximately 100-fold by protonation of a single ribosomal group with a pK(a) of 7.5, indicating general acid-base catalysis and/or a pH-dependent conformational change within the active site. According to the kinetics of mutant ribosomes, these effects may be attributed to a candidate catalytic base (A2451) suggested by the crystal structure.     

Rapid peptide bond formation on isolated 50S ribosomal subunits

The catalytic site of the ribosome, the peptidyl transferase centre, is located on the large (50S in bacteria) ribosomal subunit. On the basis of results obtained with small substrate analogues, isolated 50S subunits seem to be less active in peptide bond formation than 70S ribosomes by several orders of magnitude, suggesting that the reaction mechanisms on 50S subunits and 70S ribosomes may be different. Here we show that with full-size fMet-tRNA(fMet) and puromycin or C-puromycin as peptide donor and acceptor substrates, respectively, the reaction proceeds as rapidly on 50S subunits as on 70S ribosomes, indicating that the intrinsic activity of 50S subunits is not different from that of 70S ribosomes. The faster reaction on 50S subunits with fMet-tRNA(fMet), compared with oligonucleotide substrate analogues, suggests that full-size transfer RNA in the P site is important for maintaining the active conformation of the peptidyl transferase centre.     

Mutation affecting peptide bond formation in nikkomycin biosynthesis

Nikkomycin, a nucleoside-peptide analog of UDP-N-acetylglucosamine, is a potent chitin synthase inhibitor produced by the bacterium Streptomyces tendae. The HPLC profile of fermentation products in culture broths of a non-producing mutant, Nik 15, was compared with nikkomycin standards. Nikkomycin C and D, the glycone and aglycone moieties, respectively, of nikkomycin Z accumulated. This indicates the mutation affects the capacity to form a peptide bond between nikkomycin C and D, which is here proposed to be the terminal step in the synthesis of the biologically active nikkomycin Z. This is also the first documented case of a mutation affecting a specific step in nikkomycin biosynthesis.     

Kinetics of inhibition of peptide bond formation on bacterial ribosomes.

A cell-free system derived from Escherichia coli has been used in order to study the kinetics of inhibition of peptide bond formation with the aid of the puromycin reaction in solution. A similar study has been carried out earlier on a solid support matrix with the same inhibitors. We find that the overall pattern of the kinetics of inhibition is the same in the two systems. At low concentrations of inhibitor there is a competitive phase of inhibition, whereas at higher concentrations of inhibitor the type of inhibition becomes mixed noncompetitive. The values of Ki of the competitive phase in the system in solution are: 5.8 microM (amicetin), 0.2 microM (blasticidin S), 0.5 microM (chloramphenicol), and 0.5 microM (tevenel). The inhibitors amicetin, blasticidin S, and tevenel interact with the ribosome in a reaction which is slower than that of the substrate puromycin, showing clear-cut characteristics of slow-onset inhibition in both systems. Chloramphenicol, on the other hand does not easily show such a delay in solution. It interacts with the ribosome relatively faster than the other three antibiotics. Despite this, chloramphenicol too shows characteristics of time-dependent inhibition.