Structural insights into the roles of water and the 2' hydroxyl of the P site tRNA in the peptidyl transferase reaction

Peptide bond formation is catalyzed at the peptidyl transferase center (PTC) of the large ribosomal subunit. Crystal structures of the large ribosomal subunit of Haloarcula marismortui (Hma) complexed with several analogs that represent either the substrates or the transition state intermediate of the peptidyl transferase reaction show that this reaction proceeds through a tetrahedral intermediate with S chirality. The oxyanion of the tetrahedral intermediate interacts with a water molecule that is positioned by nucleotides A2637 (E. coli numbering, 2602) and (methyl)U2619(2584). There are no Mg2+ ions or monovalent metal ions observed in the PTC that could directly promote catalysis. The A76 2' hydroxyl of the peptidyl-tRNA is hydrogen bonded to the alpha-amino group and could facilitate peptide bond formation by substrate positioning and by acting as a proton shuttle between the alpha-amino group and the A76 3' hydroxyl of the peptidyl-tRNA.     

Structure and functions of 5S rRNA.

The ribosome is a macromolecular assembly that is responsible for protein biosynthesis in all organisms. It is composed of two-subunit, ribonucleoprotein particles that translate the genetic material into an encoded polypeptides. The small subunit is the site of codon-anticodon interaction between the messenger RNA (mRNA) and transfer RNA (tRNA) substrates, and the large subunit catalyses peptide bond formation. The peptidyltransferase activity is fulfilled by 23S rRNA, which means that ribosome is a ribozyme. 5S rRNA is a conserved component of the large ribosomal subunit that is thought to enhance protein synthesis by stabilizing ribosome structure. This paper shortly summarises new results obtained on the structure and function of 5S rRNA.     

The chemistry of protein synthesis and voyage through the ribosomal tunnel

High-resolution structures of ribosomal subunits and their complexes with substrates and antibiotics have revealed fundamental principles of template-directed protein synthesis. Mechanistic questions regarding ribosome function and catalysis can now be addressed with structure-based experiments. Recent studies have investigated the mechanism of peptide bond formation catalyzed by the large ribosomal subunit, the mode of protein synthesis inhibition by macrolide antibiotics, the interaction of nascent polypeptides with the ribosomal exit tunnel, and the role of ribosomal proteins in the recruitment of accessory factors that assist protein folding and targeting.     

Ribosomal crystallography: peptide bond formation and its inhibition

Ribosomes, the universal cellular organelles catalyzing the translation of genetic code into proteins, are protein/RNA assemblies, of a molecular weight 2.5 mega Daltons or higher. They are built of two subunits that associate for performing protein biosynthesis. The large subunit creates the peptide bond and provides the path for emerging proteins. The small has key roles in initiating the process and controlling its fidelity. Crystallographic studies on complexes of the small and the large eubacterial ribosomal subunits with substrate analogs, antibiotics, and inhibitors confirmed that the ribosomal RNA governs most of its activities, and indicated that the main catalytic contribution of the ribosome is the precise positioning and alignment of its substrates, the tRNA molecules. A symmetry-related region of a significant size, containing about two hundred nucleotides, was revealed in all known structures of the large ribosomal subunit, despite the asymmetric nature of the ribosome. The symmetry rotation axis, identified in the middle of the peptide-bond formation site, coincides with the bond connecting the tRNA double-helical features with its single-stranded 3' end, which is the moiety carrying the amino acids. This thus implies sovereign movements of tRNA features and suggests that tRNA translocation involves a rotatory motion within the ribosomal active site. This motion is guided and anchored by ribosomal nucleotides belonging to the active site walls, and results in geometry suitable for peptide-bond formation with no significant rearrangements. The sole geometrical requirement for this proposed mechanism is that the initial P-site tRNA adopts the flipped orientation. The rotatory motion is the major component of unified machinery for peptide-bond formation, translocation, and nascent protein progression, since its spiral nature ensures the entrance of the nascent peptide into the ribosomal exit tunnel. This tunnel, assumed to be a passive path for the growing chains, was found to be involved dynamically in gating and discrimination.     

A conserved base-pair between tRNA and 23 S rRNA in the peptidyl transferase center is important for peptide release

The 3' terminus of tRNAs has the universally conserved bases C74C75A76 that interact with the ribosomal large subunit. In the ribosomal P site, bases C74 and C75 of tRNA, form Watson-Crick base-pairs with G2252 and G2251, respectively, present in the conserved P-loop of 23 S rRNA. Previous studies have suggested that the G2252-C74 base-pair is important for peptide bond formation. Using a pure population of mutant ribosomes, we analyzed the precise role of this base-pair in peptide bond formation, elongation factor G-dependent translocation, and peptide release by release factor 1. Surprisingly, our results show that the G2252-C74 base-pair is not essential for peptide bond formation with intact aminoacyl tRNAs as substrates and for EF-G catalyzed translocation. Interestingly, however, peptide release was reduced substantially when base-pair formation between G2252 and C74 of P site tRNA was disrupted, indicating that this conserved base-pair plays an important role in ester bond hydrolysis during translation termination.     

A structural view on the mechanism of the ribosome-catalyzed peptide bond formation

The ribosome is a large ribonucleoprotein particle that translates genetic information encoded in mRNA into specific proteins. Its highly conserved active site, the peptidyl-transferase center (PTC), is located on the large (50S) ribosomal subunit and is comprised solely of rRNA, which makes the ribosome the only natural ribozyme with polymerase activity. The last decade witnessed a rapid accumulation of atomic-resolution structural data on both ribosomal subunits as well as on the entire ribosome. This has allowed studies on the mechanism of peptide bond formation at a level of detail that surpasses that for the classical protein enzymes. A current understanding of the mechanism of the ribosome-catalyzed peptide bond formation is the focus of this review. Implications on the mechanism of peptide release are discussed as well.     

On peptide bond formation, translocation, nascent protein progression and the regulatory properties of ribosomes. Derived on 20 October 2002 at the 28th FEBS Meeting in Istanbul.

High-resolution crystal structures of large ribosomal subunits from Deinococcus radiodurans complexed with tRNA-mimics indicate that precise substrate positioning, mandatory for efficient protein biosynthesis with no further conformational rearrangements, is governed by remote interactions of the tRNA helical features. Based on the peptidyl transferase center (PTC) architecture, on the placement of tRNA mimics, and on the existence of a two-fold related region consisting of about 180 nucleotides of the 23S RNA, we proposed a unified mechanism integrating peptide bond formation, A-to-P site translocation, and the entrance of the nascent protein into its exit tunnel. This mechanism implies sovereign, albeit correlated, motions of the tRNA termini and includes a spiral rotation of the A-site tRNA-3' end around a local two-fold rotation axis, identified within the PTC. PTC features, ensuring the precise orientation required for the A-site nucleophilic attack on the P-site carbonyl-carbon, guide these motions. Solvent mediated hydrogen transfer appears to facilitate peptide bond formation in conjunction with the spiral rotation. The detection of similar two-fold symmetry-related regions in all known structures of the large ribosomal subunit, indicate the universality of this mechanism, and emphasizes the significance of the ribosomal template for the precise alignment of the substrates as well as for accurate and efficient translocation. The symmetry-related region may also be involved in regulatory tasks, such as signal transmission between the ribosomal features facilitating the entrance and the release of the tRNA molecules. The protein exit tunnel is an additional feature that has a role in cellular regulation. We showed by crystallographic methods that this tunnel is capable of undergoing conformational oscillations and correlated the tunnel mobility with sequence discrimination, gating and intracellular regulation.     

Inhibition of an initiation codon function by hemin deficiency and the hemin-controlled translational repressor in the reticulocyte cell-free system

The trinucleotide codon, AUG, promotes the combination of reticulocyte 40S and 60S native ribosomal subunits as well as recombination of those derived by dissociation factor. This interaction is inhibited by hemin deficiency or the hemin-controlled repressor and results in the loss of methionine from ribosomal bound met-tRNA_f. The locus of inhibition among the partial reactions of peptide chain initiation is between met-tRNA_f and initiation codon binding to the 40S ribosomal subunit and peptide bond formation.     

The peptidyltransferase centre of the Escherichia coli ribosome. The histidine of protein L16 affects the reconstitution and control of the active centre but is not essential for release-factor-mediated peptidyl-tRNA hydrolysis and peptide bond formation

Modification of the Escherichia coli 50S ribosomal subunit with histidine-specific diethyl pyrocarbonate affects peptide bond formation and release-factor-dependent peptidyl-tRNA hydrolysis. Unmodified L16 can restore activity to a split protein fraction from the altered subunit but other proteins of the core also contain histidine residues important for the activity of the peptidyltransferase centre. When isolated and purified by centrifugation, particles reconstituted with unmodified proteins and modified L16 do not retain the altered L16. The modified protein does mediate the partial restoration of peptide bond formation and release-factor-2 activities to these particles. It must be exerting its effect during the assembly of the peptidyltransferase centre in the reconstituted particle. A particle could be reconstituted which lacks L16 and has significant activity in peptide bond formation and peptidyl-tRNA hydrolysis. L16 stimulates these activities. A tighter ribosomal binding of the release factor 2, dependent upon the absence of protein L11, can in part compensate for the loss of activity of the peptidyltransferase centre when it is assembled with either modified L16 or in the absence of L16. The protein and its histidine residue seem important, therefore, for the peptidyltransferase centre to be formed in the correct conformation but not essential for activity once the centre is assembled.     

The ribosomal peptidyl transferase center: structure, function, evolution, inhibition

The ribosomal peptidyl transferase center (PTC) resides in the large ribosomal subunit and catalyzes the two principal chemical reactions of protein synthesis: peptide bond formation and peptide release. The catalytic mechanisms employed and their inhibition by antibiotics have been in the focus of molecular and structural biologists for decades. With the elucidation of atomic structures of the large ribosomal subunit at the dawn of the new millennium, these questions gained a new level of molecular significance. The crystallographic structures compellingly confirmed that peptidyl transferase is an RNA enzyme. This places the ribosome on the list of naturally occurring ribozymes that outlived the transition from the pre-biotic RNA World to contemporary biology. Biochemical, genetic and structural evidence highlight the role of the ribosome as an entropic catalyst that accelerates peptide bond formation primarily by substrate positioning. At the same time, peptide release should more strongly depend on chemical catalysis likely involving an rRNA group of the PTC. The PTC is characterized by the most pronounced accumulation of universally conserved rRNA nucleotides in the entire ribosome. Thus, it came as a surprise that recent findings revealed an unexpected high level of variation in the mode of antibiotic binding to the PTC of ribosomes from different organisms.     

Structural dynamics of ribosomal RNA during decoding on the ribosome

Decoding is a multistep process by which the ribosome accurately selects aminoacyl-tRNA (aa-tRNA) that matches the mRNA codon in the A site. The correct geometry of the codon-anticodon complex is monitored by the ribosome, resulting in conformational changes in the decoding center of the small (30S) ribosomal subunit by an induced-fit mechanism. The recognition of aa-tRNA is modulated by changes of the ribosome conformation in regions other than the decoding center that may either affect the architecture of the latter or alter the communication of the 30S subunit with the large (50S) subunit where the GTPase and peptidyl transferase centers are located. Correct codon-anticodon complex formation greatly accelerates the rates of GTP hydrolysis and peptide bond formation, indicating the importance of crosstalk between the subunits and the role of the 50S subunit in aa-tRNA selection. In the present review, recent results of the ribosome crystallography, cryoelectron microscopy (cryo-EM), genetics, rapid kinetics and biochemical approaches are reviewed which show that the dynamics of the structure of ribosomal RNA (rRNA) play a crucial role in decoding.     

NMR studies of the mechanism of cyclic AMP-dependent protein kinase

NMR has been used to study the role of the divalent cation, the conformations, arrangement, and exchange rates of the enzyme-bound metal-ATP and peptide substrates, the mechanism of the phosphoryl transfer, and the structure and role of the regulatory subunit on type II cyclic AMP (cAMP)-dependent protein kinase from bovine heart. The active complex consists of an enzyme-ATP-metal bridge in which the metal is beta, gamma coordinated, with delta chirality at P beta, and a torsional angle at the adenine-ribose bond in the high-anti range (x approximately 80 degrees). The bound heptapeptide substrate Leu-Arg-Arg-Ala-Ser-Leu-Gly is extended in conformation, forming either a coil or, less likely, a beta turn but not an alpha helix or beta sheet. The distance from the gamma-P of bound ATP analogs to the Ser-OH of the bound peptide (5.3 +/- 0.7 A) would permit a metaphosphate or an elongated phosphorane intermediate or transition state. The regulatory subunit (R2) blocks the peptide- or protein-binding site of the catalytic subunit. The 31P chemical shift of cAMP is not greatly altered on binding to R2, but the resonance is broadened to approximately 32 Hz, which indicates no chemical change but marked immobilization of bound cAMP. A narrower (approximately 7 Hz) 31P resonance at 4.44 ppm is assigned to P-serine-95 of R2 because it disappears with catalytic subunit, Mg2+, and an ADP-generating system.     

A model for the spatial arrangement of the proteins in the large subunit of the Escherichia coli ribosome.

A three-dimensional model for the arrangement of 29 of the 33 proteins from the Escherichia coli large ribosomal subunit has been generated by interactive computer graphics. The topographical information that served as input in the model building process was obtained by combining the immunoelectron microscopically determined network of epitope-epitope distances on the surface of the large ribosomal subunit with in situ protein-protein cross-linking data. These two independent sets of data were shown to be compatible by geometric analysis, thus allowing the construction of an inherently consistent model. The model shows (i) that the lower third of the large subunit is protein-poor, (ii) that proteins known to be functionally involved in peptide bond formation and translocation are clustered in two separate regions, (iii) that proteins functionally interdependent during the self-assembly of the large subunit are close neighbours in the mature subunit and (iv) that proteins forming the early assembly nucleus are grouped together in a distinct region at the 'back' of the subunit.     

The active site of the ribosome is composed of two layers of conserved nucleotides with distinct roles in peptide bond formation and peptide release

Peptide bond formation and peptide release are catalyzed in the active site of the large subunit of the ribosome where universally conserved nucleotides surround the CCA ends of the peptidyl- and aminoacyl-tRNA substrates. Here, we describe the use of an affinity-tagging system for the purification of mutant ribosomes and analysis of four universally conserved nucleotides in the innermost layer of the active site: A2451, U2506, U2585, and A2602. While pre-steady-state kinetic analysis of the peptidyl transferase activity of the mutant ribosomes reveals substantially reduced rates of peptide bond formation using the minimal substrate puromycin, their rates of peptide bond formation are unaffected when the substrates are intact aminoacyl-tRNAs. These mutant ribosomes do, however, display substantial defects in peptide release. These results reveal a view of the catalytic center in which an inner shell of conserved nucleotides is pivotal for peptide release, while an outer shell is responsible for promoting peptide bond formation.     

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.     

The Ribosomal Peptidyl Transferase Center: Structure,Function, Evolution,Inhibition

ABSTRACT

The ribosomal peptidyl transferase center (PTC) resides in the large ribosomal subunit and catalyzes the two principal chemical reactions of protein synthesis: peptide bond formation and peptide release. The catalytic mechanisms employed and their inhibition by antibiotics have been in the focus of molecular and structural biologists for decades. With the elucidation of atomic structures of the large ribosomal subunit at the dawn of the new millennium, these questions gained a new level of molecular significance. The crystallographic structures compellingly confirmed that peptidyl transferase is an RNA enzyme. This places the ribosome on the list of naturally occurring riboyzmes that outlived the transition from the pre-biotic RNA World to contemporary biology. Biochemical, genetic and structural evidence highlight the role of the ribosome as an entropic catalyst that accelerates peptide bond formation primarily by substrate positioning. At the same time, peptide release should more strongly depend on chemical catalysis likely involving an rRNA group of the PTC. The PTC is characterized by the most pronounced accumulation of universally conserved rRNA nucleotides in the entire ribosome. Thus, it came as a surprise that recent findings revealed an unexpected high level of variation in the mode of antibiotic binding to the PTC of ribosomes from different organisms.     

ClpP hydrolyzes a protein substrate processively in the absence of the ClpA ATPase: mechanistic studies of ATP-independent proteolysis

ATP-dependent proteases are processive, meaning that they degrade full-length proteins into small peptide products without releasing large intermediates along the reaction pathway. In the case of the bacterial ATP-dependent protease ClpAP, ATP hydrolysis by the ClpA component has been proposed to be required for processive proteolysis of full-length protein substrates. We present here data showing that in the absence of the ATPase subunit ClpA, the protease subunit ClpP can degrade full-length protein substrates processively, albeit at a greatly reduced rate. Moreover, the size distribution of peptide products from a ClpP-catalyzed digest is remarkably similar to the size distribution of products from a ClpAP-catalyzed digest. The ClpAP- and ClpP-generated peptide product size distributions are fitted well by a sum of multiple underlying Gaussian peaks with means at integral multiples of approximately 900 Da (7-8 amino acids). Our results are consistent with a mechanism in which ClpP controls product sizes by alternating between translocation in steps of 7-8 (+/-2-3) amino acid residues and proteolysis. On the structural and molecular level, the step size may be controlled by the spacing between the ClpP active sites, and processivity may be achieved by coupling peptide bond hydrolysis to the binding and release of substrate and products in the protease chamber.     

Structures of five antibiotics bound at the peptidyl transferase center of the large ribosomal subunit

Structures of anisomycin, chloramphenicol, sparsomycin, blasticidin S, and virginiamycin M bound to the large ribosomal subunit of Haloarcula marismortui have been determined at 3.0A resolution. Most of these antibiotics bind to sites that overlap those of either peptidyl-tRNA or aminoacyl-tRNA, consistent with their functioning as competitive inhibitors of peptide bond formation. Two hydrophobic crevices, one at the peptidyl transferase center and the other at the entrance to the peptide exit tunnel play roles in binding these antibiotics. Midway between these crevices, nucleotide A2103 of H.marismortui (2062 Escherichia coli) varies in its conformation and thereby contacts antibiotics bound at either crevice. The aromatic ring of anisomycin binds to the active-site hydrophobic crevice, as does the aromatic ring of puromycin, while the aromatic ring of chloramphenicol binds to the exit tunnel hydrophobic crevice. Sparsomycin contacts primarily a P-site bound substrate, but also extends into the active-site hydrophobic crevice. Virginiamycin M occupies portions of both the A and P-site, and induces a conformational change in the ribosome. Blasticidin S base-pairs with the P-loop and thereby mimics C74 and C75 of a P-site bound tRNA.     

Protein S20 Binds Two 16S rRNA Sites as Assembly Is Initiated

Ribosomal protein S20 is a primary binding protein that bridges the 5′ domain and the 3′ minor domain of the 16S ribosomal RNA (rRNA) in the 30S ribosomal subunit. Using time-dependent dimethyl sulfate modification, we have determined that as it is bound to 16S rRNA, protein S20 causes rapid protection of bases A246, A274, A279, and A282 in the stem region of helix 11 in the 5′ domain and moderately fast modifications of helix 44 bases A1433 and A1434 in the 3′ minor domain. At a later time, enhancements occur with bases A181and A190 in helix 9, bases A325 and A327 in helix 13, and base C264 at the distal end of helix 11 in the 5′ domain of 16S rRNA. The modifications that occur in the stem region of helix 11 are distant from the binding site of protein S20, as determined from the crystal structure. Simultaneous addition of protein S17 with S20 to the complex significantly alters the modifications caused by protein S20 in the stem region of helix 11 but does not alter the remaining modifications. Our results indicate that protein S20 is binding to at least two alternate 16S rRNA sites during the early assembly process.     

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.