The Experts below are selected from a list of 165 Experts worldwide ranked by ideXlab platform
Dennis Synetos - One of the best experts on this subject based on the ideXlab platform.
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A dispensable yeast ribosomal protein optimizes Peptidyltransferase activity and affects translocation.
The Journal of biological chemistry, 2002Co-Authors: John Dresios, Panagiotis Panopoulos, Katsuyuki Suzuki, Dennis SynetosAbstract:Abstract Yeast ribosomal protein L41 is dispensable in the yeast. Its absence had no effect on polyphenylalanine synthesis activity, and a limited effect on growth, translational accuracy, or the resistance toward the antibiotic paromomycin. Removal of L41 did not affect the 60:40 S ratio, but it reduced the amount of 80 S, suggesting that L41 is involved in ribosomal subunit association. However, the two most important effects of L41 were on Peptidyltransferase activity and translocation. Peptidyltransferase activity was measured as a second-order rate constant (k cat/K s) corresponding to the rate of peptide bond formation; thisk cat/K s was lowered 3-fold to 1.15 min−1 mm −1 in the L41 mutant compared with 3.46 min−1mm −1 in the wild type. Translocation was also affected by L41. Elongation factor 2 (EF2)-dependent (enzymatic) translocation of Ac-Phe-tRNA from the A- to P-site was more efficient in the absence of L41, because 50% translocation was achieved at only 0.004 μm EF2 compared with 0.02 μm for the wild type. Furthermore, the EF2-dependent translocation was inhibited by 50% at 2.5 μm of the translocation inhibitor cycloheximide in the L41 mutant compared with 1.2 μm in the wild type. Finally, the rate of EF2-independent (spontaneous) translocation was increased in the absence of L41.
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Yeast ribosomal protein deletion mutants possess altered Peptidyltransferase activity and different sensitivity to cycloheximide.
Biochemistry, 2001Co-Authors: John Dresios, Panagiotis Panopoulos, Christina P. Frantziou, Dennis SynetosAbstract:The major function of the ribosome is its ability to catalyze formation of peptide bonds, and it is carried out by the ribosomal Peptidyltransferase. Recent evidence suggests that the catalyst of peptide bond formation is the 23S rRNA of the large ribosomal subunit. We have developed an in vitro system for the determination of Peptidyltransferase activity in yeast ribosomes. Using this system, a kinetic analysis of a model reaction for Peptidyltransferase is described with Ac-Phe-tRNA as the peptidyl donor and puromycin as the acceptor. The Ac-Phe-tRNA-poly(U)-80S ribosome complex (complex C) was isolated and then reacted with excess puromycin to give Ac-Phe-puromycin. This reaction (puromycin reaction) followed first-order kinetics. At saturating concentrations of puromycin, the first-order rate constant (k(3)) is identical to the catalytic rate constant (k(cat)) of Peptidyltransferase. This k(cat) from wild-type yeast strains was equal to 2.18 min(-1) at 30 degrees C. We now present for the first time kinetic evidence that yeast ribosomes lacking a particular protein of the 60S subunit may possess significantly altered peptide bond-forming ability. The k(cat) of Peptidyltransferase from mutants lacking ribosomal protein L24 was decreased 3-fold to 0.69 min(-1), whereas the k(cat) from mutants lacking L39 was slightly increased to 3.05 min(-1) and that from mutants lacking both proteins was 1.07 min(-1). These results suggest that the presence of ribosomal proteins L24 and, to a lesser extent, L39 is required for exhibition of the normal catalytic activity of the ribosome. Finally, the L24 or L39 mutants did not affect the rate or the extent of the translocation phase of protein synthesis. However, the absence of L24 caused increased resistance to cycloheximide, a translocation inhibitor. Translocation of Ac-Phe-tRNA from the A- to P-site was inhibited by 50% at 1.4 microM cycloheximide for the L24 mutant compared to 0.7 microM for the wild type.
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Kinetics of Inhibition of Rabbit Reticulocyte Peptidyltransferase by Anisomycin and Sparsomycin
Molecular pharmacology, 1998Co-Authors: Margarita Ioannou, Charalambos Coutsogeorgopoulos, Dennis SynetosAbstract:A detailed kinetic study was carried out on the inhibitory mechanisms of two eukaryotic Peptidyltransferase drugs (I), anisomycin and sparsomycin. In an in vitro system from rabbit reticulocytes, AcPhe-puromycin is produced in a pseudo-first-order reaction from the preformed AcPhe-tRNA/poly(U)/80S ribosome complex (complex C) and excess puromycin (S). This reaction is inhibited by anisomycin and sparsomycin through different mechanisms. Anisomycin acts as a mixed noncompetitive inhibitor. The product, AcPhe-puromycin, is derived only from C according to the puromycin reaction. On the other hand, sparsomycin reacts with complex C in a two-step reaction, C + I K i ⇌ CI k 6 ⇌ k 7 C ∗ I An initial rapid binding of the drug produces the encounter complex CI. During this step and before conversion of CI to C*I, sparsomycin behaves as a competitive inhibitor. The rapidly produced CI is isomerized slowly to a conformationally altered species C*I in which I is bound more tightly. The rate constants of this step arek6 = 2.1 min−1 andk7 = 0.095 min−1. Moreover, the low value of the association rate constantk7/Ki′ (2 × 105m−1sec−1), provides insight into the rates of possible conformational changes occurring during protein synthesis and supports the proposal that sparsomycin is the first example of a slow-binding inhibitor of eukaryotic Peptidyltransferase. When complex C is preincubated with concentrations of sparsomycin of >8Ki and then reacts with a mixture of puromycin and sparsomycin, the inhibition becomes linear mixed noncompetitive and involves C*I instead of CI. During this phase, AcPhe-puromycin is produced from a new, modified ribosomal complex with a lower catalytic rate constant. Thus, sparsomycin also acts as a modifier of eukaryotic Peptidyltransferase activity.
Jonathan D Dinman - One of the best experts on this subject based on the ideXlab platform.
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Structure/function analysis of yeast ribosomal protein L2
Nucleic acids research, 2008Co-Authors: Arturas Meskauskas, Johnathan R. Russ, Jonathan D DinmanAbstract:Ribosomal protein L2 is a core element of the large subunit that is highly conserved among all three kingdoms. L2 contacts almost every domain of the large subunit rRNA and participates in an intersubunit bridge with the small subunit rRNA. It contains a solvent-accessible globular domain that interfaces with the solvent accessible side of the large subunit that is linked through a bridge to an extension domain that approaches the Peptidyltransferase center. Here, screening of randomly generated library of yeast RPL2A alleles identified three translationally defective mutants, which could be grouped into two classes. The V48D and L125Q mutants map to the globular domain. They strongly affect ribosomal A-site associated functions, Peptidyltransferase activity and subunit joining. H215Y, located at the tip of the extended domain interacts with Helix 93. This mutant specifically affects peptidyl-tRNA binding and Peptidyltransferase activity. Both classes affect rRNA structure far away from the protein in the A-site of the Peptidyltransferase center. These findings suggest that defective interactions with Helix 55 and with the Helix 65-66 structure may indicate a certain degree of flexibility in L2 in the neck region between the two other domains, and that this might help to coordinate tRNA-ribosome interactions.
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rRNA mutants in the yeast Peptidyltransferase center reveal allosteric information networks and mechanisms of drug resistance
Nucleic acids research, 2008Co-Authors: Rasa Rakauskaite, Jonathan D DinmanAbstract:To ensure accurate and rapid protein synthesis, nearby and distantly located functional regions of the ribosome must dynamically communicate and coordinate with one another through a series of information exchange networks. The ribosome is »2/3 rRNA and information should pass mostly through this medium. Here, two viable mutants located in the Peptidyltransferase center (PTC) of yeast ribosomes were created using a yeast genetic system that enables stable production of ribosomes containing only mutant rRNAs. The specific mutants were C2820U (Escherichia coli C2452) and )2922C (E. coli U2554). Biochemical and genetic analyses of these mutants suggest that they may trap the PTC in the ‘open’ or aa-tRNA bound conformation, decreasing peptidyl-tRNA binding. We suggest that these structural changes are manifested at the biological level by affecting large ribosomal subunit biogenesis, ribosomal subunit joining during initiation, susceptibility/resistance to Peptidyltransferase inhibitors, and the ability of ribosomes to properly decode termination codons. These studies also add to our understanding of how information is transmitted both locally and over long distances through allosteric networks of rRNA–rRNA and rRNA–protein interactions.
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Decreased Peptidyltransferase activity correlates with increased programmed -1 ribosomal frameshifting and viral maintenance defects in the yeast Saccharomyces cerevisiae.
RNA (New York N.Y.), 2003Co-Authors: Arturas Meskauskas, Jason W. Harger, Kristi L. Muldoon Jacobs, Jonathan D DinmanAbstract:Increased efficiencies of programmed −1 ribosomal frameshifting in yeast cells expressing mutant forms of ribosomal protein L3 are unable to maintain the dsRNA “Killer” virus. Here we demonstrate that changes in frameshifting and virus maintenance in these mutants correlates with decreased Peptidyltransferase activities. The mutants did not affect Ty1-directed programmed +1 ribosomal frameshifting or nonsense-mediated mRNA decay. Independent experiments demonstrate similar programmed −1 ribosomal frameshifting specific defects in cells lacking ribosomal protein L41,which has previously been shown to result in Peptidyltransferase defects in yeast. These findings are consistent with the hypothesis that decreased Peptidyltransferase activity should result in longer ribosome pause times after the accommodation step of the elongation cycle,allowing more time for ribosomal slippage at programmed −1 ribosomal frameshift signals.
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Ribosomal Protein L3 Mutants Alter Translational Fidelity and Promote Rapid Loss of the Yeast Killer Virus
Molecular and cellular biology, 1999Co-Authors: Stuart W. Peltz, Amy B. Hammell, Ying Cui, Jason Yasenchak, Lara Puljanowski, Jonathan D DinmanAbstract:Programmed −1 ribosomal frameshifting is utilized by a number of RNA viruses as a means of ensuring the correct ratio of viral structural to enzymatic proteins available for viral particle assembly. Altering frameshifting efficiencies upsets this ratio, interfering with virus propagation. We have previously demonstrated that compounds that alter the kinetics of the peptidyl-transfer reaction affect programmed −1 ribosomal frameshift efficiencies and interfere with viral propagation in yeast. Here, the use of a genetic approach lends further support to the hypothesis that alterations affecting the ribosome’s Peptidyltransferase activity lead to changes in frameshifting efficiency and virus loss. Mutations in the RPL3 gene, which encodes a ribosomal protein located at the Peptidyltransferase center, promote approximately three- to fourfold increases in programmed −1 ribosomal frameshift efficiencies and loss of the M1 killer virus of yeast. The mak8-1 allele of RPL3 contains two adjacent missense mutations which are predicted to structurally alter the Mak8-1p. Furthermore, a second allele that encodes the N-terminal 100 amino acids of L3 (called L3Δ) exerts a trans-dominant effect on programmed −1 ribosomal frameshifting and killer virus maintenance. Taken together, these results support the hypothesis that alterations in the Peptidyltransferase center affect programmed −1 ribosomal frameshifting.
Dimitrios L. Kalpaxis - One of the best experts on this subject based on the ideXlab platform.
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Deacylated tRNA is released from the E site upon A site occupation but before GTP is hydrolyzed by EF-Tu
Nucleic Acids Research, 2005Co-Authors: George P. Dinos, Daniel N. Wilson, Dimitrios L. Kalpaxis, Knud H. NierhausAbstract:The presence or absence of deacylated tRNA at the E site sharply influences the activation energy required for binding of a ternary complex to the ribosomal A site indicating the different conformations that the E-tRNA imparts on the ribosome. Here we address two questions: (i) whether or not Peptidyltransferase—the essential catalytic activity of the large ribosomal subunit—also depends on the occupancy state of the E site and (ii) at what stage the E-tRNA is released during an elongation cycle. Kinetics of the puromycin reaction on various functional states of the ribosome indicate that the A-site substrate of the Peptidyltransferase center, puromycin, requires the same activation energy for peptide-bond formation under all conditions tested. We further demonstrate that deacylated tRNA is released from the E site by binding a ternary complex aminoacyl-tRNA•EF-Tu•GDPNP to the A site. This observation indicates that the E-tRNA is released after the decoding step but before both GTP hydrolysis by EF-Tu and accommodation of the A-tRNA. Collectively these results reveal that the reciprocal linkage between the E and A sites affects the decoding center on the 30S subunit, but does not influence the rate of peptide-bond formation at the active center of the 50S subunit.
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Effect of polyamines on the inhibition of Peptidyltransferase by antibiotics: revisiting the mechanism of chloramphenicol action
Nucleic acids research, 2003Co-Authors: Maria A. Xaplanteri, George P. Dinos, Athanasios Andreou, Dimitrios L. KalpaxisAbstract:Chloramphenicol is thought to interfere competitively with the binding of the aminoacyl-tRNA 3′-terminus to ribosomal A-site. However, noncompetitive or mixed-noncompetitive inhibition, often observed to be dependent on chloramphenicol concentration and ionic conditions, leaves some doubt about the precise mode of action. Here, we examine further the inhibition effect of chloramphenicol, using a model system derived from Escherichia coli in which a peptide bond is formed between puromycin and AcPhe-tRNA bound at the P-site of poly(U)-programmed ribosomes, under ionic conditions (6 mM Mg2+, 100 mM NH4+, 100 µM spermine) more closely resembling the physiological status. Kinetics reveal that chloramphenicol (I) reacts rapidly with AcPhe-tRNA·poly(U)·70S ribosomal complex (C) to form the encounter complex CI which is then isomerized slowly to a more tight complex, C*I. A similar inhibition pattern is observed, if complex C modified by a photoreactive analogue of spermine, reacts in buffer free of spermine. Spermine, either reversibly interacting with or covalently attached to ribosomes, enhances the Peptidyltransferase activity and increases the chloramphenicol potency, without affecting the isomerization step. As indicated by photoaffinity labeling, the Peptidyltransferase center at which chloramphenicol binds, is one of the preferred cross-linking sites for polyamines. This fact may explain the effect of spermine on chloramphenicol binding to ribosomes.
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New aspects on the kinetics of activation of ribosomal Peptidyltransferase-catalyzed peptide bond formation by monovalent ions and spermine
Biochimica et biophysica acta, 1997Co-Authors: Maria Michelinaki, Charalambos Coutsogeorgopoulos, Anestis Spanos, Dimitrios L. KalpaxisAbstract:Abstract The effect of NH4+ and K+ ions on the activity of ribosomal Peptidyltransferase was investigated in a model system derived from Escherichia coli, in which AcPhe-puromycin is produced by a pseudo-first-order reaction between the preformed AcPhe-tRNA-poly(U)-ribosome complex (complex C) and excess puromycin. Detailed kinetic analysis suggests that both NH4+ and K+ ions act as essential activators of Peptidyltransferase by filling randomly, but not cooperatively, multiple sites on the ribosome. With respect to the NH4+ effect at 25°C, the values of the molecular interaction coefficient (n), the dissociation constant (KA), and the apparent catalytic rate constant (kmax) of Peptidyltransferase at saturating levels of NH4+ and puromycin are 1.99, 268.7 mM and 24.8 min−1, respectively. The stimulation of Peptidyltransferase by K+ ions at 25°C (n=4.38, KA=95.5 mM, kmax=9.6 min−1) is not as marked as that caused by NH4+ ions. Furthermore, it is evident that NH4+ at high concentration (200 mM) is effective in filling regulatory sites of complex C, which are responsible for the modulatory effect of spermine. The combination of NH4+ ions (200 mM) with spermine (300 μM) produces an additive increase in Peptidyltransferase activity. Taken together, these findings suggest the involvement of two related pathways in the regulation of Peptidyltransferase activity, one mediated by specific monovalent cations and the other mediated by spermine.
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Growth phase and growth rate dependence of ribosomal Peptidyltransferase activity status in Escherichia coli.
Biochimie, 1995Co-Authors: Dimitrios L. Kalpaxis, Panagiotis Karahalios, M. PapapetropoulouAbstract:Ribosomes from a clinical isolate of E coli were purified and characterized. The structural features of these ribosomes were identical to wild-type E coli ribosomes, with the exception that rRNA in general, but especially 23S rRNA, was degraded as a result of the transition from early to late logarithmic growth phase, on different growth media. Analysis of the ribosomal protein by gel electrophoresis indicated that the L12/L7 molar ratio increases during early logarithmic phase, reaching a maximum value of about 1.6 at midlogarithmic phase, and then falling to 0.7 in late logarithmic phase. Concomitantly with L12/L7 alterations, the activity status of ribosomal Peptidyltransferase was found to undergo a striking shift. Reconstitution experiments demonstrated that the two effects are closely related. Moreover, L12/L7 molar ratio as well as Peptidyltransferase activity increased with increasing growth rate. In the latter case, however, the acetylation level of L12 protein per se seemed to be inadequate to modulate the Peptidyltransferase activity.
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Slow-onset inhibition of ribosomal Peptidyltransferase by lincomycin.
Archives of biochemistry and biophysics, 1992Co-Authors: Sofia Kallia-raftopoulos, Dimitrios L. Kalpaxis, Charalambos CoutsogeorgopoulosAbstract:Abstract In a system derived from Escherichia coli, we carried out a detailed kinetic analysis of the inhibition of the puromycin reaction by lincomycin. N-Acetylphenylalanyl-tRNA (Ac-Phe-tRNA; the donor) reacts with excess puromycin (S) according to reaction [1], C + S ⇌ K S CS → C ′+ P where C is the Ac-Phe-tRNA-poly(U)-ribosome ternary complex (complex C). The entire course of reaction [1] appears as a straight line when the reaction is analyzed as pseudo-first-order and the data are plotted in a logarithmic form (logarithmic time plot). The slope of this straight line gives the apparent k S obs = k 3 [ S ] (K s + [ S ]) . In the presence of lincomycin the logarithmic time plot is not a straight line, but becomes biphasic, giving an early slope ** K e = k 3 [ S ] {K s (1+[ I ] K 1 ) and a late slope ** k 1 = k 3 [ S ]{ K s (1+[ I ] k′ 1 +[ S ]} . Kinetic analysis of the early slopes at various concentrations of S and I shows competitive inhibition with Ki = 10.0 μM. The late slopes also give competitive inhibition with a distinct inhibition constant K'i = 2.0 μM. Excluding alternative models, the two phases of inhibition are compatible with a model in which reaction [1] is coupled with reaction [2], C + I ⇌ k 5 k 4 CI ⇌ k 7 k 6 C ∗ I where the isomerization step CI ⇌ CI∗ is slower than the first step C + I ⇌ CI, K i = k 5 k 4 and K′ i = K′ i [ k 7 (k 6 + k 7 ) ] . Corroborative evidence for this model comes from the examination of reaction [2] alone in the absence of S. This reaction is analyzed as pseudo-first-order going toward equilibrium with eq I = k 6 + k 6 [I] (K i + [ I] ) . The plot of keqI versus [I] is not linear. This plot supports the two-step mechanism of reaction [2] in which k = 5.2 min−1 and k7 = 1.3 min−1. This is the first example of slow-onset inhibition of ribosomal Peptidyltransferase which follows a simple model leading to the determination of the isomerization constants k6 and k7. We suggest that lincomycin inhibits protein synthesis by binding initially to the ribosome in competition with aminoacyl-tRNA. Subsequently, as a result of a conformational change, an isomerization occurs (CI ⇌ C∗I), after which lincomycin continues to interfere with the binding of aminoacyl-tRNA to the isomerized complex.
Charalambos Coutsogeorgopoulos - One of the best experts on this subject based on the ideXlab platform.
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Kinetics of Inhibition of Rabbit Reticulocyte Peptidyltransferase by Anisomycin and Sparsomycin
Molecular pharmacology, 1998Co-Authors: Margarita Ioannou, Charalambos Coutsogeorgopoulos, Dennis SynetosAbstract:A detailed kinetic study was carried out on the inhibitory mechanisms of two eukaryotic Peptidyltransferase drugs (I), anisomycin and sparsomycin. In an in vitro system from rabbit reticulocytes, AcPhe-puromycin is produced in a pseudo-first-order reaction from the preformed AcPhe-tRNA/poly(U)/80S ribosome complex (complex C) and excess puromycin (S). This reaction is inhibited by anisomycin and sparsomycin through different mechanisms. Anisomycin acts as a mixed noncompetitive inhibitor. The product, AcPhe-puromycin, is derived only from C according to the puromycin reaction. On the other hand, sparsomycin reacts with complex C in a two-step reaction, C + I K i ⇌ CI k 6 ⇌ k 7 C ∗ I An initial rapid binding of the drug produces the encounter complex CI. During this step and before conversion of CI to C*I, sparsomycin behaves as a competitive inhibitor. The rapidly produced CI is isomerized slowly to a conformationally altered species C*I in which I is bound more tightly. The rate constants of this step arek6 = 2.1 min−1 andk7 = 0.095 min−1. Moreover, the low value of the association rate constantk7/Ki′ (2 × 105m−1sec−1), provides insight into the rates of possible conformational changes occurring during protein synthesis and supports the proposal that sparsomycin is the first example of a slow-binding inhibitor of eukaryotic Peptidyltransferase. When complex C is preincubated with concentrations of sparsomycin of >8Ki and then reacts with a mixture of puromycin and sparsomycin, the inhibition becomes linear mixed noncompetitive and involves C*I instead of CI. During this phase, AcPhe-puromycin is produced from a new, modified ribosomal complex with a lower catalytic rate constant. Thus, sparsomycin also acts as a modifier of eukaryotic Peptidyltransferase activity.
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New aspects on the kinetics of activation of ribosomal Peptidyltransferase-catalyzed peptide bond formation by monovalent ions and spermine
Biochimica et biophysica acta, 1997Co-Authors: Maria Michelinaki, Charalambos Coutsogeorgopoulos, Anestis Spanos, Dimitrios L. KalpaxisAbstract:Abstract The effect of NH4+ and K+ ions on the activity of ribosomal Peptidyltransferase was investigated in a model system derived from Escherichia coli, in which AcPhe-puromycin is produced by a pseudo-first-order reaction between the preformed AcPhe-tRNA-poly(U)-ribosome complex (complex C) and excess puromycin. Detailed kinetic analysis suggests that both NH4+ and K+ ions act as essential activators of Peptidyltransferase by filling randomly, but not cooperatively, multiple sites on the ribosome. With respect to the NH4+ effect at 25°C, the values of the molecular interaction coefficient (n), the dissociation constant (KA), and the apparent catalytic rate constant (kmax) of Peptidyltransferase at saturating levels of NH4+ and puromycin are 1.99, 268.7 mM and 24.8 min−1, respectively. The stimulation of Peptidyltransferase by K+ ions at 25°C (n=4.38, KA=95.5 mM, kmax=9.6 min−1) is not as marked as that caused by NH4+ ions. Furthermore, it is evident that NH4+ at high concentration (200 mM) is effective in filling regulatory sites of complex C, which are responsible for the modulatory effect of spermine. The combination of NH4+ ions (200 mM) with spermine (300 μM) produces an additive increase in Peptidyltransferase activity. Taken together, these findings suggest the involvement of two related pathways in the regulation of Peptidyltransferase activity, one mediated by specific monovalent cations and the other mediated by spermine.
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Kinetic Studies on the Activation of Eukaryotic Peptidyltransferase by Potassium
Archives of biochemistry and biophysics, 1997Co-Authors: Margarita Ioannou, Charalambos CoutsogeorgopoulosAbstract:Abstract In an effort to elucidate the role of potassium ions in the formation of peptide bond, we have used the reaction between puromycin and a ribosomal complex (from rabbit reticulocytes) bearing the donor substrate, AcPhe-tRNA, prebound at the so-called P site (puromycin-reactive state). This reaction can be analyzed as a first-order reaction. At saturating concentrations of puromycin (S) the first-order rate constant ( k max S ) is a measure of the apparent catalytic rate constant of Peptidyltransferase in the puromycin reaction. This k max S depends on the concentration of potassium ions and increases when the concentration of K + is increased. The data suggest a kinetic model in which potassium acts as an essential activator in the puromycin reaction. A single molecule of potassium participates in the mechanism of activation. The kinetics correspond to a sequential addition of potassium and puromycin to two separate and independent sites on the ribosome. At saturating levels of both K + and S the maximal value for the catalytic rate constant of Peptidyltransferase ( k p ) is equal to 20 min −1 at 25°C.
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Determination of Eukaryotic Peptidyltransferase Activity by Pseudo-First-Order Kinetic Analysis
Analytical biochemistry, 1997Co-Authors: Margarita Ioannou, Charalambos Coutsogeorgopoulos, Denis DrainasAbstract:Abstract We have developed an in vitro system for the determination of Peptidyltransferase activity in rabbit reticulocyte ribosomes. Using this system, a detailed kinetic analysis of a model reaction for Peptidyltransferase is described, with AcPhe-tRNA as the peptidyl donor and puromycin as the acceptor. The [AcPhe-tRNA–poly(U)–80S ribosome] complex (complex C) is isolated and then reacted with excess puromycin to give AcPhe-puromycin. This reaction (puromycin reaction) follows first-order kinetics at all concentrations of puromycin tested. At saturating concentrations of puromycin, the first-order rate ( k 3 ) constant is identical to the catalytic rate constant ( k cat ) of Peptidyltransferase. This k 3 of Peptidyltransferase is equal to 2.9 min −1 at 37°C. Moreover, the ratio k 3 / K s , which is an accurate measure of Peptidyltransferase activity, was increased 80-fold when salt-washed ribosomes were replaced by unwashed ribosomes. Finally, the puromycin reaction was inhibited by several well-known antibiotics acting on the eukaryotic Peptidyltransferase.
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Slow-onset inhibition of ribosomal Peptidyltransferase by lincomycin.
Archives of biochemistry and biophysics, 1992Co-Authors: Sofia Kallia-raftopoulos, Dimitrios L. Kalpaxis, Charalambos CoutsogeorgopoulosAbstract:Abstract In a system derived from Escherichia coli, we carried out a detailed kinetic analysis of the inhibition of the puromycin reaction by lincomycin. N-Acetylphenylalanyl-tRNA (Ac-Phe-tRNA; the donor) reacts with excess puromycin (S) according to reaction [1], C + S ⇌ K S CS → C ′+ P where C is the Ac-Phe-tRNA-poly(U)-ribosome ternary complex (complex C). The entire course of reaction [1] appears as a straight line when the reaction is analyzed as pseudo-first-order and the data are plotted in a logarithmic form (logarithmic time plot). The slope of this straight line gives the apparent k S obs = k 3 [ S ] (K s + [ S ]) . In the presence of lincomycin the logarithmic time plot is not a straight line, but becomes biphasic, giving an early slope ** K e = k 3 [ S ] {K s (1+[ I ] K 1 ) and a late slope ** k 1 = k 3 [ S ]{ K s (1+[ I ] k′ 1 +[ S ]} . Kinetic analysis of the early slopes at various concentrations of S and I shows competitive inhibition with Ki = 10.0 μM. The late slopes also give competitive inhibition with a distinct inhibition constant K'i = 2.0 μM. Excluding alternative models, the two phases of inhibition are compatible with a model in which reaction [1] is coupled with reaction [2], C + I ⇌ k 5 k 4 CI ⇌ k 7 k 6 C ∗ I where the isomerization step CI ⇌ CI∗ is slower than the first step C + I ⇌ CI, K i = k 5 k 4 and K′ i = K′ i [ k 7 (k 6 + k 7 ) ] . Corroborative evidence for this model comes from the examination of reaction [2] alone in the absence of S. This reaction is analyzed as pseudo-first-order going toward equilibrium with eq I = k 6 + k 6 [I] (K i + [ I] ) . The plot of keqI versus [I] is not linear. This plot supports the two-step mechanism of reaction [2] in which k = 5.2 min−1 and k7 = 1.3 min−1. This is the first example of slow-onset inhibition of ribosomal Peptidyltransferase which follows a simple model leading to the determination of the isomerization constants k6 and k7. We suggest that lincomycin inhibits protein synthesis by binding initially to the ribosome in competition with aminoacyl-tRNA. Subsequently, as a result of a conformational change, an isomerization occurs (CI ⇌ C∗I), after which lincomycin continues to interfere with the binding of aminoacyl-tRNA to the isomerized complex.
Albert E Dahlberg - One of the best experts on this subject based on the ideXlab platform.
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the a2453 c2499 wobble base pair in escherichia coli 23s ribosomal rna is responsible for ph sensitivity of the Peptidyltransferase active site conformation
Nucleic Acids Research, 2004Co-Authors: Mark A Bayfield, Jill Thompson, Albert E DahlbergAbstract:Peptide bond formation, catalyzed by the ribosomal Peptidyltransferase, has long been known to be sensitive to monovalent cation concentrations and pH. More recently, we and others have shown that residue A2451 in the Peptidyltransferase center of the Escherichia coli 50S ribosomal subunit changes conformation in response to alterations in pH, depending on ionic conditions and temperature. Two wobble pairs, A2453-C2499 and A2450-C2063, have been proposed as potential candidates to convey pH-dependent flexibility to the Peptidyltransferase center. Each is presumed to possess a near-neutral pKa, and both lie in proximity to A2451. We show through mutagenesis and chemical probing that the identity of the A2453-C2499 base pair, but not the A2450-C2063 base pair, is critical for the pH-dependent structural rearrangement of A2451. We conclude that, while the A2453-C2499 base pair may be important for maintaining the structure of the active site in the E.coli Peptidyltransferase center, its lack of conservation makes it, and consequently its near-neutral pKa, unlikely to contribute to function during peptide bond formation.
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analysis of mutations at residues a2451 and g2447 of 23s rrna in the Peptidyltransferase active site of the 50s ribosomal subunit
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Jill Thompson, Steven T. Gregory, Mark A Bayfield, Daniel F Kim, Michael Oconnor, Kate R Lieberman, Rachel Green, Harry F Noller, Albert E DahlbergAbstract:On the basis of the recent atomic-resolution x-ray structure of the 50S ribosomal subunit, residues A2451 and G2447 of 23S rRNA were proposed to participate directly in ribosome-catalyzed peptide bond formation. We have examined the Peptidyltransferase and protein synthesis activities of ribosomes carrying mutations at these nucleotides. In Escherichia coli, pure mutant ribosome populations carrying either the G2447A or G2447C mutations maintained cell viability. In vitro, the G2447A ribosomes supported protein synthesis at a rate comparable to that of wild-type ribosomes. In single-turnover Peptidyltransferase assays, G2447A ribosomes were shown to have essentially unimpaired Peptidyltransferase activity at saturating substrate concentrations. All three base changes at the universally conserved A2451 conferred a dominant lethal phenotype when expressed in E. coli. Nonetheless, significant amounts of 2451 mutant ribosomes accumulated in polysomes, and all three 2451 mutations stimulated frameshifting and readthrough of stop codons in vivo. Furthermore, ribosomes carrying the A2451U transversion synthesized full-length β-lactamase chains in vitro. Pure mutant ribosome populations with changes at A2451 were generated by reconstituting Bacillus stearothermophilus 50S subunits from in vitro transcribed 23S rRNA. In single-turnover Peptidyltransferase assays, the rate of peptide bond formation was diminished 3- to 14-fold by these mutations. Peptidyltransferase activity and in vitro β-lactamase synthesis by ribosomes with mutations at A2451 or G2447 were highly resistant to chloramphenicol. The significant levels of Peptidyltransferase activity of ribosomes with mutations at A2451 and G2447 need to be reconciled with the roles proposed for these residues in catalysis.