Peptidyl Transferase Activity Research Articles

RAPP-containing arrest peptides induce translational stalling by short circuiting the ribosomal peptidyltransferase activity

Arrest peptides containing RAPP (ArgAlaProPro) motifs have been discovered in both Gram-positive and Gram-negative bacteria, where they are thought to regulate expression of important protein localization machinery components. Here we determine cryo-EM structures of ribosomes stalled on RAPP arrest motifs in both Bacillus subtilis and Escherichia coli. Together with molecular dynamics simulations, our structures reveal that the RAPP motifs allow full accommodation of the A-site tRNA, but prevent the subsequent peptide bond from forming. Our data support a model where the RAP in the P-site interacts and stabilizes a single hydrogen atom on the Pro-tRNA in the A-site, thereby preventing an optimal geometry for the nucleophilic attack required for peptide bond formation to occur. This mechanism to short circuit the ribosomal peptidyltransferase activity is likely to operate for the majority of other RAPP-like arrest peptides found across diverse bacterial phylogenies.

Puromycin reveals a distinct conformation of neuronal ribosomes

Puromycin is covalently added to the nascent chain of proteins by the peptidyl transferase activity of the ribosome and the dissociation of the puromycylated peptide typically follows this event. It was postulated that blocking the translocation of the ribosome with emetine could retain the puromycylated peptide on the ribosome, but evidence against this has recently been published [Hobson et al., Elife 9, e60048 (2020); and Enam et al., Elife 9, e60303 (2020)]. In neurons, puromycylated nascent chains remain in the ribosome even in the absence of emetine, yet direct evidence for this has been lacking. Using biochemistry and cryoelectron microscopy, we show that the puromycylated peptides remain in the ribosome exit channel in the large subunit in a subset of neuronal ribosomes stalled in the hybrid state. These results validate previous experiments to localize stalled polysomes in neurons and provide insight into how neuronal ribosomes are stalled. Moreover, in these hybrid-state neuronal ribosomes, anisomycin, which usually blocks puromycylation, competes poorly with puromycin in the puromycylation reaction, allowing a simple assay to determine the proportion of nascent chains that are stalled in this state. In early hippocampal neuronal cultures, over 50% of all nascent peptides are found in these stalled polysomes. These results provide insights into the stalling mechanisms of neuronal ribosomes and suggest that puromycylated peptides can be used to reveal subcellular sites of hybrid-state stalled ribosomes in neurons.

Functional involvement of a conserved motif in the middle region of the human ribosomal protein eL42 in translation

Ribosomal protein eL42 (formerly known as L36A), a small protein of the large (60S) subunit of the eukaryotic ribosome, is a component of its exit (E) site. The residue K53 of this protein resides within the motif QSGYGGQTK mainly conserved in eukaryotes, and it is located in the immediate vicinity of the CCA-terminus of the ribosome-bound tRNA in the hybrid P/E state. To examine the role of this eL42 motif in translation, we obtained HEK293T cells producing the wild-type FLAG-tagged protein or its mutant forms with either single substitutions of conserved amino acid residues in the above motif, or simultaneous replacements in positions 45 and 51 or 45 and 53. Examination of the level of exogenous eL42 in fractions of polysome profiles from the target protein-producing cells by the Western blotting revealed that neither single substitution affects the assembly of 60S ribosomal subunits and 80S ribosomes or critically decreases the level of polysomes, but the latter was observed with the double replacements. Analysis of tRNAs bound to 80S ribosomes containing eL42 with double substitutions and examination their peptidyl transferase activity enabled estimation the stage of the elongation cycle, in which amino acid residues of the conserved eL42 motif are involved. We clearly show that cooperative interactions implicating the eL42 residues Q45, Q51, and K53 play a critical role in the ability of the human ribosome to perform properly elongation cycle at the step of deacylated tRNA dissociation from the E site in the human cell.

The Quality Control of Midecamycin and the Predictive Demarcation between Its Impurities and Components

Midecamycin is a 16-membered macrolide antibiotic. It can inhibit the synthesis of bacterial proteins by blocking up the activity of peptidyl transferase in the 50S ribosome. We used high-resolution mass spectrometry to analyze midecamycin, and quantitatively analyzed of each component of midecamycin produced by 18 different companies. The developed methods were validated by assessing linearity, limit of quantitation (LOQ), accuracy, precision, and robustness. Good separations were achieved for all components. Ten components of midecamycin were identified, and the contents of these components were determined in midecamycin produced by different companies. The demarcation between impurities and components of midecamycin was not clear. A ligand-docking model was used for predicting the impurities and components of midecamycin. Components and impurities were docked with the target. The results reported in this article may be important for quality control and the predictive demarcation between impurities and components of midecamycin.

REVIEW OF HPLC METHODS FOR DETERMINATION OF AZITHROMYCIN IN DIFFERENT SAMPLES

Azithromycin is a semi-synthetic macrolide antibiotic of the azalide groups. It inhibits bacterial protein synthesis by binding to the 50S ribosomal subunit of the bacterial 70S ribosome. It inhibits peptidyl transferase activity and interferes with amino acid translocation during the process of translation. Its effect may be bacteriostatic or bactericidal depending on the organism and the drug concentration. Azithromycin is one of the famous and important antibiotics agents and the determination methods of azithromycin in this article were tabulated with lots of chemical and instrumental methods that used in different parameters. Different high performance chromatographic methods have been reviewed in this paper. Keywords: Azithromycin, HPLC Citation: Taqi RMM, Hammoudy SR, Hamzah MJ. Review of HPLC methods for determination of azithromycin in different samples. Iraqi JMS. 2022; 20(1): 77-82. doi: 10.22578/IJMS.20.1.10

Ribosome inhibition by C9ORF72-ALS/FTD-associated poly-PR and poly-GR proteins revealed by cryo-EM

Toxic dipeptide-repeat (DPR) proteins are produced from expanded G4C2 repeats in the C9ORF72 gene, the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Two DPR proteins, poly-PR and poly-GR, repress cellular translation but the molecular mechanism remains unknown. Here we show that poly-PR and poly-GR of ≥20 repeats inhibit the ribosome’s peptidyl-transferase activity at nanomolar concentrations, comparable to specific translation inhibitors. High-resolution cryogenic electron microscopy (cryo-EM) reveals that poly-PR and poly-GR block the polypeptide tunnel of the ribosome, extending into the peptidyl-transferase center (PTC). Consistent with these findings, the macrolide erythromycin, which binds in the tunnel, competes with poly-PR and restores peptidyl-transferase activity. Our results demonstrate that strong and specific binding of poly-PR and poly-GR in the ribosomal tunnel blocks translation, revealing the structural basis of their toxicity in C9ORF72-ALS/FTD.

Plasticity and conditional essentiality of modification enzymes for domain V of Escherichia coli 23S ribosomal RNA.

Escherichia coli rRNAs are post-transcriptionally modified at 36 positions but their modification enzymes are dispensable individually for growth, bringing into question their significance. However, a major growth defect was reported for deletion of the RlmE enzyme, which abolished a 2'O methylation near the peptidyl transferase center (PTC) of the 23S rRNA. Additionally, an adjacent 80-nt "critical region" around the PTC had to be modified to yield significant peptidyl transferase activity in vitro. Surprisingly, we discovered that an absence of just two rRNA modification enzymes is conditionally lethal (at 20°C): RlmE and RluC. At a permissive temperature (37°C), this double knockout was shown to abolish four modifications and be defective in ribosome assembly, though not more so than the RlmE single knockout. However, the double knockout exhibited an even lower rate of tripeptide synthesis than did the single knockout, suggesting an even more defective ribosomal translocation. A combination knockout of the five critical-region-modifying enzymes RluC, RlmKL, RlmN, RlmM, and RluE (not RlmE), which synthesize five of the seven critical-region modifications and 14 rRNA and tRNA modifications altogether, was viable (minor growth defect at 37°C, major at 20°C). This was surprising based on prior in vitro studies. This five-knockout combination had minimal effects on ribosome assembly and frameshifting at 37°C, but greater effects on ribosome assembly and in vitro peptidyl transferase activity at cooler temperatures. These results establish the conditional essentiality of bacterial rRNA modification enzymes and also reveal unexpected plasticity of modification of the PTC region in vivo.

The Peptidyl Transferase Center: a Window to the Past.

In his 2001 article, "Translation: in retrospect and prospect," the late Carl Woese made a prescient observation that there was a need for the then-current view of translation to be "reformulated to become an all-embracing perspective about which 21st century Biology can develop" (RNA 7:1055-1067, 2001, https://doi.org/10.1017/s1355838201010615). The quest to decipher the origins of life and the road to the genetic code are both inextricably linked with the history of the ribosome. After over 60 years of research, significant progress in our understanding of how ribosomes work has been made. Particularly attractive is a model in which the ribosome may facilitate an ∼180° rotation of the CCA end of the tRNA from the A-site to the P-site while the acceptor stem of the tRNA would then undergo a translation from the A-site to the P-site. However, the central question of how the ribosome originated remains unresolved. Along the path from a primitive RNA world or an RNA-peptide world to a proto-ribosome world, the advent of the peptidyl transferase activity would have been a seminal event. This functionality is now housed within a local region of the large-subunit (LSU) rRNA, namely, the peptidyl transferase center (PTC). The PTC is responsible for peptide bond formation during protein synthesis and is usually considered to be the oldest part of the modern ribosome. What is frequently overlooked is that by examining the origins of the PTC itself, one is likely going back even further in time. In this regard, it has been proposed that the modern PTC originated from the association of two smaller RNAs that were once independent and now comprise a pseudosymmetric region in the modern PTC. Could such an association have survived? Recent studies have shown that the extant PTC is largely depleted of ribosomal protein interactions. It is other elements like metallic ion coordination and nonstandard base/base interactions that would have had to stabilize the association of RNAs. Here, we present a detailed review of the literature focused on the nature of the extant PTC and its proposed ancestor, the proto-ribosome.

A putative mechanism underlying secondary metabolite overproduction by Streptomyces strains with a 23S rRNA mutation conferring erythromycin resistance.

Mutations in rrn encoding ribosomal RNA (rRNA) and rRNA modification often confer resistance to ribosome-targeting antibiotics by altering the site of their interaction with the small (30S) and large (50S) subunits of the bacterial ribosome. The highly conserved central loop of domain V of 23S rRNA (nucleotides 2042-2628 in Escherichia coli; the exact position varies by species) of the 50S subunit, which is implicated in peptidyl transferase activity, is known to be important in macrolide interactions and resistance. In this study, we identified an A2302T mutation in the rrnA-23S rRNA gene and an A2281G mutation in the rrnC-23S rRNA gene that were responsible for resistance to erythromycin in the model actinomycete Streptomyces coelicolor A3(2) and its close relative Streptomyces lividans 66, respectively. Interestingly, genetic and phenotypic characterization of the erythromycin-resistant mutants indicated a possibility that under coexistence of the 23S rRNA mutation and mutations in other genes, S. coelicolor A3(2) and S. lividans 66 can produce abundant amounts of the pigmented antibiotics actinorhodin and undecylprodigiosin depending on the combinations of mutations. Herein, we report the unique phenomenon occurring by unexpected characteristics of the 23S rRNA mutations that can affect the emergence of additional mutations probably with an upswing in spontaneous mutations and enrichment in their variations in Streptomyces strains. Further, we discuss a putative mechanism underlying secondary metabolite overproduction by Streptomyces strains with a 23S rRNA mutation conferring erythromycin resistance.

The Effects of Deoxynivalenol (DON) on the Gut Microbiota, Morphology and Immune System of Chicken – A Review

Abstract Feed contamination is a major cause of diseases outbreak in the poultry industry. There is a direct relationship between feeding, the intestinal microbiota and how the immune system responds to disease infestation. Cereals which form the bulk of poultry feed are mostly contaminated by mycotoxins of Fusarium origin. Adequate knowledge of mycotoxins and their effects on animals is necessary. Deoxynivalenol (DON) is a major contaminant of poultry feed. DON has the ability to bind with a large number of eukaryotic ribosomal subunits because of the presence of an epoxide group and these disrupt the activity of peptidyl transferase and the elongation or shortening of peptide chains. Deoxynivalenol has varying effect ranging from acute, overt diseases with high morbidity and death to chronic disease, decreased resistance to pathogens and reduced animal productivity. Deoxynivalenol also impairs the intestinal morphology, nutrient absorption, barrier function, and the innate immune response in chickens. This review highlights the impacts of deoxynivalenol on the immune system, intestinal microbiota composition and the morphology of chicken.

Ribosomal protein eL42 contributes to the catalytic activity of the yeast ribosome at the elongation step of translation

The GGQ minidomain of the ribosomal protein eL42 was previously shown to contact the CCA-arm of P-site bound tRNA in human ribosome, indicating a possible involvement of the protein in the catalytic activity. Here, using Schizosaccharomyces pombe (S. pombe) cells, we demonstrate that the GGQ minidomain and neighboring region of eL42 is critical for the ribosomal function. Mutant eL42 proteins containing amino acid substitutions within or adjacent to the GGQ minidomain failed to complement the function of wild-type eL42, and expression of the mutant eL42 proteins led to severe growth defects. These results suggest that the mutations in eL42 interfere with the ribosomal function in vivo. Furthermore, we show that some of the mutations associated with the conserved GGQ region lead to reduced activities in the poly(Phe) synthesis and/or in the peptidyl transferase reaction with respect to puromycin, as compared with those of the wild-type ribosomes. A pK value of 6.95 was measured for the side chain of Lys-55/Arg-55, which is considerably less than that of a Lys or Arg residue. Altogether, our findings suggest that eL42 contributes to the 80S ribosome's peptidyl transferase activity by promoting the course of the elongation cycle.

Open-Source High-Throughput Phenomics of Bacterial Promoter-Reporter Strains.

Open-source electronics are becoming more prevalent in biological sciences, enabling novel and unique means of data acquisition. Here, we present 3D-printed, open-source tools to acquire fluorescence phenotypes with high temporal resolution. Printed fluorescence imaging boxes (PFIboxes) cost approximately 200US dollars to assemble, can be placed in incubators or hypoxic chambers, and accurately read high-density colony arrays of microorganisms. We demonstrate the utility of PFIboxes using a timecourse gene expression approach, examining globalEscherichia coli promoter activity using a fluorescent reporter library across a diverse panel of 15 antibiotics, each at several concentrations. Many secondary and indirect effects were observed when E.coli was challenged with various drugs, including increased gene expression in carbon metabolism processes. Further, kinetic data acquisition enabled non-destructive time course gene expression, clustering of which revealed patterns of co-expression. In all, PFIboxes provide an open solution to gene expression, for about 2US dollars per treatment condition, including technical replicates.

Antibiotic resistance ABCF proteins reset the peptidyl transferase centre of the ribosome to counter translational arrest.

Several ATPases in the ATP-binding cassette F (ABCF) family confer resistance to macrolides, lincosamides and streptogramins (MLS) antibiotics. MLS are structurally distinct classes, but inhibit a common target: the peptidyl transferase (PTC) active site of the ribosome. Antibiotic resistance (ARE) ABCFs have recently been shown to operate through direct ribosomal protection, but the mechanistic details of this resistance mechanism are lacking. Using a reconstituted translational system, we dissect the molecular mechanism of Staphylococcus haemolyticus VgaALC and Enterococcus faecalis LsaA on the ribosome. We demonstrate that VgaALC is an NTPase that operates as a molecular machine strictly requiring NTP hydrolysis (not just NTP binding) for antibiotic protection. Moreover, when bound to the ribosome in the NTP-bound form, hydrolytically inactive EQ2 ABCF ARE mutants inhibit peptidyl transferase activity, suggesting a direct interaction between the ABCF ARE and the PTC. The likely structural candidate responsible for antibiotic displacement by wild type ABCF AREs, and PTC inhibition by the EQ2 mutant, is the extended inter-ABC domain linker region. Deletion of the linker region renders wild type VgaALC inactive in antibiotic protection and the EQ2 mutant inactive in PTC inhibition.

The force-sensing peptide VemP employs extreme compaction and secondary structure formation to induce ribosomal stalling.

Interaction between the nascent polypeptide chain and the ribosomal exit tunnel can modulate the rate of translation and induce translational arrest to regulate expression of downstream genes. The ribosomal tunnel also provides a protected environment for initial protein folding events. Here, we present a 2.9 Å cryo-electron microscopy structure of a ribosome stalled during translation of the extremely compacted VemP nascent chain. The nascent chain forms two α-helices connected by an α-turn and a loop, enabling a total of 37 amino acids to be observed within the first 50-55 Å of the exit tunnel. The structure reveals how α-helix formation directly within the peptidyltransferase center of the ribosome interferes with aminoacyl-tRNA accommodation, suggesting that during canonical translation, a major role of the exit tunnel is to prevent excessive secondary structure formation that can interfere with the peptidyltransferase activity of the ribosome.

Florfenicol induces more severe hemotoxicity and immunotoxicity than equal doses of chloramphenicol and thiamphenicol in Kunming mice

Amphenicols are effective, broad-spectrum antibiotics that function by inhibiting the peptidyl transferase activity of bacteria, while the drugs can also inhibit mitochondrial protein synthesis in eukaryotes through the same mechanism, which leads to multi-organ toxicity. Some side effects of each drug have been studied, while differences in the severity of the hemotoxicities and immunotoxicities of amphenicols have not been reported. Thus, it is important to identify, evaluate, and compare the potential hemotoxicities and immunotoxicities to guide their proper use in humans and animals, which will guarantee food safety and animal welfare. Ovalbumin-immunized Kunming mice were gavaged daily with amphenicols for seven days. Blood samples were collected for hematology analysis, and measuring anti-ovalbumin antibody levels and serum intereukin-2 concentrations. The bone marrow, spleen and thymus were collected for histopathology and apoptosis analyzes. Bone marrow nucleated cells (BMNCs) and splenocytes were harvested to determine their cell cycle stages and to analyze lymphocyte proliferation. The results demonstrated that amphenicols, especially florfenicol (FLO), induced cell cycle arrest and apoptosis of hematopoietic cells, and it changed the bone marrow hematopoietic microenvironment by decreasing the number of peripheral blood cells. Moreover, amphenicols, especially FLO, induced hypoplasia and atrophy of the spleen and thymus, induced cell cycle arrest, as well as splenocyte apoptosis, and decreased the proliferation and viability of lymphocytes and the humoral and cellular immunity of the treated mice. These results suggest that amphenicols induce hemotoxicity and immunotoxicity to some extent, and that FLO induces more severe toxicity than equal doses of chloramphenicol (CAP) and thiamphenicol (TAP).

Solvent and Water Mediated Structural Variations in Deoxynivalenol and Their Potential Implications on the Disruption of Ribosomal Function.

Fusarium head blight (FHB) is a disease of cereal crops caused by trichothecene producing Fusarium species. Trichothecenes, macrocylicic fungal metabolites composed of three fused rings (A–C) with one epoxide functionality, are a class of mycotoxins known to inhibit protein synthesis in eukaryotic ribosomes. These toxins accumulate in the kernels of infected plants rendering them unsuitable for human and animal consumption. Among the four classes of trichothecenes (A–D) A and B are associated with FHB, where the type B trichothecene deoxynivalenol (DON) is most relevant. While it is known that these toxins inhibit protein synthesis by disrupting peptidyl transferase activity, the exact mechanism of this inhibition is poorly understood. The three-dimensional structures and H-bonding behavior of DON were evaluated using one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy techniques. Comparisons of the NMR structure presented here with the recently reported crystal structure of DON bound in the yeast ribosome reveal insights into the possible toxicity mechanism of this compound. The work described herein identifies a water binding pocket in the core structure of DON, where the 3OH plays an important role in this interaction. These results provide preliminary insights into how substitution at C3 reduces trichothecene toxicity. Further investigations along these lines will provide opportunities to develop trichothecene remediation strategies based on the disruption of water binding interactions with 3OH.

Stall no more at polyproline stretches with the translation elongation factors EF-P and IF-5A.

Synthesis of polyproline proteins leads to translation arrest. To overcome this ribosome stalling effect, bacteria depend on a specialized translation elongation factor P (EF-P), being orthologous and functionally identical to eukaryotic/archaeal elongation factor e/aIF-5A (recently renamed 'EF5'). EF-P binds to the stalled ribosome between the peptidyl-tRNA binding and tRNA-exiting sites, and stimulates peptidyl-transferase activity, thus allowing translation to resume. In their active form, both EF-P and e/aIF-5A are post-translationally modified at a positively charged residue, which protrudes toward the peptidyl-transferase center when bound to the ribosome. While archaeal and eukaryotic IF-5A strictly depend on (deoxy-) hypusination (hypusinylation) of a conserved lysine, bacteria have evolved diverse analogous modification strategies to activate EF-P. In Escherichia coli and Salmonella enterica a lysine is extended by β-lysinylation and subsequently hydroxylated, whereas in Pseudomonas aeruginosa and Shewanella oneidensis an arginine in the equivalent position is rhamnosylated. Inactivation of EF-P, or the corresponding modification systems, reduces not only bacterial fitness, but also impairs virulence. Here, we review the function of EF-P and IF-5A and their unusual posttranslational protein modifications.

Crystallographic characterization of the ribosomal binding site and molecular mechanism of action of Hygromycin A.

Hygromycin A (HygA) binds to the large ribosomal subunit and inhibits its peptidyl transferase (PT) activity. The presented structural and biochemical data indicate that HygA does not interfere with the initial binding of aminoacyl-tRNA to the A site, but prevents its subsequent adjustment such that it fails to act as a substrate in the PT reaction. Structurally we demonstrate that HygA binds within the peptidyl transferase center (PTC) and induces a unique conformation. Specifically in its ribosomal binding site HygA would overlap and clash with aminoacyl-A76 ribose moiety and, therefore, its primary mode of action involves sterically restricting access of the incoming aminoacyl-tRNA to the PTC.

Identification, molecular characterization and expression analysis of RPL24 genes in three Cucurbitaceae family members: cucumber, melon and watermelon

Ribosomal proteins are crucial for the proper growth and development of any organism, including plants. The ribosomal protein L24 (RPL24) is found in the large subunit of the ribosome and is responsible for the stabilization of the peptidyl transferase activity. Although RPL24 gene has been individually identified in different organisms, little is known about the genome-wide survey and expression patterns of R24 genes in Cucurbitaceae family members. We identified seven Cucurbitaceae RPL24 genes from cucumber, melon and watermelon. They were phylogenetically clustered into seven major groups. Gene structure and motif composition are relatively conserved in each group. Three-dimensional homology modelling of RPL24 proteins was performed with higher confidence level. CmRPL24-01 was isolated from melon and characterized at a molecular level. The regulation of ribosomal proteins in melon under drought stress conditions was also studied. The expression of CmRPL24-01 gene increased in melon leaf tissue at 3 h upon polyethylene glycol treatment and showed a gradual induction after 12 h. Our study provided a very useful reference for identification and functional analysis of RPL24 protein members in different plants. In addition, this research indicated a potential usage of ribosomal proteins in response to drought stress.

Reflections on the 20th anniversary of RNA.

Reflections on the 20th anniversary of RNA.