Round Of Elongation Research Articles

Quality control of protein synthesis in the early elongation stage

In the early stage of bacterial translation, peptidyl-tRNAs frequently dissociate from the ribosome (pep-tRNA drop-off) and are recycled by peptidyl-tRNA hydrolase. Here, we establish a highly sensitive method for profiling of pep-tRNAs using mass spectrometry, and successfully detect a large number of nascent peptides from pep-tRNAs accumulated in Escherichia coli pthts strain. Based on molecular mass analysis, we found about 20% of the peptides bear single amino-acid substitutions of the N-terminal sequences of E. coli ORFs. Detailed analysis of individual pep-tRNAs and reporter assay revealed that most of the substitutions take place at the C-terminal drop-off site and that the miscoded pep-tRNAs rarely participate in the next round of elongation but dissociate from the ribosome. These findings suggest that pep-tRNA drop-off is an active mechanism by which the ribosome rejects miscoded pep-tRNAs in the early elongation, thereby contributing to quality control of protein synthesis after peptide bond formation.

Peptidyl transferase center decompaction and structural constraints during early protein elongation on the ribosome

Peptide bond formation on the ribosome requires that aminoacyl-tRNAs and peptidyl-tRNAs are properly positioned on the A site and the P site of the peptidyl transferase center (PTC) so that nucleophilic attack can occur. Here we analyse some constraints associated with the induced-fit mechanism of the PTC, that promotes this positioning through a compaction around the aminoacyl ester orchestrated by U2506. The physical basis of PTC decompaction, that allows the elongated peptidyl-tRNA to free itself from that state and move to the P site of the PTC, is still unclear. From thermodynamics considerations and an analysis of published ribosome structures, the present work highlights the rational of this mechanism, in which the free-energy released by the new peptide bond is used to kick U2506 away from the reaction center. Furthermore, we show the evidence that decompaction is impaired when the nascent peptide is not yet anchored inside the exit tunnel, which may contribute to explain why the first rounds of elongation are inefficient, an issue that has attracted much interest for about two decades. Results in this field are examined in the light of the present analysis and a physico-chemical correlation in the genetic code, which suggest that elementary constraints associated with the size of the side-chain of the amino acids penalize early elongation events.

Self-assembly of a polythymine embedded activatable molecular beacon for one-step quantification of terminal deoxynucleotidyl transferase activity

We describe an isothermal, single-reaction, and one-step method for signal-on quantification of terminal deoxynucleotidyl transferase (TdT) activity based on the periodic elongation and assembly of polythymine embedded activatable molecular beacon (PTA-MB) into DNA nanostructures. PTA-MB is easily designed according to the rule of the conventional molecular beacon (MB) but engineered with a polyT composed loop. Upon exposure to the specific target TdT, the MB is first elongated with an adenine-rich (A-rich) long chain so that it can then act as the anchoring substrate to capture many original PTA-MBs along its strand. Their unfolding contributes to preliminary fluorescence emission. Significantly, the assembled PTA-MBs can also be elongated and hybridized with residual free PTA-MBs for the second round of signal amplification. Accordingly, multiple rounds of elongation, assembly, and activation of initial PTA-MBs can lead to the formation of DNA nanostructures and induce a dramatically enhanced fluorescence signal for qualitative and quantitative evaluation of TdT activity. The final assay indicated a limit of detection (LOD) of 0.042 U mL−1 TdT and showed excellent selectivity for TdT versus other common enzymes. Moreover, the practical applicability was validated by direct/absolute quantification of TdT in real biological specimens and accurate monitoring of the activity of TdT pretreated by low/high temperature and heavy metal ions. These findings demonstrated that this functional PTA-MB and its unique assembly behavior is most likely to promote the study of oligonucleotide probe-based DNA assembly, providing a reliable, convenient, and universal platform for precise and point-of-care monitoring of various biomolecules.

Loss of a single methylation in 23S rRNA delays 50S assembly at multiple late stages and impairs translation initiation and elongation

Ribosome biogenesis is a complex process, and dozens of factors are required to facilitate and regulate the subunit assembly in bacteria. The 2'-O-methylation of U2552 in 23S rRNA by methyltransferase RrmJ is a crucial step in late-stage assembly of the 50S subunit. Its absence results in severe growth defect and marked accumulation of pre50S assembly intermediates. In the present work, we employed cryoelectron microscopy to characterize a set of late-stage pre50S particles isolated from an Escherichia coli ΔrrmJ strain. These assembly intermediates (solved at 3.2 to 3.8 Å resolution) define a collection of late-stage particles on a progressive assembly pathway. Apart from the absence of L16, L35, and L36, major structural differences between these intermediates and the mature 50S subunit are clustered near the peptidyl transferase center, such as H38, H68-71, and H89-93. In addition, the ribosomal A-loop of the mature 50S subunit from ΔrrmJ strain displays large local flexibility on nucleotides next to unmethylated U2552. Fast kinetics-based biochemical assays demonstrate that the ΔrrmJ 50S subunit is only 50% active and two times slower than the WT 50S subunit in rapid subunit association. While the ΔrrmJ 70S ribosomes show no defect in peptide bond formation, peptide release, and ribosome recycling, they translocate with 20% slower rate than the WT ribosomes in each round of elongation. These defects amplify during synthesis of the full-length proteins and cause overall defect in protein synthesis. In conclusion, our data reveal the molecular roles of U2552 methylation in both ribosome biogenesis and protein translation.

Structural and Biochemical Analysis of Protein–Protein Interactions Between the Acyl‐Carrier Protein and Product Template Domain

AbstractIn fungal non‐reducing polyketide synthases (NR‐PKS) the acyl‐carrier protein (ACP) carries the growing polyketide intermediate through iterative rounds of elongation, cyclization and product release. This process occurs through a controlled, yet enigmatic coordination of the ACP with its partner enzymes. The transient nature of ACP interactions with these catalytic domains imposes a major obstacle for investigation of the influence of protein–protein interactions on polyketide product outcome. To further our understanding about how the ACP interacts with the product template (PT) domain that catalyzes polyketide cyclization, we developed the first mechanism‐based crosslinkers for NR‐PKSs. Through in vitro assays, in silico docking and bioinformatics, ACP residues involved in ACP–PT recognition were identified. We used this information to improve ACP compatibility with non‐cognate PT domains, which resulted in the first gain‐of‐function ACP with improved interactions with its partner enzymes. This advance will aid in future combinatorial biosynthesis of new polyketides.

Structural and Biochemical Analysis of Protein-Protein Interactions Between the Acyl-Carrier Protein and Product Template Domain.

In fungal non-reducing polyketide synthases (NR-PKS) the acyl-carrier protein (ACP) carries the growing polyketide intermediate through iterative rounds of elongation, cyclization and product release. This process occurs through a controlled, yet enigmatic coordination of the ACP with its partner enzymes. The transient nature of ACP interactions with these catalytic domains imposes a major obstacle for investigation of the influence of protein-protein interactions on polyketide product outcome. To further our understanding about how the ACP interacts with the product template (PT) domain that catalyzes polyketide cyclization, we developed the first mechanism-based crosslinkers for NR-PKSs. Through in vitro assays, in silico docking and bioinformatics, ACP residues involved in ACP-PT recognition were identified. We used this information to improve ACP compatibility with non-cognate PT domains, which resulted in the first gain-of-function ACP with improved interactions with its partner enzymes. This advance will aid in future combinatorial biosynthesis of new polyketides.

Improved Ribosome-Footprint and mRNA Measurements Provide Insights into Dynamics and Regulation of Yeast Translation

Ribosome-footprint profiling provides genome-wide snapshots of translation, but technical challenges can confound its analysis. Here, we use improved methods to obtain ribosome-footprint profiles and mRNA abundances that more faithfully reflect geneexpression in Saccharomyces cerevisiae. Our results support proposals that both the beginning of coding regions and codons matching rare tRNAs are more slowly translated. They also indicate that emergent polypeptides with as few as three basic residues within a ten-residue window tend to slow translation. With the improved mRNA measurements, the variation attributable to translational control in exponentially growing yeast was less than previously reported, and most of this variation could be predicted with a simple model that considered mRNA abundance, upstream open reading frames, cap-proximal structure and nucleotide composition, and lengths of the coding and 5' UTRs. Collectively, our results provide a framework for executing and interpreting ribosome-profiling studies and reveal key features of translational control in yeast.

Single-Molecule Profiling of Ribosome Translational Phenomena

Translation elongation is a heterogeneous process, involving multiple compositional factors stochastically binding to the ribosome to direct protein synthesis, which in turn regulates the conformation of the ribosome. The rate of translation is often regulated by the underlying messenger RNA (mRNA) sequence and structure. Here, we use single-molecule fluorescence resonant energy transfer (FRET) and colocalization with zero-mode waveguides (ZMWs) to correlate directly ribosome conformations and compositions of thousands of ribosomes simultaneously during multiple rounds of elongation. This allows us to profile global translational rates while delineating mechanistic details of the dynamics with codon resolution. We first studied translation of a canonical mRNA with uniform translation rates to establish the tight interplay between compositional factors and conformational dynamics of the ribosome during elongation. We then determined how mRNA sequences and structures, such as hairpins and possible ribosome-mRNA base pairing, as expressed in the the dnaX −1 frameshifting sequence, perturb the basal elongation process. Our results show how mRNAs can modulate and uncouple ribosomal conformational and compositional dynamics.

Resistance to AFN-1252 Arises from Missense Mutations in Staphylococcus aureus Enoyl-acyl Carrier Protein Reductase (FabI)

AFN-1252 is a potent antibiotic against Staphylococcus aureus that targets the enoyl-acyl carrier protein reductase (FabI). A thorough screen for AFN-1252-resistant strains was undertaken to identify the spectrum of mechanisms for acquired resistance. A missense mutation in fabI predicted to encode FabI(M99T) was isolated 49 times, and a single isolate was predicted to encode FabI(Y147H). AFN-1252 only bound to the NADPH form of FabI, and the close interactions between the drug and Met-99 and Tyr-147 explained how the mutations would result in resistant enzymes. The clone expressing FabI(Y147H) had a pronounced growth defect that was rescued by exogenous fatty acid supplementation, and the purified protein had less than 5% of the enzymatic activity of FabI. FabI(Y147F) was also catalytically defective but retained its sensitivity to AFN-1252, illustrating the importance of the conserved Tyr-147 hydroxyl group in FabI function. The strains expressing FabI(M99T) exhibited normal growth, and the biochemical properties of the purified protein were indistinguishable from those of FabI. The AFN-1252 Ki(app) increased from 4 nm in FabI to 69 nm in FabI(M99T), accounting for the increased resistance of the corresponding mutant strain. The low activity of FabI(Y147H) precluded an accurate Ki measurement. The strain expressing FabI(Y147H) was also resistant to triclosan; however, the strain expressing FabI(M99T) was more susceptible. Strains with higher levels of AFN-1252 resistance were not obtained. The AFN-1252-resistant strains remained sensitive to submicromolar concentrations of AFN-1252, which blocked growth through inhibition of fatty acid biosynthesis at the FabI step.

From models to pathogens: how much have we learned aboutStreptococcus pneumoniaecell division?

Streptococcus pneumoniae is an oval-shaped Gram-positive coccus that lives in intimate association with its human host, both as a commensal and pathogen. The seriousness of pneumococcal infections and the spread of multi-drug resistant strains call for new lines of intervention. Bacterial cell division is an attractive target to develop antimicrobial drugs. This review discusses the recent advances in understanding S. pneumoniae growth and division, in comparison with the best studied rod-shaped models, Escherichia coli and Bacillus subtilis. To maintain their shape, these bacteria propagate by peripheral and septal peptidoglycan synthesis, involving proteins that assemble into distinct complexes called the elongasome and the divisome, respectively. Many of these proteins are conserved in S. pneumoniae, supporting the notion that the ovococcal shape is also achieved by rounds of elongation and division. Importantly, S. pneumoniae and close relatives with similar morphology differ in several aspects from the model rods. Overall, the data support a model in which a single large machinery, containing both the peripheral and septal peptidoglycan synthesis complexes, assembles at midcell and governs growth and division. The mechanisms generating the ovococcal or coccal shape in lactic-acid bacteria have likely evolved by gene reduction from a rod-shaped ancestor of the same group.

Coordinated conformational and compositional dynamics drive ribosome translocation

During translation elongation, the compositional factors, elongation factor G (EF-G; encoded by fusA) and transfer RNA (tRNA), alternately bind to the ribosome to direct protein synthesis, in turn regulating the conformation of the ribosome. Here, we use single-molecule fluorescence with zero-mode waveguides to correlate directly ribosome conformations and compositions during multiple rounds of elongation at high factor concentrations in Escherichia coli. Our results show that EF-G-GTP continuously samples both rotational sates of the ribosome, binding with higher affinity to the rotated state. Upon successful accommodation into the rotated ribosome, the EF-G-ribosome complex evolves through several rate-limiting conformational changes and the hydrolysis of GTP, which results in a transition back to the non-rotated state, in turn driving translocation and facilitating both EF-G-GDP and E-site tRNA release. These experiments highlight the power of tracking single-molecule conformation and composition simultaneously in real-time.

Ultra-sensitive detection of Ag+ ions based on Ag+-assisted isothermal exponential degradation reaction

Ag+ ions are greatly toxic to a lot of algae, fungi, viruses and bacteria, which can also induce harmful side-effects to environments and human health. Herein we report an ultra-sensitive method for the selective detection of Ag+ ions with electrochemical technique based on Ag+-assisted isothermal exponential degradation reaction. In the presence of Ag+, mismatched trigger DNA can transiently bind to template DNA immobilized on an electrode surface through the formation of C–Ag+–C base pair, which then initiates the isothermal exponential degradation reaction. As a result, the mismatched trigger DNA may melt off the cleaved template DNA to trigger rounds of elongation and cutting. After the cyclic degradation reactions, removal of the template DNA immobilized on the electrode surface can be efficiently monitored by using electrochemical technique to show the status of the electrode surface, which can be then used to determine the presence of Ag+. Further studies reveal that the proposed method can be ultra-sensitive to detect Ag+ at a picomolar level. The selectivity of the detection can also be satisfactory, thus the proposed method for the Ag+ ions detection may be potentially useful in the future.

Distortion of tRNA upon Near-cognate Codon Recognition on the Ribosome

The accurate decoding of the genetic information by the ribosome relies on the communication between the decoding center of the ribosome, where the tRNA anticodon interacts with the codon, and the GTPase center of EF-Tu, where GTP hydrolysis takes place. In the A/T state of decoding, the tRNA undergoes a large conformational change that results in a more open, distorted tRNA structure. Here we use a real-time transient fluorescence quenching approach to monitor the timing and the extent of the tRNA distortion upon reading cognate or near-cognate codons. The tRNA is distorted upon codon recognition and remains in that conformation until the tRNA is released from EF-Tu, although the extent of distortion gradually changes upon transition from the pre- to the post-hydrolysis steps of decoding. The timing and extent of the rearrangement is similar on cognate and near-cognate codons, suggesting that the tRNA distortion alone does not provide a specific switch for the preferential activation of GTP hydrolysis on the cognate codon. Thus, although the tRNA plays an active role in signal transmission between the decoding and GTPase centers, other regulators of signaling must be involved.

Following the intersubunit conformation of the ribosome during translation in real time

We report the direct observation of conformational rearrangements of the ribosome during multiple rounds of elongation. Using single-molecule fluorescence resonance energy transfer, we monitor the intersubunit conformation of the ribosome – in real time – as it proceeds from codon to codon. During each elongation cycle, the ribosome unlocks upon peptide bond formation, followed by reversion to the locked state upon translocation onto the next codon. Our data reveal both the specific and cumulative effects of antibiotics on individual steps of translation, and uncover the processivity of the ribosome as it elongates. Our approach interrogates the precise molecular events occurring at each codon of the mRNA within the full context of ongoing translation.

Irreversible chemical steps control intersubunit dynamics during translation

The ribosome, a two-subunit macromolecular machine, deciphers the genetic code and catalyzes peptide bond formation. Dynamic rotational movement between ribosomal subunits is likely required for efficient and accurate protein synthesis, but direct observation of intersubunit dynamics has been obscured by the repetitive, multistep nature of translation. Here, we report a collection of single-molecule fluorescence resonance energy transfer assays that reveal a ribosomal intersubunit conformational cycle in real time during initiation and the first round of elongation. After subunit joining and delivery of correct aminoacyl-tRNA to the ribosome, peptide bond formation results in a rapid conformational change, consistent with the counterclockwise rotation of the 30S subunit with respect to the 50S subunit implied by prior structural and biochemical studies. Subsequent binding of elongation factor G and GTP hydrolysis results in a clockwise rotation of the 30S subunit relative to the 50S subunit, preparing the ribosome for the next round of tRNA selection and peptide bond formation. The ribosome thus harnesses the free energy of irreversible peptidyl transfer and GTP hydrolysis to surmount activation barriers to large-scale conformational changes during translation. Intersubunit rotation is likely a requirement for the concerted movement of tRNA and mRNA substrates during translocation.

Cryptosporidium parvum Long-Chain Fatty Acid Elongase

We report the presence of a new fatty acyl coenzyme A (acyl-CoA) elongation system in Cryptosporidium and the functional characterization of the key enzyme, a single long-chain fatty acid elongase (LCE), in this parasite. This enzyme contains conserved motifs and predicted transmembrane domains characteristic to the elongase family and is placed within the ELO6 family specific for saturated substrates. CpLCE1 gene transcripts are present at all life cycle stages, but the levels are highest in free sporozoites and in stages at 36 h and 60 h postinfection that typically contain free merozoites. Immunostaining revealed localization to the outer surface of sporozoites and to the parasitophorous vacuolar membrane. Recombinant CpLCE1 displayed allosteric kinetics towards malonyl-CoA and palmitoyl-CoA and Michaelis-Menten kinetics towards NADPH. Myristoyl-CoA (C14:0) and palmitoyl-CoA (C16:0) display the highest activity when used as substrates, and only one round of elongation occurs. CpLCE1 is fairly resistant to cerulenin, an inhibitor for both type I and II fatty acid synthases (i.e., maximum inhibitions of 20.5% and 32.7% were observed when C16:0 and C14:0 were used as substrates, respectively). These observations ultimately validate the function of CpLCE1.

Glucosinolate biosynthesis: demonstration and characterization of the condensing enzyme of the chain elongation cycle in Eruca sativa

Glucosinolates are a group of sulfur-rich thioglucoside natural products common in the Brassicaceae and related plant families. The first phase in the formation of many glucosinolates involves the chain extension of the amino acid methionine. Additional methylene groups are inserted into the side chain of methionine by a three-step elongation cycle involving 2-oxo acid intermediates. This investigation demonstrated the first step of this chain elongation cycle in a partially-purified preparation from arugula ( Eruca sativa). The 2-oxo acid derived from methionine, 4-methylthio-2-oxobutanoic acid, was shown to condense with acetyl-CoA to form 2-(2′-methylthioethyl)malate. The catalyst, designated as a 2-(ω-methylthioalkyl)malate synthase, belongs to a family of enzymes that mediate the condensation of acyl-CoAs with 2-oxo acids, including citrate synthase of the citric acid cycle, and 2-isopropylmalate synthase of leucine biosynthesis. The 2-(ω-methylthioalkyl)malate synthase studied here shares properties with other enzymes of this class, but appears chromatographically distinct and is found only in extracts of plant species producing glucosinolates from chain-elongated methionine derivatives. Although the principal glucosinolates of arugula are formed from methionine that has undergone two rounds of chain elongation to form dihomomethionine, studies with substrates and substrate analogs of different chain lengths showed that the isolated enzyme is responsible only for the condensation step of the first round of elongation.

Mycolic acid biosynthesis and enzymic characterization of the beta-ketoacyl-ACP synthase A-condensing enzyme from Mycobacterium tuberculosis.

Mycolic acids consist of long-chain alpha-alkyl-beta-hydroxy fatty acids that are produced by successive rounds of elongation catalysed by a type II fatty acid synthase (FAS-II). A key feature in the elongation process is the condensation of a two-carbon unit from malonyl-acyl-carrier protein (ACP) to a growing acyl-ACP chain catalysed by a beta-ketoacyl-ACP synthase (Kas). In the present study, we provide evidence that kasA from Mycobacterium tuberculosis encodes an enzyme that elongates in vivo the meromycolate chain, in both Mycobacterium smegmatis and Mycobacterium chelonae. We demonstrate that KasA belongs to the FAS-II system, which utilizes primarily palmitoyl-ACP rather than short-chain acyl-ACP primers. Furthermore, in an in vitro condensing assay using purified recombinant KasA, palmitoyl-AcpM and malonyl-AcpM, KasA was found to express Kas activity. Also, mutated KasA proteins, with mutation of Cys(171), His(311), Lys(340) and His(345) to Ala abrogated the condensation activity of KasA in vitro completely. Finally, purified KasA was highly sensitive to cerulenin, a well-known inhibitor of Kas, which may lead to the development of novel anti-mycobacterial drugs targeting KasA.

Isolation of transcriptionally active chromatin from human breast cancer cells using Sulfolink coupling gel chromatography.

The process of transcription unfolds the nucleosome. The unfolded nucleosome structure will be maintained as long as the histones are in a highly acetylated state. Typically the cysteine residue at position 110 of histone H3 is buried in the interior of the nucleosome. However, the transcribed unfolded nucleosome has its H3 cysteine exposed, offering a tag to isolate and study transcribed nucleosomes. In this study, we applied Sulfolink Coupling Gel chromatography to isolate unfolded nucleosomes from estrogen dependent human cancer T5 cells. Inhibition of histone deacetylase activity did not enhance the yield of unfolded nucleosomes from these cells. We show that the estrogen receptor and c-myc transcribed DNA sequences are associated with unfolded nucleosomes. In chromatin immunoprecipitation (ChIPs) assays, we found that the coding regions of the estrogen receptor and c-myc genes are bound to highly acetylated H3 and H4 in cultured T5 Cells. We conclude that in cultured T5 breast cancer cells H3 and H4 are in highly acetylated states maintaining the unfolded structure of the transcribed nucleosome and facilitating subsequent rounds of elongation.

Different modes of de novo telomere formation by plant telomerases.

The telomerase reverse transcriptase can recognize broken chromosome ends and add new telomeres de novo in a reaction termed "chromosome healing". Here we investigate new telomere formation in vitro by telomerases from a variety of flowering plant species. Comparing the electrophoretic mobilities and nucleotide sequences of the products, we uncovered three different modes of new telomere formation. The soybean telomerase, designated a Class I enzyme, only elongated DNA primers ending in telomeric nucleotides. Arabidopsis and maize telomerases, designated Class II enzymes, efficiently extended completely non-telomeric sequences by positioning the 3' terminus at a preferred site on the RNA template. Silene latifolia and sorghum telomerases constituted class III enzymes that elongated non-telomeric DNA primers by annealing them at alternative sites on the RNA template. For all enzymes, errors were prevalent during synthesis of the first two repeats, likely reflecting lateral instability of the primer 3' terminus on the template during the initial rounds of elongation. Class III telomerases, however, were five- to 13-fold more error prone than class II, generating more mistakes in distal repeats added to the primers. This remarkable variability in enzyme-DNA interactions among plant telomerases does not reflect phylogenetic relationships, and therefore implies that the telomerase active site can evolve rapidly.