An Alternative Role of RluD in the Fidelity of Translation Initiation in Escherichia coli

The fidelity of initiator tRNA (i-tRNA) selection in the ribosomal P-site is a key step in translation initiation. The highly conserved three consecutive G:C base pairs (3GC pairs) in the i-tRNA anticodon stem play a crucial role in its selective binding in the P-site. Mutations in the 3GC pairs (3GC mutant) render the i-tRNA inactive in initiation. Here, we show that a mutation (E265K) in the unique C-terminal tail domain of RluD, a large ribosomal subunit pseudouridine synthase, results in compromised fidelity of initiation and allows initiation with the 3GC mutant i-tRNA. RluD modifies the uridine residues in H69 to pseudouridines. However, the role of its C-terminal tail domain remained unknown. The E265K mutation does not diminish the pseudouridine synthase activity of RluD, or the growth phenotype of Escherichia coli, or cause any detectable defects in the ribosomal assembly in our assays. However, in our in vivo analyses, we observed that the E265K mutation resulted in increased retention of the ribosome binding factor A (RbfA) on 30S suggesting a new role of RluD in contributing to RbfA release, a function which may be attributed to its (RluD) C-terminal tail domain. The studies also reveal that deficiency of RbfA release from 30S compromises the fidelity of i-tRNA selection in the ribosomal P-site.     

Molecular insights into protein synthesis with proline residues

Proline is an amino acid with a unique cyclic structure that facilitates the folding of many proteins, but also impedes the rate of peptide bond formation by the ribosome. As a ribosome substrate, proline reacts markedly slower when compared with other amino acids both as a donor and as an acceptor of the nascent peptide. Furthermore, synthesis of peptides with consecutive proline residues triggers ribosome stalling. Here, we report crystal structures of the eukaryotic ribosome bound to analogs of mono‐ and diprolyl‐tRNAs. These structures provide a high‐resolution insight into unique properties of proline as a ribosome substrate. They show that the cyclic structure of proline residue prevents proline positioning in the amino acid binding pocket and affects the nascent peptide chain position in the ribosomal peptide exit tunnel. These observations extend current knowledge of the protein synthesis mechanism. They also revise an old dogma that amino acids bind the ribosomal active site in a uniform way by showing that proline has a binding mode distinct from other amino acids.     

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.     

Fidelity of Initiation of Protein Synthesis After Premature Chain Termination in Polarity Mutants

beta-Isopropylmalate dehydrogenase, the product of the second cistron of the leucine operon in Salmonella typhimurium, produced by strains bearing nonsense or frameshift mutations in the first cistron of the operon was shown to be homogeneous as judged by electrophoretic and immunological techniques. Amino terminal analyses suggest that the enzyme produced by the mutant strains is identical with the wild-type enzyme. This view is supported by the observation that a nonsense mutant strain beta-isopropylmalate dehydrogenase copurifies with the wild-type enzyme. The results suggest that the uncoupling of normal chain termination and reinitiation does not interfere with the fidelity of subsequent polypeptide chain initiation in a polycistronic messenger ribonucleic acid.     

Modulating the activity of the peptidyl transferase center of the ribosome

The peptidyl transferase (PT) center of the ribosome catalyzes two nucleophilic reactions, peptide bond formation between aminoacylated tRNA substrates and, together with release factor, peptide release. Structure and function of the PT center are modulated by binding of aminoacyl-tRNA or release factor, thus providing the basis for the specificity of catalysis. Another way by which the function of the PT center is controlled is signaling from the peptide exit tunnel. The SecM nascent peptide induces ribosome stalling, presumably by inhibition of peptide bond formation. Similarly, the release factor-induced hydrolytic activity of the PT center can be suppressed by the TnaC nascent peptide contained in the exit tunnel. Thus, local and long-range conformational rearrangements can lead to changes in the reaction specificity and catalytic activity of the PT center.     

真核翻译起始因子5A在蛋白质合成中的功能及机制

在蛋白质合成过程中,除核糖体、氨酰 tRNA和mRNA外,还有多种翻译因子参与其中。真核翻译起始因子5A（eukaryotic translation initiation factor 5A, eIF5A）是维持细胞活性必不可少的翻译因子,在进化上高度保守。eIF5A是真核细胞中唯一含有羟腐胺赖氨酸（hypusine）的蛋白质,该翻译后修饰对eIF5A的活性至关重要。1978年,人们首次鉴定出eIF5A,认为它在翻译起始阶段促进第1个肽键的形成。直到2013年才证实它主要在翻译延伸阶段调控含多聚脯氨酸基序蛋白质的翻译。在经过四十多年研究后,人们对eIF5A的功能有了新的认识。近期基于核糖体图谱数据的分析表明,eIF5A能够缓解翻译延伸过程中核糖体在多种基序处的停滞,并不局限于多聚脯氨酸基序,并且它还能够通过促进肽链的释放增强翻译终止。此外,eIF5A还可以通过调控某些蛋白质的翻译,间接影响细胞内的各种生命活动。本文综述了eIF5A的多种翻译后修饰、在蛋白质合成和细胞自噬过程中的调控作用以及与人类疾病的关系,并与细菌及古细菌中的同源蛋白质进行了比较,探讨了该因子在进化中的保守性,以期为相关领域的研究提供一定的理论基础。     

The C-Terminal Portion of an Archaeal Toxin,aRelE, Plays a Crucial Role in Protein Synthesis Inhibition

Mutations of amino acids in the C-terminal region of an archaeal toxin, aRelE, from Pyrococcus horikoshii were characterized with respect to protein synthesis inhibitory activity and 70S ribosome-binding activity. The results suggest that basic residues at the C-terminal region in aRelE play a crucial role both in 70S ribosome binding and in protein synthesis inhibition activities.     

Activities of the peptidyl transferase center of ribosomes lacking protein L27

The ribosome is the molecular machine responsible for protein synthesis in all living organisms. Its catalytic core, the peptidyl transferase center (PTC), is built of rRNA, although several proteins reach close to the inner rRNA shell. In the Escherichia coli ribosome, the flexible N-terminal tail of the ribosomal protein L27 contacts the A- and P-site tRNA. Based on computer simulations of the PTC and on previous biochemical evidence, the N-terminal α-amino group of L27 was suggested to take part in the peptidyl-transfer reaction. However, the contribution of this group to catalysis has not been tested experimentally. Here we investigate the role of L27 in peptide-bond formation using fast kinetics approaches. We show that the rate of peptide-bond formation at physiological pH, both with aminoacyl-tRNA or with the substrate analog puromycin, is independent of the presence of L27; furthermore, translation of natural mRNAs is only marginally affected in the absence of L27. The pH dependence of the puromycin reaction is unaltered in the absence of L27, indicating that the N-terminal α-amine is not the ionizing group taking part in catalysis. Likewise, L27 is not required for the peptidyl-tRNA hydrolysis during termination. Thus, apart from the known effect on subunit association, which most likely explains the phenotype of the deletion strains, L27 does not appear to be a key player in the core mechanism of peptide-bond formation on the ribosome.     

Peptidyl-CCA deacylation on the ribosome promoted by induced fit and the O3'-hydroxyl group of A76 of the unacylated A-site tRNA

The last step in ribosome-catalyzed protein synthesis is the hydrolytic release of the newly formed polypeptide from the P-site bound tRNA. Hydrolysis of the ester link of the peptidyl-tRNA is stimulated normally by the binding of release factors (RFs). However, an unacylated tRNA or just CCA binding to the ribosomal A site can also stimulate deacylation under some nonphysiological conditions. Although the sequence of events is well described by biochemical studies, the structural basis of the mechanism underlying this process is not well understood. Two new structures of the large ribosomal subunit of Haloarcula marismortui complexed with a peptidyl-tRNA analog in the P site and two oligonucleotide mimics of unacylated tRNA, CCA and CA, in the A site show that the binding of either CA or CCA induces a very similar conformational change in the peptidyl-transferase center as induced by aminoacyl-CCA. However, only CCA positions a water molecule appropriately to attack the carbonyl carbon of the peptidyl-tRNA and stabilizes the proper orientation of the ester link for hydrolysis. We, thus, conclude that both the ability of the O3′-hydroxyl group of the A-site A76 to position the water and the A-site CCA induced conformational change of the PTC are critical for the catalysis of the deacylation of the peptidyl-tRNA by CCA, and perhaps, an analogous mechanism is used by RFs.     

蛋白质翻译过程中的忠实性机制

蛋白质合成的忠实性对细胞活力非常重要,否则会干扰细胞的生理过程,甚至导致疾病. 生物已经进化出多种机制以维持翻译的准确性,包括底物选择、校对和转肽后的质量控制机制.这些机制在氨基酸活化、翻译起始、延伸和终止等不同阶段发挥作用. 现在,对蛋白质合成的研究已经延伸到了其它领域,如病原体致病机制、耐药性,以及药物开发等. 本文主要综述了蛋白质合成起始、延伸和终止过程的忠实性机制,以及mRNA的质量控制方式,并对相关研究在抗生素药物及药物靶点开发方面的应用前景做了展望.     

蛋白质生物合成的第四步：翻译终止后复合物的解体

蛋白质合成过程一般被归纳为由合成的起始、肽链的延伸和合成的终止组成的三步曲 . 然而,随着对核糖体再循环因子 (ribosome recycling factor , RRF) 在蛋白质合成过程中作用的深入研究,人们提出了蛋白质生物合成应是四步曲, 这第四步就是翻译终止后核糖体复合物的解体 , 也就是通常说的核糖体循环再利用 . 简要地介绍了翻译终止后复合物解体的可能机制：核糖体再循环因子和蛋白质合成延伸因子 G 在核糖体上协同作用催化这一过程的完成 .     

Effect of spermine on peptide-bond formation,catalyzed by ribosomal peptidyltransferase

The effect of spermine on the binding of AcPhe-tRNA to poly(U)-programmed ribosomes (step 1) and on the puromycin reaction (step 2) has been studied in a cell-free system, derived from E. coli.In the absence of ribosomal wash (FWR fraction) and at suboptimal concentration of Mg⁺⁺ (6 mM), spermine stimulated the binding of AcPhe-tRNA at least five fold, while at 10 mM Mg⁺⁺ there was a three fold stimulation. The above stimulatory effect was decreased at 6 mM Mg⁺⁺, or was abolished at 10 mM Mg⁺⁺ by the presence of FWR during the binding. Beside the stimulatory effect, spermine enhanced the stability of initiation complex AcPhe-tRNA-poly(U)-ribosome.In step 2, spermine affected the final degree of puromycin reaction and the activity status of peptidyltransferase. Both stimulatory and inhibitory effects have been observed, depending on the experimental conditions followed during the binding of the donor and during the peptide bond formation.     

Effect of Exercise on Tissue Protein Synthesis in Rats

The effect of exercise on the protein metabolism in skeletal muscles (gastrocnemius and soleus), liver and small intestine was investigated in rats. Treadmill treatment for 7 d resulted in atrophy of the liver and small intestine, which was associated with a reduction in protein content. The rates of protein synthesis in the liver and small intestine were significantly suppressed in rats subjected to exercise. The change in protein synthesis in the visceral organs was mediated by the change in RNA activity (protein synthesis per unit RNA) but not by the change in RNA concentration. The tissue weight and the rate of protein synthesis in the gastrocnemius and soleus muscles were not affected by exercise. The results suggest that these changes in protein synthesis in the liver and small intestine may explain, at least partly, the atrophy of these organs which was observed after 7 d of exercise.     

Depression of Neuronal Protein Synthesis Initiation by Protein Tyrosine Kinase Inhibitors

Abstract— Growth factors stimulate cellular protein synthesis, but the intracellular signaling mechanisms that regulate initiation of mRNA translation in neurons have not been clarified. A rate-limiting step in the initiation of protein synthesis is the formation of the ternary complex among GTP, eukaryotic initiation factor 2 (elF-2), and the initiator tRNA. Here we report that genistein, a specific tyrosine kinase inhibitor, decreases tyrosine kinase activity and the content of phosphotyrosine proteins in cultured primary cortical neurons. Genistein inhibits protein synthesis by >80% in a dose-dependent manner (10–80 μg/ml) and concurrently decreases ternary complex formation by 60%. At the doses investigated, genistein depresses tyrosine kinase activity and concomitantly stimulates PKC activity. We propose that a protein tyrosine kinase participates in the initiation of protein synthesis in neurons, by affecting the activity of elF-2 directly or through a protein kinase cascade.     

Distinct functional classes of ram mutations in 16S rRNA

During decoding, the ribosome selects the correct (cognate) aminoacyl-tRNA (aa-tRNA) from a large pool of incorrect aa-tRNAs through a two-stage mechanism. In the initial selection stage, aa-tRNA is delivered to the ribosome as part of a ternary complex with elongation factor EF-Tu and GTP. Interactions between codon and anticodon lead to activation of the GTPase domain of EF-Tu and GTP hydrolysis. Then, in the proofreading stage, aa-tRNA is released from EF-Tu and either moves fully into the A/A site (a step termed “accommodation”) or dissociates from the ribosome. Cognate codon-anticodon pairing not only stabilizes aa-tRNA at both stages of decoding but also stimulates GTP hydrolysis and accommodation, allowing the process to be both accurate and fast. In previous work, we isolated a number of ribosomal ambiguity (ram) mutations in 16S rRNA, implicating particular regions of the ribosome in the mechanism of decoding. Here, we analyze a representative subset of these mutations with respect to initial selection, proofreading, RF2-dependent termination, and overall miscoding in various contexts. We find that mutations that disrupt inter-subunit bridge B8 increase miscoding in a general way, causing defects in both initial selection and proofreading. Mutations in or near the A site behave differently, increasing miscoding in a codon-anticodon-dependent manner. These latter mutations may create spurious favorable interactions in the A site for certain near-cognate aa-tRNAs, providing an explanation for their context-dependent phenotypes in the cell.     

Regioselective Disulfide Solid Phase Synthesis, Chemical Characterization and In Vitro Receptor Binding Activity of Equine Relaxin

In the equine industry, pregnancy loss during the third trimester constitutes a large percentage of fetal and neonatal mortality and represents a major financial loss and time investment for the breeder. Early identification of placental insufficiency would, in some cases, make it possible to sustain the pregnancy through medical intervention. Recent work suggests that relaxin is a valuable clinical tool for diagnosing placental insufficiency and monitoring treatment efficacy in mares. Relaxin is a polypeptide member of the insulin superfamily that consists of a two-chain structure and three disulfide bonds in a disposition identical to that of insulin. It is typically produced in the ovary during pregnancy and has primary roles in maintaining mammalian pregnancy and facilitating the delivery of the young via remodelling of the reproductive tract. The placenta is the primary source of relaxin in the mare during pregnancy. Its primary structure has been determined and shown to be the smallest of the known mammalian relaxins. It consists of a 20 residue A-chain and a 28-residue B-chain. To undertake detailed biophysical and biological characterization of the peptide, its chemical synthesis was undertaken using regioselective disulfide formation methods. The synthetic equine relaxin showed typical α-helical structure under physiological conditions. The peptide was found to bind to the relaxin receptor, LGR7, in vitro, and its binding affinity was found to be higher than that of the “gold standard”, porcine relaxin, and similar to that of the human relaxin-2 (H2 relaxin).     

Initiation of ribosome degradation during starvation in Escherichia coli

Ribosomes are known to be degraded under conditions of nutrient limitation. However, the mechanism by which a normally stable ribosome becomes a substrate for the degradation machinery has remained elusive. Here, we present in vitro and in vivo data demonstrating that free ribosome subunits are the actual targets of the degradative enzymes, whereas 70S particles are protected from such degradation. Conditions that increase the formation of subunits both in vitro and in vivo lead to enhanced degradation, while conditions favoring the presence of intact 70S ribosomes prevent or reduce breakdown. Thus, the simple formation of free 50S and 30S subunits is sufficient to serve as the initiation mechanism that allows endoribonuclease cleavage and subsequent ribosome breakdown.