Aminoacylation of tRNA 2′- or 3′-hydroxyl by phosphoseryl- and pyrrolysyl-tRNA synthetases

Class I and II aminoacyl-tRNA synthetases (AARSs) attach amino acids to the 2′- and 3′-OH of the tRNA terminal adenosine, respectively. One exception is phenylalanyl-tRNA synthetase (PheRS), which belongs to Class II but attaches phenylalanine to the 2′-OH. Here we show that two Class II AARSs, O-phosphoseryl- (SepRS) and pyrrolysyl-tRNA (PylRS) synthetases, aminoacylate the 2′- and 3′-OH, respectively. Structure-based-phylogenetic analysis reveals that SepRS is more closely related to PheRS than PylRS, suggesting that the idiosyncratic feature of 2′-OH acylation evolved after the split between PheRS and PylRS. Our work completes the understanding of tRNA aminoacylation positions for the 22 natural AARSs.     

Source of amino acids for tRNA acylation in growing chicks

Summary Specific radioactivity in three amino acid compartments was examined in broiler chicks following a flooding dose of leucine or phenylalanine. In general, specific radioactivity of leucine and phenylalanine in deproteinated plasma (SA_e) and tissue (SA_i) compartments, exceeded that in acylated-tRNA (SA_t). In most tissues, SA_e and SA_i rapidly reached a similar peak level by 5 min followed by a slow decline for the next 30 minutes. Many tissues (eg. GI tract, liver, skin, and thigh) failed to maintain equilibrium between SA_e and SA_i over time. More metabolically active tissues, such as GI and liver had the greatest differences between these compartments. The difference between SA_e and SA_i for both leucine and phenylalanine were due to SA_i decreasing faster than SA_e, indicating dilution with unlabelled amino acids from proteolysis. Plasma and tissue specific radioactivity overestimated tRNA specific radioactivity by as much as 5 and 2.8 fold using leucine or 2.7 and 1.4 fold using phenylalanine, respectively. These data suggest that intracellular compartmentation of protein metabolism and the coupling of protein degradation and synthesis occur, in vivo.     

Kinetic partitioning between synthetic and editing pathways in class I aminoacyl-tRNA synthetases occurs at both pre-transfer and post-transfer hydrolytic steps

Comprehensive steady-state and transient kinetic studies of the synthetic and editing activities of Escherichia coli leucyl-tRNA synthetase (LeuRS) demonstrate that the enzyme depends almost entirely on post-transfer editing to endow the cell with specificity against incorporation of norvaline into protein. Among the three class I tRNA synthetases possessing a dedicated post-transfer editing domain (connective peptide 1; CP1 domain), LeuRS resembles valyl-tRNA synthetase in its reliance on post-transfer editing, whereas isoleucyl-tRNA synthetase differs in retaining a distinct tRNA-dependent synthetic site pre-transfer editing activity to clear noncognate amino acids before misacylation. Further characterization of the post-transfer editing activity in LeuRS by single-turnover kinetics demonstrates that the rate-limiting step is dissociation of deacylated tRNA and/or amino acid product and highlights the critical role of a conserved aspartate residue in mediating the first-order hydrolytic steps on the enzyme. Parallel analyses of adenylate and aminoacyl-tRNA formation reactions by wild-type and mutant LeuRS demonstrate that the efficiency of post-transfer editing is controlled by kinetic partitioning between hydrolysis and dissociation of misacylated tRNA and shows that trans editing after rebinding is a competent kinetic pathway. Together with prior analyses of isoleucyl-tRNA synthetase and valyl-tRNA synthetase, these experiments provide the basis for a comprehensive model of editing by class I tRNA synthetases, in which kinetic partitioning plays an essential role at both pre-transfer and post-transfer steps.     

A new mechanism of post-transfer editing by aminoacyl-tRNA synthetases: catalysis of hydrolytic reaction by bacterial-type prolyl-tRNA synthetase

Aminoacyl tRNA synthetases are enzymes that specifically attach amino acids to cognate tRNAs for use in the ribosomal stage of translation. For many aminoacyl tRNA synthetases, the required level of amino acid specificity is achieved either by specific hydrolysis of misactivated aminoacyl-adenylate intermediate (pre-transfer editing) or by hydrolysis of the mischarged aminoacyl-tRNA (post-transfer editing). To investigate the mechanism of post-transfer editing of alanine by prolyl-tRNA synthetase from the pathogenic bacteria Enterococcus faecalis, we used molecular modeling, molecular dynamic simulations, quantum mechanical (QM) calculations, site-directed mutagenesis of the enzyme, and tRNA modification. The results support a new tRNA-assisted mechanism of hydrolysis of misacylated Ala-tRNA^Pro. The most important functional element of this catalytic mechanism is the 2′-OH group of the terminal adenosine 76 of Ala-tRNA^Pro, which forms an intramolecular hydrogen bond with the carbonyl group of the alanine residue, strongly facilitating hydrolysis. Hydrolysis was shown by QM methods to proceed via a general acid-base catalysis mechanism involving two functionally distinct water molecules. The transition state of the reaction was identified. Amino acid residues of the editing active site participate in the coordination of substrate and both attacking and assisting water molecules, performing the proton transfer to the 3′-O atom of A76.     

tRNA丰度与基因表达的关系

tRNA作为蛋白质合成过程中重要参与成分之一 ,其种类和含量的多少会在一定程度上直接或间接地影响到基因表达的速率和时空性 ,是生物在长期进化过程中调节和控制基因表达的主要手段之一。对tRNA丰度与基因表达的关系进行了综述 ,并指出其在基因工程中的重要地位。     

Impact of separating amino acids between plasma, extracellular and intracellular compartments on estimating protein synthesis in rodents

Summary. Three models representing different separations of amino acid sources were used to simulate experimental specific radioactivity data and to predict protein fractional synthesis rate (FSR). Data were from a pulse dose of ¹⁴C-U Leu given to a non-growing 20 g mouse and a flooding dose of ³H Phe given to a non-growing 200 g rat. Protein synthesis rates estimated using the combined extracellular and intracellular (Ec + Ic) source pool and extracellular and plasma (Ec + Pls) source pool mouse models were 78 and 120% d⁻¹ in liver, 14 and 16% d⁻¹ in brain and 15 and 14% d⁻¹ in muscle. Predicted protein synthesis rates using the Ec + Ic, Ec + Ic + Tr (combined extracellular, intracellular and aminoacyl tRNA source pool) and Ec + Pls rat models were 57, 3.4 and 57% d⁻¹ in gastrocnemius, 58, 71 and 62% d⁻¹ in gut, 8.3, 8.4 and 7.9% d⁻¹ in heart, 32, 23 and 25% d⁻¹ in kidney, 160, 90 and 80% d⁻¹ in liver, 57, 5.5 and 57% d⁻¹ in soleus and 56, 3.4 and 57% d⁻¹ in tibialis. The Ec + Ic + Tr model underestimated protein synthesis rates in mouse tissues (5.0, 27 and 2.5% d⁻¹ for brain, liver and muscle) and rat muscles (3.4, 5.5 and 3.4% d⁻¹ for gastrocnemius, soleus and tibialis). The Ec + Pls model predicted the mouse pulse dose data best and the Ec + Ic model predicted the rat flooding dose data best. Model predictions of FSR imply that identification and separation of the source specific radioactivity is critical to accurately estimate FSR. Received June 11, 2000 Accepted September 26, 2000     

Development of tRNA synthetases and connection to genetic code and disease

The genetic code is established by the aminoacylation reactions of aminoacyl tRNA synthetases, where amino acids are matched with triplet anticodons imbedded in the cognate tRNAs. The code established in this way is so robust that it gave birth to the entire tree of life. The tRNA synthetases are organized into two classes, based on their active site architectures. The details of this organization, and other considerations, suggest how the synthetases evolved by gene duplications, and how early proteins may have been statistical in nature, that is, products of a primitive code where one of several similar amino acids was used at a specific position in a polypeptide. The emergence of polypeptides with unique, defined sequences--true chemical entities--required extraordinary specificity of the aminoacylation reaction. This high specificity was achieved by editing activities that clear errors of aminoacylation and thereby prevent mistranslation. Defects in editing activities can be lethal and lead to pathologies in mammalian cells in culture. Even a mild defect in editing is casually associated with neurological disease in the mouse. Defects in editing are also mutagenic in an aging organism and suggest how mistranslation can lead to mutations that are fixed in the genome. Thus, clearance of mischarged tRNAs by the editing activities of tRNA synthetases was essential for development of the tree of life and has a role in the etiology of diseases that is just now being understood.     

Two tyrosine residues outside the editing active site in Giardia lamblia leucyl-tRNA synthetase are essential for the post-transfer editing

Leucyl-tRNA synthetase (LeuRS) is responsible for the Leu-tRNA^Leu synthesis. The connective peptide 1 (CP1) domain inserted into the Rossmann nucleotide binding fold possesses editing active site to hydrolyze the mischarged tRNA^Leu with noncognate amino acid, then to ensure high fidelity of protein synthesis. A few co-crystal structures of LeuRS with tRNA^Leu in different conformations revealed that tRNA^Leu 3′ end shuttled between synthetic and editing active sites dynamically with direct and specific interaction with the CP1 domain. Here, we reported that Y515 and Y520 outside the editing active site of CP1 domain of Giardia lamblia LeuRS (GlLeuRS) are crucial for post-transfer editing by influencing the binding affinity with mischarged tRNA^Leu. Mutations on Y515 and Y520 also decreased tRNA^Leu charging activity to various extents but had no effect on leucine activation. Our results gave some biochemical knowledge about interaction of tRNA^Leu 3′ end with the CP1 domain in archaeal/eukaryotic LeuRS.     

tRNA as an active chemical scaffold for diverse chemical transformations

During protein synthesis, tRNA serves as the intermediary between cognate amino acids and their corresponding RNA trinucleotide codons. Aminoacyl-tRNA is also a biosynthetic precursor and amino acid donor for other macromolecules. AA-tRNAs allow transformations of acidic amino acids into their amide-containing counterparts, and seryl-tRNA^Ser donates serine for antibiotic synthesis. Aminoacyl-tRNA is also used to cross-link peptidoglycan, to lysinylate the lipid bilayer, and to allow proteolytic turnover via the N-end rule. These alternative functions may signal the use of RNA in early evolution as both a biological scaffold and a catalyst to achieve a wide variety of chemical transformations.     

Plant base editing and prime editing: The current status and future perspectives

Precise replacement of an allele with an elite allele controlling an important agronomic trait in a predefined manner by gene editing technologies is highly desirable in crop improvement.Base editing and prime editing are two newly developed precision gene editing systems which can introduce the substitution of a single base and install the desired short indels to the target loci in the absence of double-strand breaks and donor repair templates,respectively.Since their discoveries,various strate...     

Amino acid transport and protein synthesis in induced ectoderm of amphibian embryos

Summary Isolated gastrula ectoderm ofTriturus alpestris orAmbystoma mexicanum was induced by the vegetalizing factor. Protein synthesis in the induced and uninduced control explants was measured by double labelling with³H-and¹⁴C-amino acids after different periods of cultivation. Slight differences were observed in the pattern of nuclear proteins after 12 h of cultivation and in the pattern of cytoplasmic proteins after 48 h of cultivation.The uptake of leucine started to increase in induced explants after 48 h of cultivation and after 96 h was about 50 times greater than in uninduced control explants. The uptake is reduced under partially anaerobic conditions. Ouabain inhibits the uptake by about 50%.     

The effect of canavanine on protein synthesis and protein degradation in IMR-90 fibroblasts

Proteins of IMR-90 fibroblasts incorporating [³⁵S]methionine during a 1 h labelling period in the presence of the arginine analogue canavanine were degraded twice as rapidly in the cells as were proteins similarly made in the presence of arginine. Using both isoelectric focusing and SDS-polyacrylamide gel electrophoretic analyses, the banding patterns of proteins labelled in the presence of canavanine and arginine were found to differ. This banding difference was detected as early as 15 min after canavanine treatment. With the exception of one minor band in isoelectric focusing gel, the relative intensity of labelled protein bands for the control samples remained unchanged during the 2 h period of protein degradation being investigated. This was also true for the proteins labelled in the presence of canavanine, despite the increase in their rate of degradation. Banding difference between canavanine and arginine treatment was also detected in an in vitro reticulocyte lysate translation system dependent on fibroblast mRNA. Proteins labelled in the presence of a different analogue, p-fluorophenylalanine instead of phenylalanine, however, had similar banding patterns as the control both in the lysate system and in intact cells.     

Identification of spin diffusion pathways in proteins by isotope-assisted NMR cross- relaxation network editing

A new isotope-assisted cross-relaxation editing experiment, [1H-13C]DINE-NOESY[1H-15N]HSQC (DINE = Double INEPT Edited), is proposed. It is based on the selectiveinversion of CH/CH3 or CH2 protons in the middle of the mixing time. The experiment sortsout the spin diffusion paths according to the principal mediators, either the CH/CH3 or theCH2 protons. This is useful in the structure refinement process, as it enables proper alignmentof the aliphatic protons in the vicinity of NH protons.     

Homology between prokaryotic and eukaryotic ribonucleases

Summary There is homology between the amino acid sequences of the extracellular ribonucleases T1 and St, from the eukaryoteAspergillus oryzae and the prokaryoteStreptomyces erythreus, respectively. Together with other extracellular ribonucleases homologous to each, these enzymes make up a family of interest to evolutionary biology and useful in studies of protein structure and function.     

Origin of amino acid homochirality: relationship with the RNA world and origin of tRNA aminoacylation

The origin of homochirality of l-amino acids has long been a mystery. Aminoacylation of tRNA might have provided chiral selectivity, since it is the first process encountered by amino acids and RNA. An RNA minihelix (progenitor of the modern tRNA) was aminoacylated by an aminoacyl phosphate oligonucleotide that exhibited a clear preference for l- as opposed to d-amino acids. A mirror-image RNA system with l-ribose exhibited the opposite selectivity, i.e., it exhibited an apparent preference for the d-amino acid. The selectivity for l-amino acids is based on the stereochemistry of RNA. The side chain of d-amino acids is located much closer to the terminal adenosine of the minihelix, causing them collide and interfere during the amino acid-transfer step. These results suggest that the putative RNA world that preceded the protein theatre determined the homochirality of l-amino acids through tRNA aminoacylation.     

The C-terminal end of the Trypanosoma brucei editing deaminase plays a critical role in tRNA binding

Adenosine to inosine editing at the wobble position allows decoding of multiple codons by a single tRNA. This reaction is catalyzed by adenosine deaminases acting on tRNA (ADATs) and is essential for viability. In bacteria, the anticodon-specific enzyme is a homodimer that recognizes a single tRNA substrate (tRNA(Arg)(ACG)) and can efficiently deaminate short anticodon stem-loop mimics of this tRNA in vitro. The eukaryal enzyme is composed of two nonidentical subunits, ADAT2 and ADAT3, which upon heterodimerization, recognize seven to eight different tRNAs as substrates, depending on the organism, and require a full-length tRNA for activity. Although crystallographic data have provided clues to why the bacterial deaminase can utilize short substrates, residues that provide substrate binding and recognition with the eukaryotic enzymes are not currently known. In the present study, we have used a combination of mutagenesis, binding studies, and kinetic analysis to explore the contribution of individual residues in Trypanosoma brucei ADAT2 (TbADAT2) to tRNA recognition. We show that deletion of the last 10 amino acids at the C terminus of TbADAT2 abolishes tRNA binding. In addition, single alanine replacements of a string of positively charged amino acids (KRKRK) lead to binding defects that correlate with losses in enzyme activity. This region, which we have termed the KR-domain, provides a first glance at key residues involved in tRNA binding by eukaryotic tRNA editing deaminases.     

The tRNA A76 Hydroxyl Groups Control Partitioning of the tRNA-dependent Pre- and Post-transfer Editing Pathways in Class I tRNA Synthetase

Aminoacyl-tRNA synthetases catalyze ATP-dependent covalent coupling of cognate amino acids and tRNAs for ribosomal protein synthesis. Escherichia coli isoleucyl-tRNA synthetase (IleRS) exploits both the tRNA-dependent pre- and post-transfer editing pathways to minimize errors in translation. However, the molecular mechanisms by which tRNA^Ile organizes the synthetic site to enhance pre-transfer editing, an idiosyncratic feature of IleRS, remains elusive. Here we show that tRNA^Ile affects both the synthetic and editing reactions localized within the IleRS synthetic site. In a complex with cognate tRNA, IleRS exhibits a 10-fold faster aminoacyl-AMP hydrolysis and a 10-fold drop in amino acid affinity relative to the free enzyme. Remarkably, the specificity against non-cognate valine was not improved by the presence of tRNA in either of these processes. Instead, amino acid specificity is determined by the protein component per se, whereas the tRNA promotes catalytic performance of the synthetic site, bringing about less error-prone and kinetically optimized isoleucyl-tRNA^Ile synthesis under cellular conditions. Finally, the extent to which tRNA^Ile modulates activation and pre-transfer editing is independent of the intactness of its 3′-end. This finding decouples aminoacylation and pre-transfer editing within the IleRS synthetic site and further demonstrates that the A76 hydroxyl groups participate in post-transfer editing only. The data are consistent with a model whereby the 3′-end of the tRNA remains free to sample different positions within the IleRS·tRNA complex, whereas the fine-tuning of the synthetic site is attained via conformational rearrangement of the enzyme through the interactions with the remaining parts of the tRNA body.     

Synthesis of protein,nucleosides and other organic compounds from cyanamide and potassium nitrite under possible primitive earth conditions

The mixing of cyanamide and KNO₂ produced changes from white solids to yellow liquid and then to orange solid. The gases cyanogen and ammonia were formed. No external energy was used. The reactions were carried out with a small amount of O₂. The presence of proteins in the reaction product formed 13 months after the mixing was indicated by the positive reactions of the cyanamide-KNO₂ reaction product with ninhydrin, microbiuret, and Folin reagent; the ultraviolet absorption at about 280 nm; the yield of 24% of 15 amino acids; and molecular weight measurements of more than 160 000. The presence of nucleosides, nucleic acid bases, hydrocarbons, and organic esters in the reaction product formed 2 months after the mixing was indicated by ultraviolet absorption at about 260 nm, and the results of ligand-exchange chromatography, paper chromatography, infrared analysis, mass spectral analysis, and NMR spectroscopy. Possible cyanamide-mediated dehydration reactions and mechanisms are discussed.     

Analysis of cysteine residues in peptides and proteins alkylated with volatile reagents

Summary Conditions are described for the reduction and alkylation of cysteines in peptides and proteins with volatile reagents by use of triethylphosphine as reductant, bromopropane as alkylating reagent and triethylamine as base. Alkylated samples need only be vacuum dried prior to subsequent analysis steps. Alkylated samples have been acid hydrolyzed and analyzed on an amino acid analyzer with recoveries of cysteine within 10% of the expected value. Alkylated samples have been directly applied to a sequencer membrane, dried on the surface and cysteines identified by sequence analysis without additional wash steps. In addition proteins blotted onto PVDF have been alkylatedin situ and sequenced with identification of cysteines. On the analyzer and sequencer the S-propylcysteine derivative elutes at a unique position allowing for the unambiguous identification of cysteine. Cysteine residues are quantitativly alkylated under the conditions developed. The ease of this procedure allows the routine analysis of cysteine in peptides and proteins without additional, time consuming repurification or dialysis steps.Abbreviations dptu diphenylthiourea - dmptu dimethylphenylthiourea - prop-cys S-propylcysteine