Yeast retrotransposons and tRNAs

The role of tRNAs in protein synthesis seems routine when compared with the novel ways in which the Ty retrotransposons of Saccharomyces cerevisiae use these interpreters of the genetic code. tRNAs and tRNA genes control essential steps in the retrotransposon life cycle by regulating protein expression, priming DNA synthesis and specifying integration target sites.     

Cytoplasmic free calcium concentration in porcine platelets: Regulation by an intracellular nonmitochondrial calcium pump and increase after thrombin stimulation

Mechanisms are assumed to exist in the resting platelet which maintain the concentration of cytoplasmic free calcium below that level required to activate cellular responses. To assess such processes the porcine platelet plasma membrane was selectively lysed with digitonin and the uptake (or flux) of free calcium monitored by an extracellular calcium electrode. Lysis resulted in an immediate lowering of the extracellular free calcium, due to the action of intracellular organelle(s) acting on the extracellular space through the permeabilized plasma membrane. In resting platelets, the rate of calcium uptake was first order with respect to the extracellular prelytic calcium concentration, and hence the cytoplasmic free concentration was found to be 1·10^?7 M by extrapolation to a point of zero flux (i.e., the null point). This approach could not be used with thrombin-stimulated platelets, as external calcium was required for both secretion of ATP + ADP and aggregation. Nevertheless, evidence for an increase in cytoplasmic free calcium after thromin stimulation was obtained. Metabolic inhibitors and agents known to inhibit calcium uptake by mitochondria had no effect on the calcium flux following lysis, indicating different mechanisms for calcium homeostasis in the platelet when compared with other cell types (e.g., liver). Levels of ionophore A23187, which caused platelet aggregation, gave a massive release of the nonmitochondrial pool of calcium into the cytoplasmic space. Thus, in porcine platelets an intracellular energy-requiring calcium pump, which sequesters calcium in a nonmitochondrial membranous compartment, is crucial for intracellular calcium homeostasis.     

体内微环境对B7-2EC克隆细胞株分化的影响

在发育生物学领域中,微环境对早期胚胎纽胞的分化,对造血系统干细胞的分化都能产生影响,这已是人所共知的事实。小鼠胚胎性癌细胞(EC细胞)是一种研究细胞恶变和细胞分化很好的材料,它可以在某些品系小鼠中自发地产生,也可由小鼠早期胚胎细胞或原始生殖嵴细胞经异位移植而获得,但移植的位置不同,生瘤率也不同。EC细胞一般分为无能和多能性两种:无能EC细胞如F9在体内接种后不能分化为各种体细胞,保持着癌细胞恶性生长的特点,而多能EC细胞接种到     

Isolation of isoaccepting tRNAs

The N-hydroxysuccinimide ester of succinated polyethylene oxide (polyethylene glycol 6000) has been prepared. The ester has been used to make the N-acyl derivatives of valyl-tRNA and phenylalanyl-tRNA from E. coli K-12. Because of the large molecular weight, high solubility in phenol, and the binding to Corning porous glass of polyethylene oxide, the acyl derivative, N-(succinated polyethylene oxide)-aminoacyl-tRNA, has been separated from unreacted tRNA. Since the reaction is reasonably specific for the amino group of the amino acid, large purifications have been obtained for tRNA_val and tRNA_phe. Evidence is presented to show that the ester can react with tRNA at a slow rate. The limitations on the purification due to this reaction are quantitatively evaluated. The highest ratios, pmoles aminoacyl-tRNA/ OD₂₆₀, obtained for valyl-tRNA and phenylalanyl-tRNA were 800 and 360.     

Preparation and evaluation of acylated tRNAs

In the cell, the activity of tRNA is governed by its acylation state. Interactions with the ribosome, translation factors, and regulatory elements are strongly influenced by the acyl group, and presumably other cellular components that interact with tRNA also use the acyl group as a specificity determinant. Thus, those using biochemical approaches to study any aspect of tRNA biology should be familiar with effective methods to prepare and evaluate acylated tRNA reagents. Here, methods to prepare aminoacyl-tRNA, N-acetyl-aminoacyl-tRNA, and fMet-tRNA(fMet) and to assess their homogeneity are described. Using these methods, acylated tRNAs of high homogeneity can be reliably obtained.     

Initiation with Elongator tRNAs

In all domains of life, initiator tRNA functions exclusively at the first step of protein synthesis while elongator tRNAs extend the polypeptide chain. Unique features of initiator tRNA enable it to preferentially bind the ribosomal P site and initiate translation. Recently, we showed that the abundance of initiator tRNA also contributes to its specialized role. This motivates the question, can a cell also use elongator tRNA to initiate translation under certain conditions? To address this, we introduced non-AUG initiation codons CCC (Pro), GAG (Glu), GGU (Gly), UCU (Ser), UGU (Cys), ACG (Thr), AAU (Asn), and AGA (Arg) into the uracil DNA glycosylase gene (ung) used as a reporter gene. Enzyme assays from log-phase cells revealed initiation from non-AUG codons when intracellular initiator tRNA levels were reduced. The activity increased significantly in stationary phase. Further increases in initiation from non-AUG codons occurred in both growth phases upon introduction of plasmid-borne genes of cognate elongator tRNAs. Since purine-rich Shine-Dalgarno sequences occur frequently on mRNAs (in places other than the canonical AUG codon initiation contexts), initiation with elongator tRNAs from the alternate contexts may generate proteome diversity under stress without compromising genomic integrity. Thus, by changing the relative amounts of initiator and elongator tRNAs within the cell, we have blurred the distinction between the two classes of tRNAs thought to be frozen through years of evolution.     

Ribosomal Discrimination of tRNAs

Mutations in two proteins of the 30S ribosomal subunit indicate that the ribosome provides a recognition screen for tRNAs before, or simultaneous with, their interaction with mRNA.     

Screening system for orthogonal suppressor tRNAs based on the species-specific toxicity of suppressor tRNAs

Incorporation of unnatural amino acids into proteins in vivo, known as expanding the genetic code, is a useful technology in the pharmaceutical and biotechnology industries. This procedure requires an orthogonal suppressor tRNA that is uniquely acylated with the desired unnatural amino acid by an orthogonal aminoacyl-tRNA synthetase. In order to enhance the numbers and types of suppressor tRNAs available for engineering genetic codes, we have developed a convenient screening system to generate suppressor tRNAs with good orthogonality from the available library of suppressor tRNA mutants. While developing an amber suppressor tRNA, we discovered that amber suppressor tRNA with poor orthogonality inhibited the growth rate of the host, indicating that suppressor tRNA demonstrates a species-specific toxicity to host cells. We verified this species-specific toxicity using amber suppressor tRNA mutants from prokaryotes, eukaryotes, and archaea. We also confirmed that adding terminal CCA to Methanococcus jannaschii tRNA^Tyr mutant is important to its toxicity against Escherichia coli. Further, we compared the toxicity of the suppressor tRNA toward the host with differing copy numbers. Using the combined toxicity of suppressor tRNA toward the host with blue–white selection, we developed a convenient screening system for orthogonal suppressor tRNA that could serve as a general platform for generating tRNA/aaRS pairs and thereby obtained three suppressor tRNA mutants with high orthogonality from the tRNA library derived from Mj tRNA^Tyr.     

T-armless tRNAs and elongated elongation factor Tu

Most tRNAs share a common secondary structure containing a T arm, a D arm, an anticodon arm and an acceptor stem. However, there are some exceptions. Most nematode mitochondrial tRNAs and some animal mitochondrial tRNAs lack the T arm, which is necessary for binding to canonical elongation factor Tu (EF-Tu). The mitochondria of the nematode Caenorhabditis elegans have a unique EF-Tu, named EF-Tu1, whose structure has supplied clues as to how truncated tRNAs can work in translation. EF-Tu1 has a C-terminal extension of about 60 aa that is absent in canonical EF-Tu. Recent data from our laboratory strongly suggests that EF-Tu1 recognizes the D-arm instead of the T arm by a mechanism involving this C-terminal region. Further biochemical analysis of mitochondrial tRNAs and EF-Tu from the distantly related nematode Trichinella spp. and sequence information on nuclear and mitochondrial DNA in arthropods suggest that T-armless tRNAs may have arisen as a result of duplication of the EF-Tu gene. These studies provide valuable insights into the co-evolution of RNA and RNA-binding proteins.     

Phylogeny and evolution of chloroplast tRNAs in Adoxaceae

Chloroplasts are semiautonomous organelles found in photosynthetic plants. The major functions of chloroplasts include photosynthesis and carbon fixation, which are mainly regulated by its circular genomes. In the highly conserved chloroplast genome, the chloroplast transfer RNA genes (cp tRNA) play important roles in protein translation within chloroplasts. However, the evolution of cp tRNAs remains unclear. Thus, in the present study, we investigated the evolutionary characteristics of chloroplast tRNAs in five Adoxaceae species using 185 tRNA gene sequences. In total, 37 tRNAs encoding 28 anticodons are found in the chloroplast genome in Adoxaceae species. Some consensus sequences are found within the Ψ‐stem and anticodon loop of the tRNAs. Some putative novel structures were also identified, including a new stem located in the variable region of tRNA^Tyr in a similar manner to the anticodon stem. Furthermore, phylogenetic and evolutionary analyses indicated that synonymous tRNAs may have evolved from multiple ancestors and frequent tRNA duplications during the evolutionary process may have been primarily caused by positive selection and adaptive evolution. The transition and transversion rates are uneven among different tRNA isotypes. For all tRNAs, the transition rate is greater with a transition/transversion bias of 3.13. Phylogenetic analysis of cp tRNA suggested that the type I introns in different taxa (including eukaryote organisms and cyanobacteria) share the conserved sequences “U‐U‐x2‐C” and “U‐x‐G‐x2‐T,” thereby indicating the diverse cyanobacterial origins of organelles. This detailed study of cp tRNAs in Adoxaceae may facilitate further investigations of the evolution, phylogeny, structure, and related functions of chloroplast tRNAs.     

氨酰-tRNA合成酶对tRNA的识别

氨酰-tRNA合成酶(aaRS)与tRNA的相互作用保证了蛋白质生物合成的忠实性. 氨酰-tRNA合成酶对tRNA识别的专一性依赖于aaRS特定的催化结构域和tRNA分子特异的三级结构构象. 反密码子和接受茎(包括73位)在大多数aaRS对tRNA分子的识别过程中起着关键作用, 其他部位如可变口袋、可变(茎)环等, 甚至修饰核苷酸对于一些识别过程也有重要作用.     

Pathogenic mutations in antisense mitochondrial tRNAs

Pathogenic mutations in mitochondrial tRNAs are 6.5 times more frequent than in other mitochondrial genes. This suggests that tRNA mutations perturb more than one function. A potential additional tRNA gene function is that of templating for antisense tRNAs. Pathogenic mutations weaken cloverleaf secondary structures of sense tRNAs. Analyses here show similar effects for most antisense tRNAs, especially after adjusting for associations between sense and antisense cloverleaf stabilities. These results imply translational activity by antisense tRNAs. For sense tRNAs Ala and Ser UCN, pathogenicity associates as much with sense as with antisense cloverleaf formation. For tRNA Pro, pathogenicity seems associated only with antisense, not sense tRNA cloverleaf formation. Translational activity by antisense tRNAs is expected for the 11 antisense tRNAs processed by regular sense RNA maturation, those recognized by their cognate amino acid’s tRNA synthetase, and those forming relatively stable cloverleaves as compared to their sense counterpart. Most antisense tRNAs probably function routinely in translation and extend the tRNA pool (extension hypothesis); others do not (avoidance hypothesis). The greater the expected translational activity of an antisense tRNA, the more pathogenic mutations weaken its cloverleaf secondary structure. Some evidence for RNA interference, a more classical role for antisense tRNAs, exists only for tRNA Ser UCN. Mutation pathogenicity probably frequently results from a mixture of effects due to sense and antisense tRNA translational activity for many mitochondrial tRNAs. Genomic studies should routinely explore for translational activity by antisense tRNAs.     

延伸因子复合物对真核生物tRNA的修饰作用

延伸因子复合物(Elongator complex, Elp)由6个亚基蛋白Elp1～6组成,在真核细胞生物中呈现高度的进化保守,提示其具有重要的生物学功能。研究表明,Elp涉及多种细胞行为如转录延伸、细胞外分泌、端粒基因沉默和DNA损伤修复、神经系统的发育和功能等。然而越来越多的证据显示,Elp通过介导tRNA修饰影响翻译过程,从而间接调控上述细胞行为。在人类,ELP1/IKBKAP突变可导致家族性植物神经功能障碍症,ELP2、ELP3和ELP4基因的遗传变异也可能与其他神经退行性病变相关。本文对Elp的结构、Elp修饰tRNA和Elp相关疾病等的研究现状及其进展进行综述。     

Threonine tRNAs and their genes in Escherichia coli

Summary The subject of this study was the threonine isoacceptor family of tRNAs in Escherichia coli and the genes coding for them. The goal was to identify and map all the genes and to determine the relative contribution of each gene to the tRNA pool. The mapping experiments exploited gene-dosage effects in partially diploid strains; if a strain harboring a particular F episome overproduced a particular tRNA species, it could be concluded that the gene for that tRNA was located on the chromosomal segment carried by the F. Isoacceptor tRNAs were distinguished by column fractionation. It was found that there are three major threonine tRNA species that occur in roughly equal amounts. These are tRNA ₁ ^Thr , which is encoded by a gene in the distal region of the rrnD ribosomal RNA operon, and tRNA ₃ ^Thr and tRNA ₄ ^Thr , which come from genes in the cluster thrU tyrU glyT thrT at 89 min on the map. The relative abundances of the tRNA species roughly match the reported frequencies of the codons that they recognize in mRNA. Although the tRNA ₄ ^Thr has a mismatched base pair that raised questions about its biological activity, it was found to be functional at least with respect to recognition by the threonyl-tRNA synthetase. An apparent fourth gene affecting threonine tRNA has been identified and mapped at 6–8 min; it is here designated thrW. It may be a structural gene for a minor tRNA species, present in one-third the amount of each of the major species, and chromatographically indistinguishable from tRNA ₄ ^Thr .A preliminary report of most of this work has appeared previously (M.M. Comer, Abstr. Annu. Meet. Am. Soc. Microbiol. 1980, p. 109)