Multiple Quality Control Pathways Limit Non-protein Amino Acid Use by Yeast Cytoplasmic Phenylalanyl-tRNA Synthetase.

Non-protein amino acids, particularly isomers of the proteinogenic amino acids, present a threat to proteome integrity if they are mistakenly inserted into proteins. Quality control during aminoacyl-tRNA synthesis reduces non-protein amino acid incorporation by both substrate discrimination and proofreading. For example phenylalanyl-tRNA synthetase (PheRS) proofreads the non-protein hydroxylated phenylalanine derivative m-Tyr after its attachment to tRNA(Phe) We now show in Saccharomyces cerevisiae that PheRS misacylation of tRNA(Phe) with the more abundant Phe oxidation product o-Tyr is limited by kinetic discrimination against o-Tyr-AMP in the transfer step followed by o-Tyr-AMP release from the synthetic active site. This selective rejection of a non-protein aminoacyl-adenylate is in addition to known kinetic discrimination against certain non-cognates in the activation step as well as catalytic hydrolysis of mispaired aminoacyl-tRNA(Phe) species. We also report an unexpected resistance to cytotoxicity by a S. cerevisiae mutant with ablated post-transfer editing activity when supplemented with o-Tyr, cognate Phe, or Ala, the latter of which is not a substrate for activation by this enzyme. Our phenotypic, metabolomic, and kinetic analyses indicate at least three modes of discrimination against non-protein amino acids by S. cerevisiae PheRS and support a non-canonical role for SccytoPheRS post-transfer editing in response to amino acid stress.

[Authentication of Lonicera japonica using bidirectional PCR amplification of specific alleles].

OBJECTIVE: To identify SNP in flos Lonicerae, and authenticate Lonicera japonica from its adulterants and the mixture by using bidirectional PCR amplification of specific alleles (Bi-PASA). METHOD: SNP of L. japonica and its adulterants was identified by using ClustulW to align trnL-trnF sequences of the Lonicera genus from GenBank database. Bi-PASA primer was designed and the PCR reaction systems including annealing temperature optimized. Optimized result was performed in 84 samples to authenticate L. japonica, its adulterants and the mixture. RESULT: When the annealing temperature was 61 degrees C, DNA from L. japonica would be amplified 468 bp whereas PCR products from all of the 9 adulterants were 324 bp. The established method also can detect 5% of intentional adulteration DNA into L. japonica. CONCLUSION: The Bi-SPASA could authenticate L. japonica from its adulterants and the mixture.

Polyamine derivatives as selective RNaseA mimics.

Site-selective scission of ribonucleic acids (RNAs) has attracted considerable interest, since RNA is an intermediate in gene expression and the genetic material of many pathogenic viruses. Polyamine-imidazole conjugates for site-selective RNA scission, without free imidazole, were synthesized and tested on yeast phenylalanine transfer RNA. These molecules catalyze RNA hydrolysis non-randomly. Within the polyamine chain, the location of the imidazole residue, the numbers of nitrogen atoms and their relative distances have notable influence on cleavage selectivity. A norspermine derivative reduces the cleavage sites to a unique location, in the anticodon loop of the tRNA, in the absence of complementary sequence. Experimental results are consistent with a cooperative participation of an ammonium group of the polyamine moiety, in addition to it's binding to the negatively charged ribose-phosphate backbone, as proton source, and the imidazole moiety as a base. There is correlation between the location of the magnesium binding sites and the RNA cleavage sites, suggesting that the protonated nitrogens of the polycationic chain compete with some of the magnesium ions for RNA binding. Therefore, the cleavage pattern is specific of the RNA structure. These compounds cleave at physiological pH, representing novel reactive groups for antisense oligonucleotide derivatives or to enhance ribozyme activity.

Genetic identification of cinnamon (Cinnamomum spp.) based on the trnL-trnF chloroplast DNA.

Genetic identification among cinnamon species was studied by analyzing nucleotide sequences of chloroplast DNA from four species (Cinnamomum cassia, C. zeylanicum, C. burmannii and C. sieboldii). The two regions studied were the intergenic spacer region between the trnL 3'exon and trnF exon (trnL -trnF IGS) and the trnL intron region. We found nucleotide variation at one site in the trnL-trnF IGS, and at three sites in the trnL intron. With the sequence data from analysis of these regions, the four Cinnamomum species used in this study were correctly identified. Furthermore, single-strand conformation polymorphism (SSCP) analysis of PCR products from the trnL-trnF IGS and the trnL intron resulted in different SSCP band patterns among C. cassia, C. zeylanicum and C. burmannii.

Phylogenetic analysis of chloroplast DNA variation in Coffea L.

The trnL-trnF intergenic spacer of cpDNA has been sequenced from 38 tree samples representing 23 Coffea taxa and the related genus Psilanthus. These sequences were used for phylogenetic reconstruction using parsimony analyses. The results suggest a radial mode of speciation and a recent origin in Africa for the genus Coffea. Phylogenetic relationships inferred from the cpDNA analysis suggest several major clades, which present a strong geographical correspondence (i.e., west Africa, central Africa, east Africa, and Madagascar). The overall results agree well with the phylogeny previously inferred from nuclear genome data. However, several inconsistencies are observed among taxa endemic to west Africa, suggesting the occurrence of introgressive hybridization. Evidence is also obtained for the genetic origin of the allotetraploid species C. arabica.

Ribonuclease P catalysis requires Mg2+ coordinated to the pro-RP oxygen of the scissile bond.

Ribonuclease P (RNase P) is an essential enzyme whose action produces the mature 5' termini of all cellular and organellar transfer RNA molecules. In bacteria, the catalytic subunit of RNase P is an RNA molecule which by itself can bind substrate pre-tRNA, select and hydrolyze the correct phosphodiester bond, and release product tRNA. The simple requirements of the reaction-a monovalent cation such as K+ or NH4+ and the divalent cation Mg2+ (or Mn2+)-have prompted proposals that all aspects of phosphodiester bond hydrolysis might be accomplished by one or more divalent metal cations coordinated to the enzyme or substrate. To precisely localize the ligands of catalytically-involved Mg2+, we assayed cleavage by Escherichia coli RNase P RNA of pre-tRNA in which specific pro-Rp phosphate oxygens were replaced with sulfur. RNase P cleavage was targeted to that bond, at or nearest to the normal cleavage site, at which Mg2+ or Mn2+ could be coordinated. Single-turnover kinetics demonstrated that the apparent rate constant for the hydrolysis event was determined quantitatively by the affinity of the divalent cation (Mg2+ or Mn2+) for the atom (O or S) at the pro-Rp position of the scissile phosphodiester bond. We propose a model for pre-tRNA cleavage in which an essential Mg2+ ion is coordinated directly to the pro-Rp phosphate oxygen and indirectly to two other ligands near the scissile bond: the upstream ribose 2'-hydroxyl and the downstream purine N7. This catalytic Mg2+ ion most likely positions and deprotonates a water molecule for in-line nucleophilic attack on the scissile bond phosphorus.

Characterization of the potato mitochondrial transcription unit containing 'native' trnS (GCU), trnF (GAA) and trnP (UGG).

In order to identify the sequences promoting the expression of plant mitochondrial tRNA genes, we have characterized the trnS (GCU), trnF (GAA) and trnP (UGG) transcription unit of the potato mitochondrial genome. These three tRNA genes were shown to be co-transcribed as a 1800 nt long primary transcript. The transcription initiation site located 305 to 312 nt upstream of trnS is surrounded by a purine-rich region but does not contain the consensus motif proposed as a promoter element in dicotyledonous plants. Differential labelling of potato mitochondrial RNA with either guanylyltransferase or T4 polynucleotide kinase suggests that this site corresponds to the unique functional region responsible for the transcription of the three tRNA genes. The initiation site recently found upstream of Oenothera mitochondrial trnF does not seem to be used in potato mitochondria, although a very similar sequence is present 317 nt upstream of the corresponding potato gene. Major processing sites were identified at the 3' end of each tRNA gene. Another processing site, surrounded by a double hairpin structure, is located 498 nt downstream of trnP in stretch of 10 A residues. As judged from northern experiments, this region is close to the determination site of this transcription unit.

Identification of a stable RNA encoded by the H-strand of the mouse mitochondrial D-loop region and a conserved sequence motif immediately upstream of its polyadenylation site.

By using a combination of Northern blot hybridization with strand-specific DNA probes, S1 nuclease protection, and sequencing of oligo-dT-primed cDNA clones, we have identified a 0.8 kb poly(A)-containing RNA encoded by the H-strand of the mouse mitochondrial D-loop region. The 5' end of the RNA maps to nucleotide 15417, a region complementary to the start of tRNA(Pro) gene and the 3' polyadenylated end maps to nucleotide 16295 of the genome, immediately upstream of tRNA(Phe) gene. The H-strand D-loop region encoded transcripts of similar size are also detected in other vertebrate systems. In the mouse, rat, and human systems, the 3' ends of the D-loop encoded RNA are preceded by conserved sequences AAUAAA, AAUUAA, or AACUAA, that resemble the polyadenylation signal. The steady-state level of the RNA is generally low in dividing or in vitro cultured cells, and markedly higher in differentiated tissues like liver, kidney, heart, and brain. Furthermore, an over 10-fold increase in the level of this RNA is observed during the induced differentiation of C2C12 mouse myoblast cells into myotubes. These results suggest that the D-loop H-strand encoded RNA may have yet unknown biological functions. A 20 base pair DNA sequence from the 3' terminal region containing the conserved sequence motif binds to a protein from the mitochondrial extracts in a sequence-specific manner. The binding specificity of this protein is distinctly different from the previously characterized H-strand DNA termination sequence in the D-loop or the H-strand transcription terminator immediately downstream of the 16S rRNA gene. Thus, we have characterized a novel poly(A)-containing RNA encoded by the H-strand of the mitochondrial D-loop region and also identified the putative ultimate termination site for the H-strand transcription.

Characterization of some major identity elements in plant alanine and phenylalanine transfer RNAs.

Alanine and phenylalanine tRNA sequences were amplified by PCR from Arabidopsis thaliana nuclear DNA using degenerate oligonucleotides which introduced specific mutations into the acceptor stem. The aminoacylation of T7 RNA polymerase transcripts of these sequences was investigated in vitro using partially purified bean alanyl- or phenylalanyl-tRNA synthetase. In parallel, the in vivo activity of amber suppressor derivatives of these tRNAs was investigated in transient expression assays in tobacco protoplasts using a beta-glucuronidase (GUS) reporter gene containing a premature amber stop codon. The results show that mutation of the G3:U70 base pair to G3:C70 blocks aminoacylation of plant alanine tRNA, whilst conversion of the G3:C70 pair normally found in plant tRNA(Phe) to G3:U70 enables the mutated tRNA(Phe) to be a good substrate for alanyl-tRNA synthetase and impairs its aminoacylation with phenylalanine. In addition, the amber suppressor derivative of wild-type tRNA(Phe) showed very little suppressor activity in vivo, and was poorly aminoacylated with phenylalanine in vitro, suggesting that the anticodon is a major identity determinant for tRNA(Phe) in plant cells.

Sequence analysis of three tRNA(Phe) nuclear genes and a mutated gene, and one gene for tRNA(Ala) from Arabidopsis thaliana.

Three genes and one mutant gene for tRNA(Phe) (GAA) and one gene for tRNA(Ala) (UGC) were isolated from a whole-cell DNA library of Arabidopsis thaliana. All three tRNA(Phe) genes are identical in their nucleotide sequence, but differ in their 5' and 3' flanking regions. The mutant tRNA(Phe) (GAA) gene differs from the other three genes by one nucleotide change from highly conserved G to C at the 57th nucleotide position. The primary structure of the first tRNA(Ala) gene was also determined in this experiment.

Variations during leaf development of the relative amounts of two bean (Phaseolus vulgaris) chloroplast tRNAs(Phe) which differ in their minor nucleotide content.

Bean (Phaseolus vulgaris cv. Saxa) chloroplasts contain two tRNA(Phe) species, namely tRNA(Phe)1 and tRNA(Phe)2. By sequence determination, we show that tRNA(Phe)2 is identical to the previously sequenced tRNA(Phe)1 except for two undermodified nucleotides. By reversed-phase chromatography analyses, we demonstrate that the relative amounts of these two chloroplast tRNAs(Phe) vary during leaf development: in etiolated leaves the undermodified tRNA(Phe)2 only represents 15% of total chloroplast tRNA(Phe), during development and greening it increases to reach 60% in 8-day-old leaves, and it then decreases to 9% in senescing leaves.