Splicing of Arabidopsis tRNAMet precursors in tobacco cell and wheat germ extracts

Intron-containing tRNA genes are exceptional within nuclear plant genomes. It appears that merely two tRNA gene families coding for tRNA^Tyr _{G A} and elongator tRNA^Met _CmAU contain intervening sequences. We have previously investigated the features required by wheat germ splicing endonuclease for efficient and accurate intron excision from Arabidopsis pre-tRNA^Tyr. Here we have studied the expression of an Arabidopsis elongator tRNA^Met gene in two plant extracts of different origin. This gene was first transcribed either in HeLa or in tobacco cell nuclear extract and splicing of intron-containing tRNA^Met precursors was then examined in wheat germ S23 extract and in the tobacco system. The results show that conversion of pre-tRNA^Met to mature tRNA proceeds very efficiently in both plant extracts. In order to elucidate the potential role of specific nucleotides at the 3 and 5 splice sites and of a structured intron for pre-tRNA^Met splicing in either extract, we have performed a systematic survey by mutational analyses. The results show that cytidine residues at intron-exon boundaries impair pre-tRNA^Met splicing and that a highly structured intron is indispensable for pre-tRNA^Met splicing. tRNA precursors with an extended anticodon stem of three to four base pairs are readily accepted as substrates by wheat and tobacco splicing endonuclease, whereas pre-tRNA molecules that can form an extended anticodon stem of only two putative base pairs are not spliced at all. An amber suppressor, generated from the intron-containing elongator tRNA^Met gene, is efficiently processed and spliced in both plant extracts.     

A critical role of water in the specific cleavage of the anticodon loop of some eukaryotic methionine initiator tRNAs

We have noticed that during a long storage and handling, the plant methionine initiator tRNA is spontaneously hydrolyzed within the anticodon loop at the C34-A35 phosphodiester bond. A literature search indicated that there is also the case for human initiator tRNA^Met but not for yeast tRNA^Met _i or E. coli tRNA^Met _f. All these tRNAs have an identical nucleotide sequence of the anticodon stems and loops with only one difference at position 33 within the loop. It means that cytosine 33 (C33) makes the anticodon loop of plant and human tRNA^Met _i susceptible to the specific cleavage reaction. Using crystallographic data of tRNA^Met _f of E. coli with U33, we modeled the anticodon loop of this tRNA with C33. We found that C33 within the anticodon loop creates a pocket that can accomodate a hydrogen bonded water molecule that acts as a general base and catalyzes a hydrolysis of C-A bond. We conclude that a single nucleotide change in the primary structure of tRNA^Met _i made changes in hydration pattern and readjustment in hydrogen bonding which lead to a cleavage of the phosphodiester bond.     

The potato mitochondrial initiator methionine tRNA gene and its flanking regions: an illustration of the diversity of mitochondrial genome rearrangements among plant species

The initiator methionine transfer RNA (tRNAf ^Met) gene was identified on a 347 bpEco RI-Hind III DNA fragment of the potato mitochondrial (mt) genome. The sequence of this gene shows 1 to 7 nucleotide differences with the other plant mt tRNAsf ^Met or tRNAf ^Met genes studied so far. Whereas the tRNAf ^Met gene is present as a single copy in the potato mt genome, a tRNA pseudogene corresponding to 60% of a complete tRNA (from the 5 end to the variable region) and located at 105 nucleotides upstream of the tRNAf ^Met gene on the opposite strand was shown to be repeated at least three times. Furthermore, the physical environment of the tRNAf ^Met gene in the mt genome is very different among plants, which suggests that the tRNAf ^Met gene region has often been implicated in recombination events of plant mt genomes leading to important rearrangements in gene order.     

Mitochondrial Genome of <Emphasis Type="Italic">Ciona savignyi</Emphasis> (Urochordata,Ascidiacea, Enterogona): Comparison of Gene Arrangement and tRNA Genes with <Emphasis Type="Italic">Halocynthia roretzi</Emphasis> Mitochondrial Genome

The complete nucleotide sequence of the urochordate Ciona savignyi (Ascidiacea, Enterogona) mitochondrial (mt) genome (14,737 bp) was determined. The Ciona mt genome does not encode a gene for ATP synthetase subunit 8 but encodes an additional tRNA^Gly gene (anticodon UCU), as is the case in another urochordate, Halocynthia roretzi (Ascidiacea, Pleurogona), mt genome. In addition, the Ciona mt genome encodes two tRNA^Met genes; anticodon CAT and anticodon TAT. The tRNA^Cys gene is thought to lack base pairs at the D-stem. Thus, the Ciona mt genome encodes 12 protein, 2 rRNA, and 24 tRNA genes. The gene arrangement of the Ciona mt genome differs greatly from those of any other metazoan mt genomes reported to date. Only three gene boundaries are shared between the Halocynthia and the Ciona mt genomes. Molecular phylogenetic analyses based on amino acid sequences of mt protein genes failed to demonstrate the monophyly of the chordates.     

Translational nonsense codon suppression as indicator for functional pre-tRNA splicing in transformed Arabidopsis hypocotyl-derived calli

The transient expression of three novel plant amber suppressors derived from a cloned Nicotiana tRNA^Ser(CGA), an Arabidopsis intron-containing tRNA^Tyr(GTA) and an Arabidopsis intron-containing tRNA^Met(CAT) gene, respectively, was studied in a homologous plant system that utilized the Agro bacterium-mediated gene transfer to Arabidopsis hypocotyl explants. This versatile system allows the detection of β-glucuronidase (GUS) activity by histochemical and enzymatic analyses. The activity of the suppressors was demonstrated by the ability to suppress a premature amber codon in a modified GUS gene. Co-transformation of Arabidopsis hypocotyls with the amber suppressor tRNA^Ser gene and the GUS reporter gene resulted in ~10% of the GUS activity found in the same tissue transformed solely with the functional control GUS gene. Amber suppressor tRNAs derived from intron-containing tRNA^Tyr or tRNA^Met genes were functional in vivo only after some additional gene manipulations. The G3:C70 base pair in the acceptor stem of tRNA^Met(CUA) had to be converted to a G3:U70 base pair, which is the major determinant for alanine tRNA identity. The inability of amber suppressor tRNA^Tyr to show any activity in vivo predominantly results from a distorted intron secondary structure of the corresponding pre-tRNA that could be cured by a single nucleotide exchange in the intervening sequence. The improved amber suppressors tRNA^Tyr and tRNA^Met were subsequently employed for studying various aspects of the plant-specific mechanism of pre-tRNA splicing as well as for demonstrating the influence of intron-dependent base modifications on suppressor activity.     

Sequences of initiator and elongator methionine tRNAs in bean mitochondria

Summary Two bean mitochondria methionine transfer RNAs, purified by RPC-5 chromatography and two-dimensional gel electrophoresis, have been sequenced usingin vitro post-labeling techniques.One of these tRNAs^Met has been identified by formylation using anE. coli enzyme as the mitochondrial tRNA_F ^Met. It displays strong structural homologies with prokaryotic and chloroplast tRNA_F ^Met sequences (70.1–83.1%) and with putative initiator tRNA_m ^Met genes described for wheat, maize andOenothera mitochondrial genomes (88.3–89.6%).The other tRNA^Met, which is the mitochondrial elongator tRNA_F ^Met, shows a high degree of sequence homology (93.3–96%& with chloroplast tRNA_m ^Met, but a weak homology (40.7%) with a sequenced maize mitochondrial putative elongator tRNA_m ^Met gene.Bean mitochondrial tRNA_F ^Met and tRNA_m ^Met were hybridized to Southern blots of the mitochondrial genomes of wheat and maize, whose maps have been recently published (15, 22), in order to locate the position of their genes.     

Sequence analysis of three tRNAPhe nuclear genes and a mutated gene,and one gene for tRNAAla 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.     

Nucleotide sequences of nuclear tRNACys genes from Nicotiana and Arabidopsis and expression in HeLa cell extract

We have recently characterized Nicotiana cytoplasmic (cyt) tRNA_GCA ^Cys as novel UGA suppressor tRNA. Here we have isolated its corresponding (NtC1) and a variant (NtC2) gene from a genomic library of Nicotiana rustica. Both tRNA^Cys genes are efficiently transcribed in HeLa cell nuclear extract and yield mature cyt tRNAs^Cys. Sequence analysis of the upstream region of the RAD51 single-copy gene of the Arabidopsis thaliana genome revealed a cluster of three tRNA^Cys genes which have the same polarity and comprise highly similar flanking sequences. Of the three Arabidopsis tRNA^Cys genes only one (i.e. AtC2) appears to code for a functional gene which exhibits an almost identical nucleotide sequence to NtC1. These are the first sequenced nuclear tDNAs^Cys of plant origin.     

Organization of the genes encoding chalcone synthase in Pisum sativum

To analyze the regulation of defense-related genes by signal molecules produced by phytopathogens, we isolated genes that encode chalcone synthase (CHS) in Pisum sativum. We have obtained seven independent genomic clones that contain at least seven classes of CHS genes, identified by the hybridization analysis to CHS cDNA and by the restriction mapping analysis. Two of the genomic clones (clone 5 and 6) each contain two CHS genes in a tandem repeat. The nucleotide sequence analysis of CHS genomic clone 5 revealed that PsCHS1 and PsCHS2 were corresponding genes of the CHS cDNA clones, pCC6 and pCC2, respectively, as reported earlier. Both genes are interrupted by a single intron of 88 nucleotides with identical sequences, although exonic sequences and 5-flanking sequences are divergent. Nucleotide sequences of the introns in five other classes of CHS genes showed that three classes had an intron of 87 nt with a striking homology to each other, but that the intron of the other two classes of CHS genes showed heterogeneity both in size and nucleotide sequence. 5-upstream regions of PsCHS1 and PsCHS2 did not show sequence homology except the 31 bp identical sequence that contains the CCTACC motif resembling the box-1 sequence. Both PsCHS1 and PsCHS2 genes are shown to be induced by fungal elicitor by a primer extension analysis and a transient transformation analysis using pea protoplasts prepared from suspension cultured-cells.     

The location of untranscribed DNA sequences within ras genes essential for eliciting plant growth suppression

Three heterologous ras DNA-coding sequences and their deletion derivatives were introduced into plant cells to investigate the role of the ras-coding sequences, especially conserved regions, in eliciting growth inhibition. All three ras-coding sequences caused a similar inhibition of plant cell growth, and it was the conserved coding regions which were responsible for this inhibitory effect. The 493 bp conserved region within the v-Ha-ras-coding sequence was studied further, and was shown to be responsible for the inhibitory effect. This region is conserved (over 44%) among the three ras genes studied and encodes a catalytic region of the Ras protein. Small deletions at either the 5 or 3 end of this 493 bp sequence could abolish or dramatically reduce the inhibitory effect. A 36 bp region at the 5 end of the 493 bp region was found to be highly conserved between v-Ha-ras and eight different plant ras or ras-related genes based upon analysis of published sequences. Small deletions affecting this highly conserved 36 bp region completely abolished the inhibitory effect, while deletion of a similar number of base pairs in adjacent regions did not. These results indicate that plant growth inhibition by ras DNA requires small regions at both ends of the 493 bp conserved region.     

Molecular analysis of the aspartate kinase-homoserine dehydrogenase gene from Arabidopsis thaliana

The gene encoding Arabidopsis thaliana aspartate kinase (ATP:L-aspartate 4-phosphotransferase, EC 2.7.2.4) was isolated from genomic DNA libraries using the carrot ak-hsdh gene as the hybridizing probe. Two genomic libraries from different A. thaliana races were screened independently with the ak probe and the hsdh probe. Nucleotide sequences of the A. thaliana overlapping clones were determined and encompassed 2 kb upstream of the coding region and 300 bp downstream. The corresponding cDNA was isolated from a cDNA library made from poly(A)⁺-mRNA extracted from cell suspension cultures. Sequence comparison between the Arabidopsis gene product and an AK-HSDH bifunctional enzyme from carrot and from the Escherichia coli thrA and metL genes shows 80%, 37.5% and 31.4% amino acid sequence identity, respectively. The A. thaliana ak-hsdh gene is proposed to be the plant thrA homologue coding for the AK isozyme feedback inhibited by threonine. The gene is present in A. thaliana in single copy and functional as evidenced by hybridization analyses.The apoprotein-coding region is interrupted by 15 introns ranging from 78 to 134 bp. An upstream chloroplast-targeting sequence with low sequence similarity with the carrot transit peptide was identified. A signal sequence is proposed starting from a functional ATG initiation codon to the first exon of the apoprotein. Two additional introns were identified: one in the 5 non-coding leader sequence and the other in the putative chloroplast targeting sequence. 5 sequence analysis revealed the presence of several possible promoter elements as well as conserved regulatory motifs. Among these, an Opaque2 and a yeast GCN4-like recognition element might be relevant for such a gene coding for an enzyme limiting the carbon-flux entry to the biosynthesis of several essential amino acids. 3 sequence analysis showed the occurrence of two polyadenylation signals upstream of the polyadenylation site.This work is the first report of the molecular cloning of a plant ak-hsdh genomic sequence. It describes a promoter element that may bring new insights to the regulation of the biosynthesis of the aspartate family of amino acids.Abbreviations AK aspartate kinase - HSDH homoserine dehydrogenase - ID intermediate domain - Tp transit peptide     

Isolation of an Arabidopsis cDNA sequence encoding a 22 kDa calcium-binding protein (CaBP-22) related to calmodulin

Complementary DNA sequences were isolated from a library of cloned Arabidopsis leaf mRNA sequences in gt10 that encoded a 21.7 kDa polypeptide (CaBP-22), which shared 66% amino acid sequence identity with Arabidopsis calmodulin. The putative Ca²⁺-binding domains of CaBP-22 and calmodulin, however, were more conserved and shared 79% sequence identity. Ca²⁺ binding by CaBP-22, which was inferred from its amino acid sequence similarity with calmodulin, was demonstrated indirectly by Ca²⁺-induced mobility shifting of in vitro translated CaBP-22 during SDS-polyacrylamide gel electrophoresis. CaBP-22 is encoded by a ca. 0.9 kb mRNA that was detected by northern blotting of leaf poly(A)⁺ RNA; this mRNA was slightly larger than the 809 bp CaBP-22 cDNA insert, indicating that the deduced amino acid sequence of CaBP-22 is near full-length. CaBP-22 mRNA was detected in RNA fractions isolated from leaves of both soil-grown and hydroponically grown Arabidopsis, but below the limits of detection in RNA isolated from roots, and developing siliques. Thus, CaBP-22 represents a new member of the EF-hand family of Ca²⁺-binding proteins with no known animal homologue and may participate in transducing Ca²⁺ signals to a specific subset of response elements.     

Nucleotide sequence of the split tRNAUAALeu gene from Vicia faba chloroplasts: evidence for structural homologies of the chloroplast tRNALeu intron with the intron from the autosplicable Tetrahymena ribosomal RNA precursor

Summary The gene encoding the tRNA _UAA ^Leu from broad bean chloroplasts has been located on a 5.1 kbp long BamHI fragment by analysis of the DNA sequence of an XbaI subfragment. This gene is 536 bp long and is split in the anticodon region. The 451 bp long intron shows high sequence homology over about 100 bp from each end with the corresponding regions of the maize chloroplast tRNA _UAA ^Leu intron. These conserved sequences are probably involved in the splicing reaction, for they can be folded into a secondary structure which is very similar to the postulated structure of the intron from the autosplicable ribosomal RNA precursor of Tetrahymena. Very little sequence conservation is found in the 5-and 3-flanking regions of the broad bean and maize chloroplast tRNA _UAA ^Leu genes.     

Genes for tRNAAsp,tRNAPro, tRNATyr and two tRNAsSer in wheat mitochondrial DNA

We have begun a systematic search for potential tRNA genes in wheat mtDNA, and present here the sequences of regions of the wheat mitochondrial genome that encode genes for tRNA^Asp (anticodon GUC), tRNA^Pro (UGG), tRNA^Tyr (GUA), and two tRNAs^Ser (UGA and GCU). These genes are all solitary, not immediately adjacent to other tRNA or known protein coding genes. Each of the encoded tRNAs can assume a secondary structure that conforms to the standard cloverleaf model, and that displays none of the structural aberrations peculiar to some of the corresponding mitochondrial tRNAs from other eukaryotes. The wheat mitochondrial tRNA sequences are, on average, substantially more similar to their eubacterial and chloroplast counterparts than to their homologues in fungal and animal mitochondria. However, an analysis of regions 150 nucleotides upstream and 100 nucleotides downstream of the tRNA coding regions has revealed no obvious conserved sequences that resemble the promoter and terminator motifs that regulate the expression of eubacterial and some chloroplast tRNA genes. When restriction digests of wheat mtDNA are probed with ³²P-labelled wheat mitochondrial tRNAs, <20 hybridizing bands are detected, whether enzymes with 4 bp or 6 bp recognition sites are used. This suggests that the wheat mitochondrial genome, despite its large size, may carry a relatively small number of tRNA genes.     

RNP-T,a ribonucleoprotein from Arabidopsis thaliana,contains two RNP-80 motifs and a novel acidic repeat arranged in an α-helix conformation

We have isolated a 1148 bp long cDNA clone encoding an RNA-binding protein in Arabidopsis. Several partial cDNA clones were isolated by screening an Arabidopsis gt11 expression library for the binding of DNA. One of these clones was used as a probe to isolate a full-length clone. The 329 amino acid protein, termed RNP-T, contains in its carboxy terminus two adjacent RNP-80 motifs, a previously described 80 amino acid long conserved putative RNA-binding domain. Each RNP-80 motif includes both consensus short sequences, RNP1 and RNP2, which are separated by 33 amino acids. We have identified an acidic domain of 54 amino acids, which is located amino-terminal to the RNP-80 motifs. Seven tandem repeats of a hexamer are present within this domain. This acidic domain has a potential -helix conformation. We propose that the acidic patch might play a role in protein-protein interaction.