Chloroplast tRNA Research Articles

Localization and nucleotide sequences of the tRNA GCC Gly , tRNA GUC Asp and tRNA GCA Cys genes from wheat chloroplast

The location on the wheat chloroplast DNA map and the nucleotide sequences of the genes coding for tRNA GCC (Gly) (trnG-GCC), tRNA GUC (Asp) (trnD-GUC) and tRNA GCA (Cys) (trnC-GCA) have been determined. These three genes are located in the large single copy region of the chloroplast genome, about half-way between one of the inverted repeats and the gene for the α subunit of ATP synthase. They are located on two Bam H1 fragments, called B6 and B18 by Bowmanet al. (1), which are separated by about 450 bp and which were cloned in our laboratory to allow sequencing. ThetrnD-GUC andtrnC-GCA sequences show 98.6 and 89% homology, respectively, with the corresponding spinach chloroplast tRNA genes sequences (2), which are the only other higher plant chloroplasttrnD-GUC andtrnC-GCA sequenced so far, while no othertrnG-GCC sequence has been published. ThetrnG-GCC sequence shows only 58% homology with the corresponding gene sequence inEuglena chloroplasts (3).

Sequence of histidyl tRNA, present as a chloroplast insert in mtDNA ofZea mays

Unfractionated tRNA, isolated from maize mitochondria, has been specifically labeled at the -CCA end and used to recover a tRNA gene-bearing fragment from a clone bank of maize mitochondrial DNA. This gene has been mapped, sequenced and found to carry the anticodon for histidine. The sequence of the gene and that of bases in its near vicinity are identical to maize chloroplast tRNA(His), although sequences more distant on the fragment are not homologous with cpDNA. The junction of the cpDNA insert has been sequenced.

Identification and mutational analysis of the promoter for a spinach chloroplast transfer RNA gene.

A transcription extract from purified spinach chloroplast was used to test chloroplast DNA sequences for their function as promoter elements. Chloroplast tRNA genes are correctly transcribed in the extract by a soluble RNA polymerase, and precursor molecules are processed into mature tRNAs. Transcription of the spinach chloroplast tRNA2Met gene (trnM2) in vitro requires 5' upstream DNA sequences. Deletion of 5' DNA sequences with exonuclease Bal31 was used to establish the 5' boundary of the promoter region. This boundary is part of a DNA sequence with partial homology to the prokaryotic -35 region. Seventeen base pairs downstream from this sequence a DNA sequence occurs which is homologous to the prokaryotic -10 region. We used synthetic oligonucleotides fused to trnM2 5' deletion mutants to create insertions, deletions and base substitutions in these regions. Internal deletion mutants demonstrated that the -10 promoter element is also required for transcription in vitro. The arrangement of DNA sequences recognised by the chloroplast RNA polymerase resembles the prokaryotic promoter organization.

Transfer RNA gene mapping studies on chloroplast DNA from Chlamydomonas reinhardii

Total tRNA of Chlamydomonas reinhardii was fractionated by 2-dimensional gel electrophoresis. Sixteen tRNAs specific for eleven amino acids could be identified by aminoacylation with Escherichia coli tRNA synthetases. Hybridization of these tRNAs with chloroplast restriction fragments allowed for the localization of the genes of tRNA Tyr, tRNA Pro, tRNA Phe (2 genes), tRNA Ile (2 genes) and tRNA His (2 genes) on the chloroplast genome of C. reinhardii. The genes for tRNA Ala (2 genes), tRNA Asn and tRNA Leu were mapped by using individual chloroplast tRNAs from higher plants as probes.

Structure of bean mitochondrial tRNA Phe and localization of the tRNA Phe gene on the mitochondrial genomes of maize and wheat

Bean mitochondrial tRNA Phe, purified by RPC-5 chromatography and two-dimensional gel electrophoresis, has been sequenced using in vitro post-labeling techniques. It is the first plant mitochondrial tRNA sequenced. It shows 76% homology with bean chloroplast tRNA Phe and has many features characteristic of prokaryotic tRNAs Phe. It was used as a probe to localize the tRNA Phe gene on the mitochondrial genomes of maize and wheat.

Distribution of transfer RNA genes in thePisum sativum chloroplast DNA

Purified chloroplast tRNAs were isolated fromPisum sativum leaves and radioactively labeled at their 3' end using tRNA nucleotidyl transferase and α(32)P-labeled CTP. Pea ctDNA was fragmented using a number of restriction endonucleases and hybridized with thein vitro labeled chloroplast tRNAs by DNA transfer method. Genes for tRNAs have been found to be dispersed throughout the chloroplast genome. A closer analysis of the several hybrid regions using recombinant DNA plasmids have shown that tRNA genes are localized in the chloroplast genome in both single and multiple arrangements. Two dimensional gel electrophoresis of total ct tRNA have identified 36 spots. All of them have been found to hybridize withPisum sativum ctDNA. Using recombinant clones, 30 of the tRNA spots have been mapped inPisum sativum ctDNA.

The nucleotide sequences of a cytoplasmic and a chloroplast tRNATyr from Scenedesmus obliquus.

The nucleotide sequences of two species of tyrosine accepting tRNA from the eukaryotic green alga Scenedesmus obliquus have been determined. The sequence of the cytoplasmic tRNATyr is: (sequence in text) This is the first chloroplast tRNATyr species to be sequenced.

Transfer RNAs and tRNA genes of Vicia faba chloroplasts

Isolated chloroplasts from broad bean and common bean were found to contain a minimum of 31 and 32 tRNA species, respectively. These individual chloroplast tRNAs were (32)P-labeled in vitro and hybridized to DNA fragments obtained upon digestion of broad bean and common bean chloroplast DNAs with various restriction endonucleases. At least 30 tRNA genes were localized on the physical maps of the two chloroplast genomes. Comparison of the broad bean tRNA gene map to that of common bean revealed DNA sequence rearrangements, such as inversions, insertions/ deletions and duplications, within these two members of the Legu minosae family.

Chloroplast tRNA Gly gene contains a long intron in the D stem: Nucleotide sequences of tobacco chloroplast genes for tRNA Gly (UCC) and tRNA Arg (UCU)

The nucleotide sequences of tobacco chloroplast genes for tRNA(Gly) (UCC) and tRNA(Arg) (UCU) have been determined. The tRNA(Gly) gene has a 691-base-pair intron located in the D stem while the tRNA(Arg) gene does not have any intron. The tRNA(Gly) and tRNA(Arg) genes are encoded on the same strand and separated by a 169-base-pair spacer. The tRNA(Gly) gene is transcribed as a 900-base precursor RNA molecule. The tRNA(Gly) and tRNA(Arg) deduced from the DNA sequences show 84% and 55% sequence homologies with Escherichia coli tRNA(Gly) (UCC) and phage T4 tRNA(Arg) (UCU), respectively.

Nucleotide sequences of three soybean chloroplast tRNAsLeu and re-examination of bean chloroplast tRNA2Leu sequence.

The nucleotide sequences of soybean chloroplast tRNAsLeu were determined using post-labeling techniques. Comparison of the primary structures of soybean chloroplast tRNAsLeu with their bean, maize and spinach counterparts only show few base differences. Contrary to previously published results (1) a re-examination of bean tRNALeu sequence shows that this tRNA resembles soybean and maize tRNA2Leu in structure.

DNA sequences for the Zea mays tRNA genes tV-UAC and tS-UGA: tV-UAC contains a large intron

The chloroplast genome contains genes for a large and probably complete set of tRNAs. These genes are unique in sharing attributes of both nuclear and bacterial tRNA genes. Two chloroplast tRNA genes from Zea mays are described here. tV-UAC, encoding a valine tRNA with the anticodon UAC, contains a 603 bp intron and is highly homologous, both in coding regions and in the intron, to the analogous gene from tobacco described by Deno et al. (Nucleic Acids Res 10:7511-7520, 1982). It is located near the gene for the beta and epsilon subunits of the CF1 complex. (Krebbers et al.: Nucleic Acids Res 10:4985-5002, 1982). The gene tS-UGA, encoding a serine tRNA with the anticodon UGA, is located 41 kbp 3' to tV-UAC. Both genes contain promoter-like sequences in their 5' flanking regions.

Mapping of transfer RNA genes on tobacco chloroplast DNA

Tobacco chloroplast tRNAs have been purified by two-dimensional polyacrylamide gel electrophoresis, identified by aminoacylation, labelled at their 3'-end and hybridized to tobacco chloroplast DNA restriction fragments, in order to establish a tRNA gene map. These hybridization studies have revealed the localization of at least seven genes in each inverted repeat region, a minimum of 22 tRNA genes in the large single copy region and one tRNA gene in the small single copy region. Comparison of the tobacco chloroplast tRNA gene map to that of maize shows many similarities, but also some differences suggesting that DNA sequence rearrangements have occurred in the chloroplast genome during evolution.

Euglena gracilis chloroplast transfer RNA transcription units. Nucleotide sequence analysis of a tRNAThr-tRNAGly-tRNAMet-tRNASer-tRNAGln gene cluster.

The arrangement and the nucleotide sequence of the tRNA genes in the 2.0-kilobase-pair EcoRI restriction fragment EcoQ of Euglena gracilis Klebs, strain Z Pringsheim chloroplast DNA have been determined. This fragment, cloned in pBR325 to form the plasmid pEZC300, contains five tRNA genes. The DNA insert of this plasmid, a known tRNA gene locus (Orozco, E.M., Jr., and Hallick, R.B. (1982) J. Biol. Chem. 257, 3258-3264) has been mapped by Southern gel analysis using a 32P-labeled oligodeoxynucleotide tRNA gene probe. The DNA sequence of 870 base pairs (bp) from EcoQ containing the entire tRNA gene locus was determined. The organization of this tRNA gene cluster on the E. gracilis chloroplast chromosome is tRNAUUGGln-14-BP spacer-RNAGCUSer-175-bp spacer-tRNACAUMet-12-bp spacer-tRNAGCCGly-5-bp spacer-tRNAUGUThr. The tRNAUUGGln and tRNAGCUSer gene sequences are of the opposite polarity as the other three gene sequences, but of the same polarity as the rRNA genes. The tRNAMet gene is a putative initiator tRNA. The five tRNA genes are separated and flanked by A-T-rich spacer sequences. This gene arrangement is consistent with the model that E. gracilis chloroplast tRNA genes are transcribed into multicistronic tRNA precursors. The DNA sequences have been used to deduce the primary and secondary structures of the tRNAs.

Nucleotide sequences of five maize chloroplast transfer RNA genes and their flanking regions.

Maize chloroplast tRNA genes encoding tRNA3Ser, tRNAPhe, tRNA2Thr, tRNA2Leu, and tRNAmMet, and their flanking regions have been sequenced. All five gene sequences show features commonly found in the few chloroplast tRNA genes sequenced so far; none of them encodes the 3'-terminal CCA triplet and none of them shows abnormal loop or stem lengths as seen in some genes for mammalian mitochondrial tRNAs. The 5'-flanking regions of some of these tRNA genes contain nucleotide stretches strongly homologous to -35 and -10 promoter regions of bacterial genes. Analysis of these conserved regions together with those seen in other plastid genes yields the consensus sequences ATTGANA at "-35" and TAAGAT at "-10." The 3'-flanking regions of some tRNA genes show dyad symmetries followed by a stretch of Thds. These and other regions may be involved in transcription termination or transcript processing.

Sexual differentiation in Mercurialis annua L. (2n=16): Evidence for a control system of nucleo-cytoplasmic relationships by the study of tyrosyl-tRNAs from ‘developmental mutants’ of male sterility

In ‘developmental mutants’ of staminogenesis carrying sensitive cytoplasm (male sterile strains of the dioecious plant Mercurialis annua), the increasing levels of tRNAs tyr aminoacylation as a function of the degree of sterility confirm the classification of strains previously obtained by means of genetics and physiology: the series begins with fertile males with normal cytoplasm (0.61), restored fertiles (0.74), semisteriles (0.74) and total steriles (0.84), (all with S cytoplasm) and ends with normal females (1.06). The fractionation of tyrosyl-tRNAs shows that two chloroplast tyrosine tRNAs isoacceptors absent in normal fertile staminogenesis and present in female organogenesis are always expressed in strains with S cytoplasm, in the presence or absence of fertility nuclear restorer genes. In S cytoplasm the chloroplast escapes from its normal regulation during sexual differentiation. In restored fertile males (S cytoplasm), the nuclear restorer genes do not suppress but only compensate the chloroplast disfunction that remains of female type.

Selective in vitro transcription of chloroplast genes.

Transcription of Euglena gracilis chloroplast genes has been investigated by using in vitro transcription systems. A DNA-dependent RNA polymerase responsible for the transcription of rRNA genes has been isolated as a nucleoprotein complex (transcriptionally active chromosome). The RNA polymerase remains tightly bound to the chloroplast DNA template and does not initiate transcription with cloned chloroplast genes. A transcriptionally active extract has been prepared from intact Euglena chloroplasts. The soluble RNA polymerase in this extract recognizes cloned chloroplast tRNA genes and tRNA-sized products have been detected after transcription. The tRNA-sized molecules specifically hybridize to the tRNA genes in the plasmid DNA. At least five tRNA-sized products have been identified from transcription of a trnY1trnH1-trnM1-trnE1-trnW1-trnG1 cluster. Evidence is also presented that processing enzymes in the chloroplast-extract can recognize a polycistronic tRNAVal-tRNAAsn-tRNAArg precursor and process it into tRNA-sized molecules. Truncated templates have been used to demonstrate that the chloroplast tRNA genes are actively transcribed. From a comparison of 5' flanking sequences in chloroplast tRNA genes, a consensus sequence which might function as a promoter, has been identified. The properties of the RNA polymerase involved in the transcription of chloroplast rRNA genes and tRNA genes have been investigated and compared.

Euglena gracilis chloroplast transfer RNA transcription units. Nucleotide sequence analysis of a tRNATyr-tRNAHis-tRNAMet-tRNATrp-tRNAGlu-tRNAGly gene cluster.

A tRNA coding locus in the Bam-Sal 9 region of Euglena gracilis Pringsheim strain Z chloroplast DNA was chosen for detailed study. This DNA contains the previously mapped tRNA coding sequences of the adjacent Euglena chloroplast EcoRI products of EcoV and EcoH (Orozco, E. M., Jr., and Hallick, R. B. (1982) J. Biol. Chem. 257, 3258-3264). The 3.2-kilobase pair Bam-Sal 9 fragment was cloned into the BamHI and SalI cut plasmid vector pBR322, resulting in the recombined plasmid pPG76. The tRNA coding locus was mapped to a region of Bam-Sal 9 that contains portions of both EcoH and EcoV. The DNA sequence of 1-kilobase pair Bam-Sal 9, containing the entire tRNA coding locus, was determined. A cluster of six tRNA genes was found. The gene organization is as follows, where bp is base pair: tRNATyrGUA-64 bp spacer-tRNAHisGUG-14 bp spacer-tRNAMetCAU-4 bp spacer-tRNATrpCCA-27 bp spacer-tRNAGluUUC-6 bp spacer-tRNAGlyUCC. The tRNAMetCAU is believed to be an elongator tRNA. The first four genes are within EcoV. The EcoRI cleavage site that separates EcoV and EcoH is in the tRNAGlu gene. The tRNAGly gene is in EcoH. This is the largest known chloroplast tRNA gene cluster.

Fractionation and identification of Euglena gracilis cytoplasmic and chloroplastic tRNAs and mapping of tRNA genes on chloroplast DNA

The cytoplasmic and chloroplast tRNAs of Euglena gracilis Z strain were fractionated by two-dimensional gel electrophoresis and identified by aminoacylation. Purified chloroplast tRNAs, labeled in vitro with |(32)P|, were hybridized to endonuclease restriction fragments of chloroplast DNA, allowing the corresponding tRNA genes to be localized on the physical map of Euglena chloroplast DNA.

An evaluation of mitochondrial tRNA gene evolution and its relation to the genetic code.

Extensive sequence data on mitochondrial (mt) tRNAs give for the first time an opportunity to evaluate tRNA gene evolution in this organelle. Deductions from these gene structures relate to the evolution of tRNA genes in other cellular systems and to the origin of the genetic code. Mt tRNAs, in contrast to the prokaryotic nature of chloroplastic tRNA structure, can not at the present time be definitely related to either prokaryotic or eukaryotic tRNAs, probably because of a higher mutation rate in mitochondria. Fungal mt tRNAs having the same anticodon and function are generally similar enough to be considered homologous. Comparisons af all mt tRNA sequences contained in the same mitochondrion indicate that some tRNAs originated by duplication of a prototypic gene which, after divergence, led to tRNAs having different amino acid specificities. The deviant mt genetic code, although admittedly permitting a simpler decoding mechanism, is not useful in determining whether the origin of mitochondria had preceded or was derived from prokaryotes or eukaryotes, since the genetic code is variable even among mitochondria. Variants of the mt genetic code lead to speculation on the nature of the primordial code and its relation to the present "universal" code.

Euglena gracilis chloroplast transfer RNA transcription units. I. Physical map of the transfer RNA gene loci.

The locations of transfer RNA genes with respect to the restriction endonuclease cleavage map of Euglena gracilis Klebs, strain Z Pringsheim chloroplast DNA have been determined. Purified chloroplast tRNAs were treated with snake venom phosphodiesterase to remove the 3'-CCA terminus, and radioactively labeled by the action of Escherichia coli tRNA nucleotidyltransferase in the presence of [alpha-32P]CTP. Chloroplast DNA was treated individually and with combinations of the enzymes Bal I, Bam HI, Eco RI, Pst I, Pvu II, Sal I, and Xho I. The location of tRNA genes with respect to the cleavage sites for these enzymes was determined by hybridization of the 32P-labeled tRNAs to membrane filter blots of the chloroplast DNA restriction nuclease fragments following gel electrophoresis. The 145-kilobase pair genome was resolved into nine areas of strong tRNA hybridization, separated by areas of weak or no tRNA hybridization. The loci of tRNA genes are within the Eco RI fragments Eco A, B, G, H, I, J', P, Q, and V.