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PubMed1995

Characterization and partial purification of two pre-tRNA 5'-processing activities from Daucus carrota (carrot) suspension cells.

Two distinct RNase P-like activities which cleave leader sequences from pre-tRNA molecules to give mature 5' ends have been identified in carrot suspension-culture cells. An Escherichia coli pre-tRNA(Phe) and a tobacco pre-tRNA(Tyr) were transcribed in vitro then used as substrates for processing reactions in a cell-free extract. The pre-tRNA(Tyr) transcript was used to establish optimal salt and divalent cation requirements for processing. Kinetic experiments were then carried out on both substrates to determine if 5' and 3' processing were ordered. Primer extension analysis of processing intermediates and stable products verified that an ammonium sulfate fraction of the extract was indeed capable of accurately processing the 5' ends of both pre-tRNAs. Subsequent fractionation of the 5' end-processing activity by chromatography on phosphocellulose revealed two distinct activities, eluting at 0.1 and 0.5 M KCI, when assayed with the tobacco pre-tRNA(Tyr) substrate. When the same fractions were assayed with the E. coli pre-tRNA(Phe), only the 0.1 M KCI fraction exhibited activity. Both of the active fraction display sensitivity to micrococcal nuclease (MN) and proteinase K indicating each is a ribonucleoprotein, a result not seen with other plant RNase Ps. Subsequent FPLC fractionation of the two activities using Mono Q and Mono S columns demonstrated that the two activities could be further distinguished on the basis of their chromatographic behavior.

Base Sequence↗

PubMed1990

Genes for tRNA(Gly), tRNA(His), tRNA(Lys), tRNA(Phe), tRNA(Ser) and tRNA(Tyr) are encoded in Oenothera mitochondrial DNA.

The genes coding for tRNA(Gly), tRNA(His), tRNA(Lys), tRNA(Phe), tRNA(Ser) and tRNA(Tyr) have been identified in Oenothera mitochondrial DNA. Sequence analysis of these genes and their surrounding sequences are presented and compared with other known tRNA genes from plant mitochondria. All six deduced tRNA sequences can be folded into the classical cloverleaf structure model. Only the tRNA(His) gene shows high homology with the corresponding chloroplast gene and thus appears to be derived from a transfer event of chloroplast sequences into the mitochondrial genome. The sequences surrounding this gene, however, show little similarity with the chloroplast genome. The other five deduced tRNAs display a much lower similarity with their chloroplast counterparts and thus appear to be genuine mitochondrial tRNAs. These tRNAs are highly conserved between monocots and dicots with maximally three nucleotides differing between the Oenothera sequences and their wheat homologues. A purine-rich sequence is found upstream of each tRNA gene in Oenothera, similar to wheat mitochondrial tRNA genes, that could be involved in transcription signalling.

Base Sequence↗

PubMed1989

Effect of spermine on the efficiency and fidelity of the codon-specific binding of tRNA to the ribosomes.

Binding of the yeast Tyr-tRNA and Phe-tRNA to the A site, and the binding of their acetyl derivatives to the P site of poly(U11,A)-programmed Escherichia coli ribosomes was studied. Spermine stimulated the rate of binding of both tRNAs at least threefold, enabling more than 90% final saturation of both ribosomal binding sites. The effect is observed when the tRNAs, but not ribosomes or poly(U11,A), are preincubated with polyamine. Regardless of the binding site, optimal saturation was reached at spermine/tRNA molar ratios of 3 for tRNA(Phe) and 5 for tRNA(Tyr). The same low spermine/tRNA ratios were previously reported to stabilize the conformation of these tRNAs in solution. On the other hand, the messenger-free, EF-Tu- and EF-G-dependent polymerization of lysine from E. coli Lys-tRNA is drastically reduced, while the poly(A)-directed polymerization is stimulated by spermine through a wide range of Mg2+ concentrations. Misreading of UUU codons as isoleucine, assayed by the A-site binding of E. coli Ile-tRNA, is also inhibited by spermine. All these results demonstrate that spermine increases the efficiency and accuracy of a series of macromolecular interactions leading to the correct incorporation of an amino acid into protein, at the same time preventing some unspecific or erroneous interactions. From the analogy with its known structural effects, it can be inferred that spermine does so by conferring on the tRNA a specific biologically functional conformation.

Binding Sites↗

PubMed1994

Nucleotide correlations that suggest tertiary interactions in the TV-replacement loop-containing mitochondrial tRNAs of the nematodes, Caenorhabditis elegans and Ascaris suum.

In the predicted secondary structures of 20 of the 22 tRNAs encoded in mitochondrial DNA (mtDNA) molecules of the nematodes, Caenorhabditis elegans and Ascaris suum, the T psi C arm and variable loop are replaced with a loop of 6 to 12 nucleotides: the TV-replacement loop. From considerations of patterns of nucleotide correlations in the central regions of these tRNAs, it seems highly likely that tertiary interactions occur within five sets of binary and ternary combinations of nucleotides that correspond in location to nucleotides known to be involved in tertiary interactions in yeast tRNA(Phe) and other standard tRNAs. These observations are consistent with the nematode TV-replacement loop-containing mt-tRNAs being folded into a similar L-shaped functional form to that demonstrated for standard tRNAs, and for the bovine DHU (dihydrouridine) arm replacement-loop-containing mt-tRNA(Ser(AGY)). However, the apparent occurrence in nematode mt-tRNAs of tertiary bonds common to standard tRNAs contrasts with the situation in bovine mt-tRNA(Ser(AGY)) where the functional form is dependent on an almost unique set of tertiary interactions. Because three of the proposed conserved tertiary interactions in the nematode mt-tRNAs involve nucleotides that occur in the variable loop in standard tRNAs, it seems more likely that in nematode mt-tRNAs it is the T psi C arm rather than the variable loop that has undergone the greatest proportional decrease in nucleotide number.

Animals↗

PubMed1996

Nonradioactive labeling of RNA.

A modified ribodinucleotide (named pSEEp) has been synthesized using commercially available components on a DNA synthesizer. The presence of a 3'-terminal primary amino group permits its coupling to a range of nonradioactive labels, exemplified here by fluorescein. The product of chemical derivatization of the parent dinucleotide is a good substrate for T4 RNA ligase-mediated coupling to RNA under very mild conditions; the target RNA is not itself subjected to chemical treatment. The well-defined product of this terminal labeling may be analyzed by, for example, the use of a fluorescence-based DNA sequencer. The applications documented serve to demonstrate the power of this approach suggesting that any procedure depending on the detection or targeted tagging of RNA may be adapted to using pSEEp and its derivatives.

Fluorescein↗

PubMed1995

Identification of base-triples in RNA using comparative sequence analysis.

Comparative sequence analysis has proven to be a very efficient tool for the determination of RNA secondary structure and certain tertiary interactions. However, base-triples, an important RNA structural element, cannot be predicted accurately from sequence data. We show here that the poor base correlations observed at base-triple positions are the result of two factors. (1) Base covariation is not as strictly required in triples as it is in Watson-Crick pairs. (2) Base-triple structures are less conserved among homologous molecules. A particularity of known triple-helical regions is the presence of multiple base correlations that do not reflect direct pairing. We suggest that natural mutations in base-triples create structural changes that require compensatory mutations in adjacent base-pairs and triples to maintain the triple-helix conformation. On the basis of these observations, we devised two new measures of association that significantly enhance the base-triple signal in correlation studies. We evaluated correlations between base-pairs and single stranded bases, and correlations between adjacent base-pairs. Positions that score well in both analyses are the best triple candidates. This procedure correctly identifies triples, or interactions very close to the proposed triples, in type I and type II tRNAs and in the group I intron.

Base Composition↗

PubMed1998

Higher-order structure and thermal instability of bovine mitochondrial tRNASerUGA investigated by proton NMR spectroscopy.

Although mammalian mitochondrial serine-specific tRNA with the anticodon UGA (tRNASerUGA) appears to possess an almost normal cloverleaf secondary structure, it exhibits an extraordinarily low melting temperature (tm). An in vitro tRNASerUGA transcript without modified nucleosides had an even lower tm and slightly less hyperchromicity, but its tertiary structure was apparently very similar to that of the native counterpart judging from its aminoacylation activity and the body of experimental evidence so far obtained for canonical tRNAs. The transcript was therefore used to investigate the higher-order structure and thermal instability of tRNASerUGA. 1H-NMR analysis of the transcript showed that it takes a nearly L-shaped tertiary structure with similar tertiary base-pairings to those found in yeast tRNAPhe, which is representative of canonical tRNAs. However, magnesium ion titration revealed that Mg2+ affected the chemical shifts of the tRNASerUGA transcript differently than those of canonical tRNAs so far studied; the former was less sensitive toward Mg2+, especially in the D-arm region. This observation was confirmed by NMR analysis with paramagnetic manganese ion titration. Hill plots derived from the CD spectral changes caused by titration with Mg2+ suggested that the tRNASerUGA transcript had fewer Mg2+ binding sites than those of yeast tRNAPhe as well as its transcript, a finding that was consistent with the NMR data. We thus surmise that the thermal instability of both the transcript and tRNASerUGA itself originated from a reduction in the number of the divalent ion binding sites within the tRNA molecule. These results suggest a new type of thermal instability for mitochondrial tRNA.

Animals↗

PubMed1999

Determination of the angle between the acceptor and anticodon stems of a truncated mitochondrial tRNA.

Significant departures from the canonical (cloverleaf) secondary structure of transfer (t)RNAs can be found among the mitochondrial (m)tRNAs of higher metazoans; these mtRNAs thus pose a challenge to the concept of an invariant, L-shaped tertiary conformation for all tRNAs. For bovine mtRNASer(AGY), which lacks the entire "dihydrouridine" (dhU) arm, two distinct tertiary models have been proposed: the first model preserves the L-shaped conformation at the expense of overall size; the second model preserves the absolute distance between the 3' terminus and the anticodon loop, while allowing the acceptor-anticodon interstem angle to vary. We have tested the central predictions of these two models by performing a series of transient electric birefringence measurements on bovine mtRNASer(AGY) constructs in which the aminoacyl-acceptor and anticodon stems were each extended by approximately 70 bp. This mtRNA species is particularly amenable to analysis, since the native bovine (heart) mtRNA is completely unmodified outside of the anticodon loop. For magnesium ion concentrations above 1 mM, the interstem angle for the extended mtRNA, 120(+/-5) degrees, is approximately 50% larger than the corresponding angle for yeast tRNAPhe (70-80 degrees) under the same ionic conditions. Furthermore, the interstem angles of the two tRNAs exhibit strikingly different responses to the addition of magnesium ions: the interstem angle for yeast tRNAPhe is reduced by nearly 50 % upon addition of 2 mM magnesium ions, whereas the angle for mtRNASer(AGY) increases by about 10%. Our data thus support a central prediction of the second model; namely, that truncated mtRNAs will possess more open interstem angles. In addition, we demonstrate that birefringence amplitude data can be used to provide model-independent estimates for the interstem angles.

Animals↗

PubMed1995

Different rates of mitochondrial DNA sequence evolution in Kirk's dik-dik (Madoqua kirkii) populations.

We have investigated evolutionary rates of the mitochondrial genome among individuals of Madoqua kirkii using the relative rate test. Our results demonstrate that individuals of two chromosome races, East African cytotype A and Southwest African cytotype D, evolve about 2.3 times faster than East African cytotype B. Cytogenetic changes, DNA repair efficiency, mutagens, and more likely, hitherto unrecognized factors will account for the rate difference we have observed. Our results suggest additional caution when using molecular clocks in the estimation of divergence time, even within lineages of closely related taxa. Rate heterogeneity in microevolutionary timescales represents a potentially important aspect of basic evolutionary processes and may provide additional insights into factors which affect genome evolution.

Animals↗

PubMed1996

Mitochondrial gene order adjacent to the control region in crocodile, turtle, and tuatara.

We used the polymerase chain reaction and sequencing of mitochondrial gene junctions adjacent to the 5' end of the control region (light strand orientation) and the 3' end of ND6 to assess whether a representative crocodilian, turtle, or tuatara shares a unique mitochondrial gene order that is found in birds but not in mammals or amphibians. Turtles and crocodiles have the same gene order as mammals, except that crocodile has a tRNAPhe gene inserted between tRNAPro and the 5' end of the control region. Two different arrangements were detected at the 5' end of the control region in the tuatara, one resembling the mammalian (but with tRNAThr absent) and one resembling the avian gene order. Our data are consistent with the hypothesis that some tuatara mtDNAs within a single individual have undergone a deletion that removed the genes coding for cytochrome b and tRNAPro as well as 87 bp of the control region.

Alligators and Crocodiles↗

PubMed2001

Molecular systematics of the western rattlesnake, Crotalus viridis (Viperidae), with comments on the utility of the D-loop in phylogenetic studies of snakes.

Crotalus viridis, the western rattlesnake, ranges throughout western North America and has been divided into at least eight subspecies. However, the validity of and relationships among these subspecies and the monophyly of C. viridis as a whole are questionable. We used mitochondrial DNA sequence data from the D-loop region and ND2 gene to examine the relationships among 26 populations of C. viridis and to test the monophyly of this species. These data were analyzed separately and combined using maximum-likelihood and maximum-parsimony. The C. viridis group was monophyletic in all combined analyses, consisting of two strongly divergent clades. We recommend that these clades be recognized as two distinct evolutionary species: C. viridis and C. oreganus. Crotalus viridis should be restricted to the subspecies viridis and nuntius and the remaining subspecies be assigned to the species C. oreganus. Our data do not allow strong evaluation of the validity of the subspecies. We found that the ND2 gene had greater sequence divergences among closely related individuals than the D-loop region, but this relationship reversed at higher levels of divergence. This pattern is apparently due to: (1) ND2 third positions evolving faster than the D-loop but becoming saturated at higher levels of divergence, and (2) the D-loop evolving faster than ND2 second (and possibly first) positions. Our results suggest that the ND2 gene is preferable for examining intraspecific relationships and the D-loop may better resolve relationships between species of snakes. The latter result is contrary to the common perception of the phylogenetic utility of the D-loop. Another unusual result is that the 145 bp spacer region, adjacent to the 5' end of the light strand of the D-loop, provides greater phylogenetic resolution than the 1030 bp D-loop.

Animals↗

PubMed1996

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.

Base Sequence↗

PubMed1996

A chloroplast-derived sequence is utilized as a source of promoter sequences for the gene for subunit 9 of NADH dehydrogenase (nad9) in rice mitochondria.

The chloroplast-derived sequence trnS-rps4/ 3'trnL-trnF-ndhJ-ndhK (4066 bases in length) is present in a region that starts 355 bases upstream of the gene for subunit 9 of NADH dehydrogenase (nad9) in the mitochondrial genome of rice. Northern blot hybridization revealed that three large transcripts of 3.05, 1.62 and 1.05 kb hybridized to strand-specific probes for both the nad9 gene and the chloroplast-derived sequence, indicating that the nad9 gene was transcribed together with the chloroplast-derived sequence. From the results of in vitro capping and ribonuclease protection experiments, as well as primer extension analysis, we identified at least seven sites for the initiation of transcription of nad9 in the chloroplast-derived sequence. All of the initiation sites for transcription of the nad9 gene were located in sequences homologous to chloroplast DNA. Two of seven initiation sites were flanked by a sequence homologous to the consensus promoter motif that includes the CRTA motif (where R is A or G) of the rice mitochondrion. However, the sequences surrounding the other five sites showed only limited similarity to the conserved sequence. It is suggested that all the promoters of the rice nad9 gene exist in a sequence that was transferred from the chloroplast during evolution. Thus, the chloroplast-derived sequence has a novel, significant function in the mitochondrial genome of this higher plant.

Base Sequence↗

PubMed1989

Solution structure of a tRNA with a large variable region: yeast tRNASer.

Different chemical reagents were used to study the tertiary structure of yeast tRNASer, a tRNA with a large variable region: ethylnitrosourea, which alkylates the phosphate groups; dimethylsulphate, which methylates N-7 of guanosine and N-3 of cytosine; and diethylpyrocarbonate, which modifies N-7 of adenine. The non-reactivity of N-3 of cytidine 47:1, 47:6, 47:7 and 47:8 and the reactivity of cytidine 47:3 confirms the existence of a variable stem of four base-pairs and a short variable loop of three residues. For the N-7 positions in purines, accessible residues are G1, G10, Gm18, G19, G30, I34, G35, A36, i6A37, G45, G47, G47:5, G47:9 and G73. The protection of N-7 atoms of residues G9, G15, A21, A22 and G47:9 reflects the tertiary folding. Strong phosphate protection was observed for P8 to P11, P20:1 to P22, P48 to P50 and for P59 and P60. A model was built on a PS300 graphic system on the basis of these data and its stereochemistry refined. While trying to keep most tertiary interactions, we adapted the tertiary folding of the known structures of tRNAAsp and tRNAPhe to the present sequence and solution data. The resulting model has the variable arm not far from the plane of the common L-shaped structure. A generalization of this model to other tRNAs with large variable regions is discussed.

Adenine↗

PubMed1994

Dual hydrolytic role for Pb(II) ions.

RNA phosphodiester bonds can be cleaved by metal ions, of which Pb2+ is one of the most effective. It can cleave both generally and site-specifically, depending on the substrate and the conditions. In addition, metal ions are also known to cleave ester bonds between amino acid and the 3'-end of transfer RNA. Here we report that in aminoacylated transfer RNA, Pb2+ ions cleave internucleotide bonds in the 3'-end of tRNA and also cleaves the bond between tRNA and its amino-acid, attached at the 3'-end via an ester bond to the terminal ribose in aminoacyl tRNA. The two reactions proceed at different rates. The rate of deacylation is significantly faster than the rate of cleavage of phosphodiester bonds, with a pH-optimum of 7. This dual hydrolytic role is not seen for other metal ions examined, namely Zn(II), Cd(II) and Mn(II). The rate of the two kinds of hydrolyses by Pb2+ ions is compared with that of other metal-ions. The mechanism of cleavage is investigated further by modification of the 3'-end of tRNA.

Acylation↗

PubMed2006

Translocation of a tRNA with an extended anticodon through the ribosome.

Coordinated translocation of the tRNA-mRNA complex by the ribosome occurs in a precise, stepwise movement corresponding to a distance of three nucleotides along the mRNA. Frameshift suppressor tRNAs generally contain an extra nucleotide in the anticodon loop and they subvert the normal mechanisms used by the ribosome for frame maintenance. The mechanism by which suppressor tRNAs traverse the ribosome during translocation is poorly understood. Here, we demonstrate translocation of a tRNA by four nucleotides from the A site to the P site, and from the P site to the E site. We show that translocation of a punctuated mRNA is possible with an extra, unpaired nucleotide between codons. Interestingly, the NMR structure of the four nucleotide anticodon stem-loop reveals a conformation different from the canonical tRNA structure. Flexibility within the loop may allow conformational adjustment upon A site binding and for interacting with the four nucleotide codon in order to shift the mRNA reading frame.

Anticodon↗

PubMed2001

Sublethal effects of ultraviolet A radiation on Enterobacter cloacae.

We report the sublethal effects of ultraviolet A (UVA) on Enterobacter cloacae in comparison with those produced in Escherichia coli. UVA-induced sublethal effects were investigated in either bacterial membrane and at tRNA level. Limited dependence on oxygen concentration for photoinduced inhibition of biochemical membrane functions and low levels of oxidative damage during the irradiation period were found in En. cloacae. On the other hand, ultraviolet spectroscopy and reversed-phase HPLC analysis of hydrolysed tRNA showed that radio induced damage to tRNA is similar in En. cloacae and E. coli. Nevertheless, growth delay induced by UVA in En. cloacae was shorter than that found in E. coli submitted to the same experimental conditions. A limited post-irradiation ppGpp accumulation and the absence of any influence of the membrane damage on the growth delay extent seem to be responsible for the shortness of this effect in En. cloacae. Most of the differences between En. cloacae and E. coli could be attributed to an increased ability of En. cloacae to overcome oxidative stress during UVA exposure.

Cell Membrane↗

PubMed2002

A study of the phylogeny of Brassica rapa, B. nigra, Raphanus sativus, and their related genera using noncoding regions of chloroplast DNA.

There are two evolutionary lineages in the genus Brassica: the rapa/oleracea lineage and the nigra lineage. Using nuclear DNA sequences such as the intergenic spacer between 5S rRNA genes and the internal transcribed spacer between 18S and 25S rRNA genes, we and others had previously demonstrated that Raphanus sativus is closely related to the nigra lineage. In the present study, we sequenced the chloroplast noncoding region between trnT and trnF and that between trnD and trnT in seven species and showed that R. sativus is more closely related to the rapa/oleracea lineage than to the nigra lineage. The conflicting results from nuclear DNA and chloroplast DNA support the hypothesis that Raphanus was derived from a hybridization between the rapa/oleracea and the nigra lineages. We estimated the date of this hybridization event to be 60% of the divergence time between the two Brassica lineages. In addition, the pattern and rate of nucleotide substitution were studied. There were more transversions than transitions in these noncoding regions, which have a high AT content. Furthermore, the proportion of transversions among the substitutions at a site increases with increasing A + T content of its two adjacent nucleotides. An influence of immediate 5(') pyrimidine on substitution pattern is also observed when both adjacent bases in the two DNA strands are A or T. The rate of nucleotide substitution in the trnL group I intron is only about one third of the rate in the nearby intergenic spacers in the trnT-trnF fragment. The rate of nucleotide substitution in the rapa/oleracea lineage is at least 1.5 times that in the nigra lineage.

Brassica rapa↗