From the Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada
Received for publication, August 23, 2000, and in revised form, October 4, 2000
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ABSTRACT |
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Despite its large size (200-2400 kilobase
pairs), the mitochondrial genome of angiosperms does not encode the
minimal set of tRNAs required to support mitochondrial protein
synthesis. Here we report the identification of cytosolic-like tRNAs in
wheat mitochondria using a method involving quantitative hybridization to distinguish among three tRNA classes: (i) those encoded by mitochondrial DNA (mtDNA) and localized in mitochondria, (ii) those
encoded by nuclear DNA and located in the cytosol, and (iii) those
encoded by nuclear DNA and found in both the cytosol and mitochondria.
The latter class comprises tRNA species that are considered to be
imported into mitochondria to compensate for the deficiency of
mtDNA-encoded tRNAs. In a comprehensive survey of the wheat
mitochondrial tRNA population, we identified 14 such imported tRNAs,
the structural characterization of which is presented here. These
imported tRNAs complement 16 mtDNA-encoded tRNAs, for a total of at
least 30 distinct tRNA species in wheat mitochondria. Considering
differences in the set of mtDNA-encoded and imported tRNAs in the
mitochondria of various land plants, the import system must be able to
adapt relatively rapidly over evolutionary time with regard to the
particular cytosolic-like tRNAs that are brought into mitochondria.
In flowering plants (angiosperms), all of the codons of the
canonical genetic code are represented in the protein genes encoded by
mtDNA,1 yet the mitochondrial
genome appears to specify fewer than the 23-24 or 33 tRNAs minimally
required to read these codons by either expanded or standard wobble
base pairing, respectively. This observation, coupled with the
identification of cytosolic-like tRNAs in mitochondrial RNA
preparations of bean (1), wheat (2), and potato (3), suggests that
tRNAs must be imported into angiosperm mitochondria (4-6), as they are
into the mitochondria of many unicellular eukaryotes (7). A requirement
for mitochondrial tRNA import in plants is rather surprising in view of
the large size of the angiosperm mitochondrial genome (200-2400
kilobase pairs), which in principle could easily accommodate a full set
of tRNA genes, as do the much more compact mtDNAs of most animals and
fungi (8).
Depending on their genome of origin, plant mitochondrial tRNAs
(mt-tRNAs) can be divided into three groups (see Refs. 2 and 4-6): (i)
native mt-tRNAs, encoded by plant mtDNA and presumed to
originate from the eubacteria-like endosymbiont that was the evolutionary source of the mitochondrion and its genome; (ii) chloroplast-like tRNAs, also encoded by plant mtDNA but
originating from promiscuous chloroplast DNA sequences that in the
course of evolution have been transferred to and are now an integral part of the mitochondrial genome; and (iii) imported tRNAs,
encoded by nDNA and localized in mitochondria as well as the cytosol. Given that tRNAs encoded by a particular genome within a plant cell may
function in more than one subcellular compartment, complete characterization of a plant tRNA requires not only determination of its
anticodon sequence and aminoacylation specificity, but also its
intracellular localization and that of the gene encoding it. Although
the set of mtDNA-encoded tRNAs or their genes has been examined in a
variety of angiosperms (3, 9, 10), only in potato (a dicotyledonous
plant) has the mt-tRNA population been extensively characterized (3).
In that plant, 31 mt-tRNAs were found, including 20 encoded by mtDNA
(15 native and 5 chloroplast-like) and 11 specified by nDNA.
The research reported here aimed to identify and comprehensively
characterize nDNA-encoded tRNAs that are imported into wheat mitochondria from the cytosol. The possibility of mitochondrial tRNA
import emerged when studies to catalog wheat mtDNA-encoded tRNA genes
revealed only 16 genes specific for 13 amino acids (2). Although we
considered that some mtDNA-encoded tRNA genes might have escaped
detection in this survey, it seemed unlikely that all of the remaining
~17 tRNA genes required by standard wobble rules could have been
missed. The apparently limited number of wheat mtDNA-encoded tRNAs
prompted a preliminary investigation of the wheat mitochondrial tRNA
population, which identified three cytosolic-like tRNA species (2);
however, the latter study did not address the issue of possible
cytosolic tRNA contamination of the wheat mitochondrial tRNA
preparation. In the present investigation, we systematically examined
the question of preparative contamination and established criteria and
procedures for the identification of tRNAs that are genuinely imported
into wheat mitochondria.
Isolation of Mitochondria--
The procedures of Spencer
et al. (11) were used for preparation of viable wheat
embryos (from pedigree seed of Triticum aestivum var.
Katepwa; Alberta Wheat Pool, Calgary, Alberta, Canada), germination of
embryos and isolation of mitochondria.
Isolation of Mitochondrial and Nuclear DNA--
Wheat mtDNA was
prepared from DNase-treated, sucrose gradient-purified mitochondria
(11) by sequential sarkosyl lysis, incubation of the lysate with
Pronase, and recovery of DNA by centrifugation in CsCl-ethidium bromide
gradients (11, 12). Wheat nDNA was isolated in a similar fashion from a
crude nuclear fraction that was further purified in a discontinuous
sucrose gradient (11), except that nuclei were extracted with a
phenol/cresol mixture rather than being treated with Pronase.
Isolation of RNA Fractions--
Mitochondrial RNA (from purified
mitochondria) and cytosolic RNA (from a post-mitochondrial supernatant)
were prepared as described previously (11). For isolation of total
cellular RNA, germinating embryos were ground in a mortar with equal
volumes of 0.05 M Tris-HCl, 0.02 M Na-EDTA (pH
8.0) and phenol/cresol mix. The homogenate was centrifuged at
18,000 × g for 20 min, an equal volume of
phenol/cresol solution was added to the supernatant, and the mixture
was shaken for 10 min at 4 °C and then centrifuged at 18,000 × g for 10 min. After addition of NaCl to 0.5 M to
the aqueous phase, RNA was purified by repeated phenol extraction and
ethanol precipitation.
Fractionation of Wheat Mitochondrial tRNAs by Polyacrylamide Gel
Electrophoresis--
To obtain a sufficient quantity of individual
tRNAs for subsequent analysis, a two-dimensional gel electrophoresis
procedure (13) was adapted. Gel solutions and electrophoretic
conditions were as described (3), except that gels were 0.75 mm thick and stacking gels were employed. Approximately 125 µg of 1 M NaCl-soluble wheat mitochondrial RNA (11) was
electrophoresed in the first dimension. The tRNAs were located by
ultraviolet shadowing, and a gel slice containing them was excised for
electrophoresis in the second dimension. The separated tRNAs were
visualized by ethidium bromide staining and recovered from individual
spots by elution (14) in the presence of 10 µg of linear
polyacrylamide carrier (15). Isolated tRNAs were 3'-end-labeled using
5'-[32P]pCp and RNA ligase (16) and electrophoresed in a
6% polyacrylamide, 7 M urea gel (33 × 40 × 0.04 cm) at 1600 V for 3 h. Following autoradiography, the
resolved tRNA species were recovered by elution (14).
Sequence Analysis--
Sequences and labeling of tRNA-specific
oligonucleotides used as primers for reverse transcriptase (RT)
sequencing (17) are described below. Reaction products from 1-2 µl
of chemical (16) or RT (avian myeloblastosis virus) sequencing
reactions (heated at 80 °C for 5 min) were electrophoresed in a 10%
polyacrylamide gel containing 7 M urea, 40% (v/v)
formamide, and 1× TBE (90 mM Tris-HCl, 90 mM
boric acid, 2 mM EDTA (pH 8.0)) at 65 W (3000-3500 V;
50 °C) until a xylene cyanol marker reached 17 cm (~2 h). Gels were pre-run for 1 h at 2000 V.
Thin-layer Chromatography--
Thin-layer chromatography was
employed to determine the anticodon sequence of a wheat
mt-tRNAArg. The tRNA was partially hydrolyzed in alkali,
and the resulting 5'-OH-terminated fragments were 5'-end-labeled,
separated by gel electrophoresis, and individually recovered, as
described (14). The isolated fragments were completely digested to
nucleoside 5'-monophosphates (pN) with nuclease P1 (18), and the
resulting 32P-labeled, 5'-terminal nucleotide of each
eluted fragment was identified by co-chromatography with markers in two
solvent systems (14).
End Labeling of tRNA-specific
Oligonucleotides--
Oligodeoxyribonucleotides (Regional DNA
Synthesis Laboratory, University of Calgary, Calgary, Alberta, Canada;
see Table I) were labeled using polynucleotide kinase and
[ Slot Blot Hybridization and Analysis--
Slot blots contained
mitochondrial, cytosolic and total cellular RNA samples. In some cases,
RNA that had been treated with DNase I served as a control.
To prepare the samples, 12 µg of RNA were dissolved in 50 µl of TE
and added to 250 µl of 20× SSC (1× SSC = 0.15 M
NaCl, 0.015 M sodium citrate), 100 µl of 37% (v/v)
formaldehyde and 100 µl of H2O. The RNA-containing
solution was heated at 65 °C for 15 min, immediately cooled on ice,
then serially diluted seven times with an equal volume of 10× SSC, to
generate 1:2- to 1:128-fold dilutions. RNA samples (6-0.047 µg) were
applied to a Biotrans nylon membrane (ICN Biomedicals) using a Bio-Dot
SF blotting apparatus (Bio-Rad) according to the supplier's
instructions, with 10× SSC as wash solution. Blots were baked for
2 h at 80 °C, then rinsed in 2× SSC. Prehybridization,
hybridization using 0.1 µg of oligonucleotide probe (5'-end-labeled
as described above), and washing of blots were conducted as described
(20). Hybridizations were performed for ~16 h at 5-7 °C lower
than the calculated dissociation temperature (Td) of the oligonucleotide probe (20). Blots
were exposed to Kodak X-Omat K x-ray film without intensifying screens,
the resulting autoradiographs were digitized, the intensity of each
band was determined using the NIH Image Analysis program, and linear
regression coefficients were calculated using MacCurveFit.
Polymerase Chain Reaction (PCR) Amplification and Cloning of
Wheat Nuclear tRNA Genes--
PCR amplification experiments employed
oligonucleotides (generally 20-mers) designed to target the 5'- and
3'-terminal regions of the desired tRNA gene, using sequence
information obtained by chemical and RT sequencing of the corresponding
tRNA. Nucleus-encoded tRNA sequences (DNA and RNA) of other plants (21)
assisted in primer construction.
Purified nDNA (11) was used either directly or after extensive shearing
by two passes at 20,000 p.s.i. through a French pressure cell. The
sheared DNA (mean size about 400 base pairs) produced few, if any,
artifactual bands and so was the preferred template. Amplification was
in a Perkin Elmer GeneAmp PCR System 2400 using Vent(exo
For cloning, blunt-ended PCR products were ligated into pT7
Blue® (Novagen) and transformed into DH5 The term "mitochondrial tRNA" (mt-tRNA) is used here to refer
generally to tRNA species present in the isolated mitochondrial tRNA
population, whether these species are encoded by the mitochondrial genome (mtDNA-encoded mt-tRNA) or nuclear genome (nDNA-encoded mt-tRNA). The term "cytosolic tRNA" (cy-tRNA) is reserved for nDNA-encoded tRNAs that are normally localized to and function in the
cytosol. The term "imported tRNA" is applied to those nDNA-encoded tRNAs that are selectively accumulated in mitochondria, presumably for
use in mitochondrial protein synthesis.
Preliminary Experiments--
When radioactively end-labeled wheat
mt-tRNAs were incubated with mtDNA or nDNA, only a fraction of the
total mt-tRNA population hybridized to mtDNA. After two-dimensional
polyacrylamide gel electrophoresis, the latter tRNAs generated a
distinctly different pattern than those hybridizing to nDNA (23). Among
the latter group were three cytosolic-like tRNAs (Gly (CCC); Leu (CAA);
Val (GAC); anticodon sequences in parentheses) previously identified in
the wheat mt-tRNA population (2). Total cy-tRNA had a more complex gel
electrophoretic profile than the mt-tRNA fraction hybridizing to nDNA,
and it contained unique species (23). This makes it unlikely that
cy-tRNA contamination of the mt-tRNA fraction is of such magnitude and
extent that those mt-tRNAs that hybridize to nDNA represent
contaminating cy-tRNAs, rather than bona fide imported
tRNAs. Nevertheless, this approach cannot unequivocally distinguish
imported nDNA-encoded tRNAs from nDNA-encoded cy-tRNA contaminants of
the mitochondrial preparation.
To assess the level of cy-tRNA co-isolating with wheat mitochondrial
RNA, a wheat cytosolic RNA preparation was incubated with
[
To determine whether the cy-tRNA associated with the mitochondrial
fraction in this way represents a specific subset of the cy-tRNA
population or is a representative sampling, mitochondrial RNA
containing radiolabeled cy-tRNA was subjected to polyacrylamide gel
electrophoresis. No differences in the electrophoretic banding or
intensity patterns of the radiolabeled cy-tRNA associated with the
mitochondrial fraction could be discerned in comparison to those of
similarly labeled cy-tRNA fractions (23).
Micrococcal nuclease treatment of isolated mitochondria has been used
in other studies to reduce contamination of mitochondrial RNA
preparations by cytosolic tRNA (24, 25). To assess the effectiveness of
this treatment, we carried out a quantitative investigation (23), which
showed that such treatment reduced the amount of radiolabeled cy-tRNA
co-isolating with mitochondrial RNA by at least 50% compared with
untreated mitochondria. However, as in other published work, the yield
of mitochondrial RNA was also substantially reduced. This meant that,
on average, micrococcal nuclease treatment lowered residual
contamination by approximately 35%, not 50%.
To ascertain whether the cy-tRNA remaining after micrococcal nuclease
treatment was a specific subset of the cy-tRNA population, the
mitochondrial RNA was electrophoresed in a 4 M urea, 10%
polyacrylamide gel. Although the signal was faint, no selection was
evident (23).
Determination of the Subcellular Location of Wheat
tRNAs--
Because micrococcal nuclease treatment of wheat
mitochondria was not effective in reducing the amount of added cy-tRNA
that co-isolated with mt-tRNA to what we would consider a negligible level (<5% of total mt-tRNA), we therefore developed an alternative approach for distinguishing bona fide, imported tRNAs from
ones nonspecifically associated with mitochondria, possibly as a result of preparative contamination by cy-tRNAs. Slot blots containing mitochondrial RNA and cytosolic RNA were hybridized with
oligonucleotides (Table I) specific for
mtDNA-encoded tRNAUGGPro (26),
the cytosolic-like tRNAGCCGly (27)
previously identified in wheat mitochondrial RNA (2), and nDNA-encoded
cy-tRNAGAAPhe (28). The latter tRNA is
not required in wheat mitochondria (i.e. is functionally
redundant) because the mtDNA encodes a
tRNAGAAPhe (2).
As expected, distinctly different hybridization patterns were obtained
in these experiments (Fig. 1). With the
tRNAUGGPro oligonucleotide, there was a
strong signal with mitochondrial RNA and a much weaker signal with
cytosolic RNA. The reverse was seen with the
tRNAGAAPhe oligonucleotide: a strong
signal with cytosolic RNA and a much weaker signal with mitochondrial
RNA. With tRNAGCCGly (a nDNA-encoded
tRNA previously considered to be imported into wheat mitochondria (Ref.
2)), the corresponding oligonucleotide hybridized equally well with
mitochondrial and cytosolic RNA.
To quantify the relative intensities of the hybridization signals,
autoradiographs were digitized and the relationship between the
strength of each signal and RNA concentration was determined in the
linear region of the plot. The slopes of the lines (regression coefficients) obtained following hybridization of an oligonucleotide to
mitochondrial RNA and cytosolic RNA were then compared, providing an
estimate of the relative intensities of the two signals (Table II).
The ratios (mitochondrial RNA/cytosolic RNA) of the regression
coefficients (RRC) for tRNAGAAPhe and
another cy-tRNA, tRNAGUCAsp (also not
required by the mitochondria because wheat mtDNA encodes a
tRNAAsp (Refs. 26 and 29)), were only 0.24 and 0.31, respectively, compared with an RRC of 8.3 for
tRNAUGGPro. As expected, the RRC for
tRNAGCCGly was different than in the
case of the other two classes of tRNA, being close to 1 (1.35). This
method provides a relative measure of the concentration of each tRNA in
the cytosolic and mitochondrial fractions and offers a way to
distinguish nDNA-encoded tRNAs that specifically accumulate in
mitochondria (i.e. imported tRNAs, by our definition) from
those cy-tRNAs that may be present solely as a result of contamination.
This slot blot procedure was used to assess the import status of 16 cytosolic-like wheat mt-tRNAs isolated by two-dimensional gel
electrophoresis, as described in the next section. Sequence analysis of
individual tRNAs provided information for synthesis of tRNA-specific
oligonucleotide probes (Table I). Discussion of the import status of
these tRNA species follows below, after presentation of data relating
to their structural characterization.
Isolation and Sequencing of Wheat Mitochondrial tRNAs--
To
isolate individual tRNAs, we adopted a procedure involving
two-dimensional polyacrylamide gel electrophoresis of a relatively large quantity (125 µg) of a 1 M NaCl-soluble fraction of
wheat mitochondrial RNA (~85% tRNA). Fig.
2 shows the resulting ethidium bromide-stained RNA profile. Heterogeneity at the metabolically labile
3'-CCAOH terminus (all or a portion of which may be lacking in an individual tRNA) was anticipated (30) and, as confirmed by
sequence analysis, is particularly evident at the periphery of the RNA
profile. In general, a single isoaccepting tRNA was resolved into three
species by this procedure.
Spots numbered 1-50 in Fig. 2 correspond to
tRNAs in the two-dimensional gel electrophoretic pattern previously
reported in Ref. 2. Prior to electrophoresis in that study, wheat
mt-tRNAs were 3'-end-labeled using wheat tRNA nucleotidyltransferase,
thereby eliminating the 3'-end heterogeneity observed here.
Lowercase letters in Fig. 2 denote putative
single tRNA species with varying degrees of completion of the
3'-terminal -CCAOH. Bands numbered 70-79 do not appear to match any of those reported in Ref.
2.
Following the initial two-dimensional polyacrylamide gel fractionation,
recovered tRNAs were radiolabeled and electrophoresed in a denaturing
polyacrylamide gel ("third dimension") (Fig.
3). Many of the tRNAs isolated from the
initial two-dimensional gel were homogeneous at this stage, whereas
others separated into two, three, and occasionally four bands. This
result is primarily a consequence of overlap of species in the
two-dimensional gel because of 3'-end heterogeneity, although it is
known that individual tRNAs can exist in distinct, separable forms due
to variation in post-transcriptional modifications (31). Species
resolved in the third dimension are identified by a suffix (1-4; see
Table III), in order of increasing
mobility.
Sequence Analysis--
To avoid sequencing previously identified
mtDNA-encoded tRNAs (2), species obviously coincident in migration
position with these tRNAs were not analyzed. These species primarily
included tRNAs on the periphery of the two-dimensional gel pattern,
which in the previous analysis (2) proved to be homogeneous.
Altogether, material was recovered from 44 spots in the two-dimensional
gel and electrophoresed in the third dimension, generating 78 species in total, all of which were subjected to chemical sequence analysis. Table III lists the wheat mt-tRNAs identified here as nDNA-encoded; previously identified mtDNA-encoded tRNAs are not included.
A limitation of the direct chemical sequencing procedure was the
appearance of a truncated C ladder in certain cases, which we attribute
to the presence in the variable loop of an unidentified modified
nucleoside with greatly enhanced reactivity in the C reaction.
Virtually complete scission of the polynucleotide chain occurred at the
residue in question, with the result that very faint or no bands
appeared in the C track of sequencing films beyond (5' of) this position.
Except for tRNAGCCGly, all of the tRNAs
listed in Table III were also sequenced using RT. In addition to
confirming chemical sequence data, RT analysis was particularly useful
in establishing sequence in the variable loop (a region often
containing a number of unassigned nucleosides in the chemical sequence
analysis) and in allowing identification of C residues unable to be
assigned on the basis of the chemical sequence analysis because of the
anomalous C-specific cleavage noted above. As well, RT sequencing
confirmed or expanded anticodon sequence information for six of the
tRNAs (Table III).
Thin-layer Chromatography--
In chemical sequencing reactions
the modified nucleoside inosine (I) is cleaved in the G-specific
reaction, generating a band in the G lane. Inosine is present in the
first position of the anticodon (position 34, the only site at which
this modification occurs in tRNA; Ref. 21) in several plant tRNAs,
including wheat germ tRNAIAUIle (32) and
tRNAICGArg (33), potato
mt-tRNAIGCAla (a nDNA-encoded species;
Ref. 3), and lupin cy-tRNAIACVal (34).
In the present study, the sequence of the anticodon loop region of the
Arg2 tRNA (anticodon "G"CG; Table III) was directly determined (see
"Experimental Procedures"), showing that inosine occupies the first
(wobble) position of the anticodon of this tRNA (23), as documented
independently elsewhere (33).
PCR Amplification of Nuclear tRNA Genes--
PCR was used in an
attempt to amplify nuclear gene sequences corresponding to all of the
16 cytosolic-like mt-tRNA species characterized here by RNA sequencing.
This approach was successful in 11 cases (Table III), with sequence
data from cloned PCR products either confirming the anticodon sequence
or clarifying ones remaining ambiguous after RNA sequencing. Although
an exhaustive attempt was made to recover the remaining five nuclear
tRNA genes using a variety of amplification conditions and primer
combinations, PCR products were not obtained in these cases.
Nucleotide Sequence Comparisons--
The wheat mt-tRNAs described
in this report were identified as cytosolic-like by comparison (35)
with homologous mt- and cy-tRNA sequences from various organisms (23).
In general, each wheat mt-tRNA identified here proved to be
substantially more similar to a corresponding plant cy-tRNA or nuclear
tRNA gene sequence (>80% identity) than to a plant mtDNA-encoded one
(<60%), in cases where such mtDNA-encoded counterparts are
available for comparison (23).
Primary and Secondary Structures of Sequenced tRNAs--
The 16 wheat tRNA sequences determined here (Table III) can be folded into the
standard cloverleaf secondary structure (Fig. 4). With few exceptions (23), these
structures contain the expected invariant and semi-invariant
nucleosides characteristic of a conventional tRNA.
For each wheat mt-tRNA sequence reported here, BLASTN searches (35)
identified a number of highly similar plant homologs that allowed
assignment of most of the remaining undetermined positions in the wheat
sequences, and which provided sufficient additional information to
permit inference of the anticodon sequence in all cases.
Alanine--
Species Ala1 and Ala3 differ slightly in nucleotide
sequence, but we conclude that they have the same anticodon, IGC. PCR amplification provided evidence of heterogeneity (C/T) at one position
in the variable loop, and homologous Arabidopsis sequences with the corresponding heterogeneity were identified in BLAST searches
(Table III and Fig. 4). PCR amplification also demonstrated that
anticodon loop positions 34 and 37 are both A in the gene sequence, as
they are in the corresponding Arabidopsis and other plant
tRNAAla sequences. Positions 34 and 37 are inosine and
1-methylinosine (m1I) in sequenced cytoplasmic
tRNAAla species, which would account for the fact that
these residues registered as G during sequencing of wheat Ala1. Like
their Arabidopsis counterparts, the wheat Ala1 and Ala3
sequences have A at position 54, rather than the almost universally
conserved 5-methyluridine (m5U).
We infer that species Ala2 (ugN) has the anticodon sequence UGC, based
on comparison with an Arabidopsis
tRNAUGCAla sequence that differs by one
compensated base pair in the T stem (Table III and Fig. 4). Because
position 37 is A rather than G in the Arabidopsis gene
sequence, it is likely that this position is occupied by
m1I in the wheat Ala2 tRNA, as we suggest above for the
Ala1 and Ala3 tRNAs.
Arginine--
The Arg1 and Arg2 tRNA species are essentially
identical to previously sequenced wheat tRNAArg species
with anticodons CCU (36) and ICG (33), respectively. PCR amplification
of the wheat Arg2 sequence revealed the presence of wobble position A;
as noted above, direct analysis identified this residue as inosine, as
in the published sequence (33).
Aspartate--
This sequence is identical to an
Arabidopsis tRNAAsp sequence also having the
anticodon GUC (Table III).
Glycine--
The sequences of Gly1 and Gly2 are identical,
respectively, to those of a previously published wheat
tRNAGCCGly (27) and an
Arabidopsis homolog with anticodon UCC (Table III). Both
Gly1 and Gly2 appear to have unmodified U rather than the usual
m5U at position 54, as noted previously for wheat
tRNAGCCGly (37).
Histidine--
The wheat sequence differs at six positions from a
sequenced lupin tRNAHis (38) but at only two positions from
several Arabidopsis tRNAHis homologs (Table III
and Fig. 4). Curiously, all of the Arabidopsis tRNAHis sequences currently in the data base (representing
genes on several different chromosomes) have C rather than T at
position 54, which is normally m5U in the mature tRNA. Both
the wheat and lupin tRNAsHis evidently contain unmodified U
at this position.
Isoleucine--
This tRNA sequence differs at several positions
from a published wheat tRNAIle sequence (32). Within the D
loop, the stretch AGUGG in our sequence is
AGDDGG in the published wheat germ sequence and
AG(C/T)TGG in homologous Marchantia and
Arabidopsis sequences. At several other positions (A49:U65,
G59, A60, G70), our sequence matches the homologous
Marchantia sequence rather than the published wheat germ
sequence. Position 34 is A in the Marchantia and
Arabidopsis sequences and inosine in the published wheat
sequence; therefore, the anticodon tentatively identified here as
"G"AU by direct sequencing is very likely IAU.
Leucine--
The wheat Leu1 (anticodon UAG) is virtually identical
to an Arabidopsis tRNAUAGLeu
sequence (Table III and Fig. 4). The Leu2 (anticodon "G"AG) is most
similar to a rice tRNALeu species with anticodon IAG.
Although no PCR data were obtained for wheat Leu2, other homologous
plant tRNA gene sequences have only A at position 34, with the
homologous lupin tRNALeu (39) also having an IAG anticodon.
Wheat Leu3 (anticodon confirmed as CAA by PCR; Table III) is virtually
identical to an Arabidopsis
tRNACAALeu gene sequence, the pair
differing by only two substitutions in the variable loop. Wheat Leu4
(anticodon UAA by PCR) is likewise highly similar to the gene sequence
for an Arabidopsis
tRNAUAALeu. In yeast, two different
tRNANAALeu species have either a
modified U (40) or a modified C (41) in the wobble position,
restricting base pairing to either UUA or UUG codons, respectively. The
same situation probably also exists in mammals (42) and plants (1, 25).
Judging by the results of direct sequencing of the wheat Leu3 and Leu4
tRNAs, the wobble nucleoside in both species also appears to be modified.
Valine--
The wheat Val1 sequence appeared to have a UAC
anticodon on the basis of direct sequence analysis but CAC (in two
independently isolated clones) by PCR amplification. With a wheat
mitochondrial tRNA preparation, sequencing of an RT product provided
evidence only of the CAC anticodon, which we therefore assign to wheat Val1. Homologous Arabidopsis sequences all have C at the
wobble position. Wheat Val2 is virtually identical over the sequenced region to a number of Arabidopsis
tRNAAACVal sequences from different
chromosomes. Although direct sequencing suggested a "G"AC
anticodon, PCR analysis gave AAC. The corresponding lupin
tRNAVal sequence has an IAC anticodon (34), which wheat
Val2 most likely has, as well.
Determination of Import Status of Cytosolic-like Mitochondrial
tRNAs--
Most of the 16 cytosolic-like tRNAs isolated from wheat
mitochondria had an RRC >0.9 (Table II), indicating that these tRNAs are present in wheat mitochondria at levels substantially exceeding those observed for cy-tRNAs that are not required by the mitochondrion for protein synthesis. Notably, the RRC for one cytosolic-like tRNA,
tRNAU*AALeu4, was significantly greater
than the usual range of 1-2, which may indicate that this tRNA is not
used or is not required in large amounts in the cytosol, and/or is
required in mitochondria in greater relative proportion than it is in
the cytosol. In a different case (cytosolic-like
tRNAIAGLeu2), the RRC was only 0.47. The
latter tRNA was isolated from a major band in the two-dimensional
polyacrylamide gel, and in an additional gel electrophoretic
purification step (see above), it produced a band similar in intensity
to that of the tRNAUAGLeu1. Several
possibilities may explain this result. (i) The oligonucleotide used in
this experiment may not have been completely specific for cytosolic
tRNAIAGLeu2. However, the fact that it
was successfully used for RT sequencing provides at least partial
confirmation of its hybridizing target in the mitochondrial RNA
fraction. (ii) The tRNAIAGLeu2-specific
oligonucleotide may have bound nonspecifically to other RNA species,
particularly cytosolic RNAs. This was a potential problem for all of
the slot blot hybridizations and was addressed in a control experiment
using a tRNAIAUIle-specific
oligonucleotide having a single nucleotide mismatch with its target
sequence. This oligonucleotide did not generate a visible signal within
the usual time period for the slot blot hybridization experiments,
although a faint signal was obtained upon much longer exposure (data
not shown). (iii) The tRNAIAGLeu2 may
actually constitute a greater proportion of the cytosolic RNA than it
does of the mitochondrial RNA. The amount of this tRNA in mitochondria
is comparable to that of other tRNAs having RCRs close to 1.0;
nevertheless, the slot blot hybridization data presented here cannot
unambiguously distinguish this mt-tRNA species from a cytosolic species
nonspecifically associated with mitochondria.
Reverse Transcriptase Sequencing of mtDNA-encoded
tRNAGUCAsp--
In the present
study, a nDNA-encoded species of wheat
mt-tRNAGUCAsp was identified on the
basis of its high sequence similarity to plant cy-tRNAs having the same
anticodon (Table III). A native tRNAAsp gene, distinct
in sequence, has been found in wheat mtDNA (26); however, a tRNA
corresponding to this gene was not encountered in the present or in a
previous (2) analysis of the wheat mitochondrial tRNA population.
To examine the possibility of low level expression of the wheat
mt-tRNAGUCAsp gene, a gene-specific
oligonucleotide was used as a primer in RT sequencing. A clear sequence
ladder was generated, with a strong stop at the position marking the
mature 5' end of the tRNA, as well as pauses within the ladder itself,
diagnostic of the presence of modified nucleosides (data not shown).
This makes it unlikely that the sequence ladder was produced from low
levels of mtDNA in purified mitochondrial RNA preparations. The
sequence for this tRNAAsp was identical to the previously
published gene sequence (26) at all positions that could be discerned.
Subcellular Localization of tRNAs--
From the data presented
here, 14 of 16 cytosolic-like mt-tRNAs are considered to be imported
into wheat mitochondria. These imported tRNAs almost entirely
complement the codon recognition and amino acid specificities of the
previously characterized mtDNA-encoded tRNA population. Moreover, the
majority of these tRNAs constitute approximately the same proportion of
either the cytosolic or mitochondrial tRNA populations, the two
exceptions being tRNAU*AALeu4, which
represents a significantly greater (~5-fold) proportion of the
mitochondrial RNA than it does of the cytosolic RNA, and tRNAIAGLeu, which comprises a much
smaller proportion of the mitochondrial RNA than of the cytosolic RNA.
From the slot blot analysis, it is not possible to assess the import
status of those nDNA-encoded tRNA species whose relative abundance in
mitochondria does not significantly exceed that of a cy-tRNA that is
not required for mitochondrial protein synthesis. However, even though
we cannot definitively classify the
tRNAIAGLeu species as an imported one,
it is presumably required by the wheat mitochondrial translation system
because no mtDNA-encoded counterpart has been found.
The mtDNA-encoded mt-tRNAAsp (detected here by RT
sequencing) also appears to be present in relatively low amounts in
wheat mitochondria, given that it eluded detection by direct sequencing
of electrophoretically separated mt-tRNAs in the present and a previous
(2) study. A similarly low level of mtDNA-encoded tRNAAsp
in potato mitochondria has been noted (3). In considering how the
evolutionary replacement of a mtDNA-encoded tRNA by a nDNA-encoded
species might occur, we anticipate a transitional stage in which both
tRNAs are functional in mitochondria, thereby allowing subsequent loss
of the mitochondrial gene. When low levels of both nDNA-encoded and
mtDNA-encoded species are detected in the mt-tRNA population, it is
conceivable that the nDNA-encoded species is in fact required because
the mtDNA-encoded tRNA is present at insufficient levels to support
mitochondrial translation on its own. The case of tRNAAsp
described here may represent an example of an intermediate stage in the
loss of expression of a mtDNA-encoded tRNA. A similar redundancy of
nDNA- and mtDNA-encoded tRNAVal species has been reported
in Marchantia mitochondria (45).
The Wheat Mitochondrial tRNA Population--
In view of the
apparent absence of any tRNALeu genes in wheat mtDNA, wheat
mitochondria may well utilize the nDNA-encoded
tRNAIAGLeu identified here. If so, the
characterized wheat mt-tRNA population would include 10 native, 6 chloroplast-like and 15 nDNA-encoded tRNAs, for a total of 31 distinct
species. The potential codon recognition pattern of these tRNAs is
presented in Table IV. All 61 sense
codons have been identified in the collection of wheat mitochondrial
protein genes sequenced to date, and there is no evidence of any
departure from the standard genetic code (46). That being the case, it
is evident that not all of the required wheat mt-tRNAs have been
identified; in particular, (a) tRNA(s) specific for threonine remain(s)
to be found.
Because the wobble pairing rules for codon/anticodon recognition have
not yet been established for the plant mitochondrial translation
system, it is unclear how many wheat mt-tRNA species are actually
required to support protein synthesis in the organelle. In vertebrate
mitochondria, expanded wobble base pairing in conjunction with a
modified genetic code reduces the number of tRNAs required for
translation to only 22-23, depending on whether the same or different
tRNAsMet are used for initiation and elongation (46). In
angiosperm chloroplasts, which like plant mitochondria use the standard
genetic code, a minimum of 32 tRNAs is required if conventional wobble base pairing occurs (47), but only 30 tRNAs have been identified. However, Pfitzinger et al. (47) have demonstrated that
chloroplast tRNAU*GCAla,
tRNAICGArg,
tRNAUAGLeu, and
tRNAU*GGPro are able to read all four
codons of the respective amino acid families, apparently employing a
"two out of three" base pair recognition mechanism.
It is possible that eight tRNAs having UNN anticodons could decipher
the 32 codons represented by the eight four-codon families of the
standard genetic code. Table IV shows that wheat mt-tRNAs of this type
have been identified for five of these eight families. However, in four
of these cases, tRNAs bearing either a GNN or INN anticodon have also
been identified, rendering U·N wobble pairing unnecessary. This might
suggest that expanded wobble pairing does not occur in wheat
mitochondria, in which case a tRNAGGGPro
would remain to be found.
Finally, it is necessary to account for recognition of CGG (Arg) and
AGA (Arg) codons, for which corresponding tRNAs have not been found.
The tRNACCUArg anticodon was
characterized here by both RT sequencing and PCR analysis, and an
unmodified wobble position C has been confirmed by direct sequence
analysis of this tRNA species. Thus, it is unlikely that
tRNACCUArg would be able to decode both
AGA and AGG codons. The remaining arginine codon (CGG) may be
recognized by tRNAICGArg, as proposed
for angiosperm chloroplasts (47).
Nuclear DNA-encoded tRNA Populations in Monocotyledon, Dicotyledon,
and Liverwort Mitochondria--
The study reported here represents the
most comprehensive sequence survey of nDNA-encoded mt-tRNA species in
any plant system. Homologs of some of these wheat nDNA-encoded mt-tRNAs
have been found in the mitochondria of maize (another monocotyledon)
(48), potato (a dicotyledon) (3), and larch (a gymnosperm) (48); however, few anticodon sequences were directly determined in these cases.
In M. polymorpha, the mitochondrial genome encodes 29 "native" tRNA genes specifying 27 distinct species (49). No
chloroplast-like tRNA genes are present in Marchantia mtDNA
(49). Compared with angiosperm and gymnosperm mitochondria, liverwort
mitochondria contain the largest number of mtDNA-encoded tRNAs.
However, even allowing for both G·U and U·N wobble base pairing,
tRNAs reading the isoleucine codons AUU and AUC and the threonine
codons ACA and ACG are still required (50). In both angiosperm and
larch mitochondria, these tRNAs are imported, and nDNA-encoded
tRNAAAUIle and
tRNAAGUThr species have since been
identified in liverwort mitochondria (51, 52).
Other similarities between the nDNA-encoded tRNA populations of
angiosperm and larch mitochondria include import of tRNAs specific for
alanine, arginine, leucine, and valine codons. However, larch
mitochondria import tRNAs that angiosperm mitochondria do not
(48), including tRNAUGGPro and
tRNAGAALys, genes for which have been
identified in angiosperm mitochondria. Although wheat mitochondria
contain the largest number of identified imported tRNAs (at least 14 species representing seven amino acids), larch mitochondria import
tRNAs specific for a greater number of amino acids (at least 11 tRNA
species corresponding to 10 amino acids).
The work presented here and elsewhere (48) determined that the wheat
mt-tRNAHis is imported. This is in contrast not only to the
situation in several dicots (53, 54) but also several other
graminaceous plants (55-57), where the mt-tRNAHis is a
chloroplast-like, mtDNA-encoded species. Similarly, there is no
tRNAPhe gene in the mitochondrial genome of A. thaliana (19); instead, a cytosolic tRNAPhe is
imported into Arabidopsis mitochondria (43), a signal
departure from other dicots. Another difference between relatively
closely related plants concerns
tRNAGCUSer and
tRNAUGASer, which are imported species
in sunflower (44) but are native mt-tRNAs in potato (3) and other dicots.
In view of the number of documented differences with respect to which
tRNA species are imported into mitochondria within the range of
monocotyledonous and dicotyledonous plants, distinctions between these two groups are becoming increasingly blurred.
Clearly, before more definitive comments can be made about the
differences in the imported tRNA populations of monocot, dicot, and
gymnosperm mitochondria, a larger number of plant species will have to
be studied. Nevertheless, the differences demonstrated to date serve to
emphasize that the plant mitochondrial translation system is quite
flexible with respect to the genetic origin of the tRNAs it uses.
Moreover, the import system must be able to adapt relatively rapidly
over evolutionary time with regard to the particular cytosolic-like tRNAs that are brought into mitochondria to function there.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP under standard conditions (17), except that
spermidine was used at a final concentration of 1 mM.
)
DNA polymerase (New England Biolabs) with 500 ng of nDNA. An optimized
regime employed 40 cycles and included a 30-s annealing at 55 °C and
a 30-s extension at 72 °C, with the ramp-up from annealing to
extension slowed to 50% of maximum. PCR was also attempted with 5%
(v/v) dimethyl sulfoxide or 1.3 M betaine in the reaction
mix; however, neither reagent supported the amplification of tRNA
sequences that were not also recovered using the standard protocol.
cells that had
been prepared by the protocol of Inoue et al. (22). Sequence
was determined using the fmol Cycle Sequencing® kit
(Promega) with 5'-32P-labeled vector-based primers.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP and tRNA nucleotidyltransferase (2), and the
resulting labeled RNA was added to homogenizing medium containing
germinating wheat embryos. Embryos were processed according to the
standard protocol for isolation of mitochondria and mitochondrial RNA
(11) and radioactivity in various preparative fractions was assessed by
liquid scintillation counting. From measurements of RNA recovered from
subcellular fractions together with calculated specific activities, we
estimated that co-isolating cy-tRNA could comprise as much as 20-25%
of the total mt-tRNA prepared by our standard protocol (23).
Sequences of tRNA-specific oligonucleotides used for slot blot
hybridization analyses
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[in a new window]
Fig. 1.
Slot blot analyses of three wheat
tRNA-specific oligonucleotide probes hybridized to wheat mitochondrial
RNA and cytosolic RNA. Autoradiographs show the hybridization of
5'-32P-labeled oligonucleotides specific for mtDNA-encoded
mt-tRNAUGGPro (A),
cy-tRNAGGAPhe (B), and
nDNA-encoded mt-tRNAGCCGly
(C), to wheat mitochondrial RNA (M), wheat
cytosolic RNA (C), total cellular RNA that had been treated
with DNase I (T1), and cytosolic RNA that had
been treated with DNase I (C1). RNA samples were
serially diluted 2-fold to produce amounts ranging from 6 µg to 0.047 µg and applied to wells of the slot blot apparatus as described under
"Experimental Procedures."
Linear regression coefficients calculated from slot blot hybridization
analyses of wheat mt-tRNAs using tRNA-specific oligonucleotide probes
View larger version (53K):
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Fig. 2.
Fractionation of wheat mt-tRNAs by
two-dimensional polyacrylamide gel electrophoresis.
Arrows indicate direction of migration in the first
(1st) and second (2nd) dimensions. Conditions of
electrophoresis are specified under "Experimental Procedures."
A, photograph of ethidium bromide-stained gel taken under
ultraviolet light, showing the tRNA fractionation pattern.
B, schematic representation of pattern shown in
A. Spots have been numbered 1-51 and
70-79 as explained in the text. Lowercase
letters denote potential resolution of a single tRNA into
separate species differing in the extent of completion of the
3'-terminal -CCAOH sequence.
View larger version (5K):
[in a new window]
Fig. 3.
Autoradiograph showing resolution of isolated
tRNAs in the third electrophoretic separation. Transfer RNAs
recovered from a two-dimensional electrophoretic separation (see Fig.
2) were 3'-end-labeled with [5'-32P]pCp and
electrophoresed in a fully denaturing polyacrylamide gel ("third
dimension"; see "Experimental Procedures"). Separated components
are designated by a number (1-4), in order of
increasing mobility.
Identity of wheat nDNA-encoded mitochondrial tRNAs resolved by
polyacrylamide gel electrophoresis
View larger version (29K):
[in a new window]
Fig. 4.
Primary sequences and potential secondary
structures of wheat nDNA-encoded mt-tRNAs (see Table III), derived from
chemical and reverse transcriptase sequencing data. Positions that
could not be identified by direct or RT sequencing or by PCR
amplification are indicated by the letter N;
those tentatively identified are denoted by lowercase
letters, with probable or possible dihydrouridine residues
indicated by d. The filled square
denotes absence of a nucleotide at that position. The sequence of the
5'-terminal region of tRNAIACVal was not
determined. A small lowercase letter
within the latter structure and beside the other structures is the
corresponding nucleotide at that position in the reference DNA sequence
listed in Table III. An uppercase letter beside a
structure indicates that the reference sequence is RNA rather than DNA,
with asterisks (*) denoting positions of
post-transcriptional modification. Positions of apparent disagreement
between direct sequencing and PCR analysis or positions of
heterogeneity in individual PCR clones are denoted by a
slash (e.g. G/A). I,
inosine.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Potential codon recognition pattern in the wheat mitochondrial
translation system
) indicates that a tRNA corresponding to that particular codon has
not been identified, whereas a number sign (#) indicates that the codon
may be recognized by a tRNA that pairs with other codons in that box.
Standard font denotes a native mtDNA-encoded tRNA; italicized font
denotes a chloroplast-like mtDNA-encoded tRNA; bold font denotes a
nDNA-encoded mt-tRNA. The deduced codon recognition pattern is based on
standard wobble rules. The mtDNA-encoded tRNAs were previously
characterized in Ref. 2 except for tRNAAsp, which along with
the nDNA-encoded tRNAs was identified in the present study (see text).
![]() |
ACKNOWLEDGEMENT |
---|
We thank M. N. Schnare for valuable advice on experimental approaches and techniques and for critical review of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Grant MT-4124 from the Medical Research Council of Canada (to M. W. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported in part by a predoctoral fellowship from the Walter C. Sumner Foundation.
§ Supported by a fellowship from the Canadian Institute for Advanced Research (Program in Evolutionary Biology). To whom correspondence should be addressed. Tel.: 902-494-2521; Fax: 902-494-1355; E-mail: m.w.gray@dal.ca.
Published, JBC Papers in Press, October 10, 2000, DOI 10.1074/jbc.M007708200
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ABBREVIATIONS |
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The abbreviations used are: mt, mitochondrial; cy, cytosolic; RT, reverse transcriptase; PCR, polymerase chain reaction; RRC, ratio of the regression coefficients.
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REFERENCES |
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