A cluster of six tRNA genes in Drosophila mitochondrial DNA that

Volume 12 Number 5 1984
Nucleic Acids Research
A duster of six tRNA genes in Drosophila mitochondrial DNA that indudes a gene for an unusual
tRNA» G Y
Douglas O.Clary and David R.Wolstenholme
Department of Biology, University of Utah, Salt Lake City, UT 84112, USA
Received 22 November 1983; Accepted 1 February 1984
ABSTRACT
Genes for URF3, tRNA ala , tRNA ar 9, tRNA asn , tRNAf^J, tRNA91u, tRNAPhe, and
the carboxyl terminal segment of the URF5 gene have been identified within a
sequenced segment of the mtDNA molecule of Drosophila yakuba. The genes occur
in the order given. The URF5 and tRNAPne genes are transcribed 1n the same
direction as replication while the URF3 and remaining five tRNA genes are
transcribed in the opposite direction. Considerable differences exist 1n the
relative arrangement of these genes 1n Q_. yakuba and mammalian mtDNA
molecules. In the tRNAjjgy gene an eleven nucleotide loop, within which
secondary structure formation seems unlikely, replaces the dihydrouridine arm,
and both the variable loop (six nucleotides) and the T'f'C loop (nine
nucleotides) are larger than 1n any other D. yakuba tRNA gene. As available
evidence is consistent with AGA codons specifying serine rather than arginine
in the Drosophila mitochondria! genetic code, the possibility 1s considered
that the 5'GCU anticodon of the D_. yakuba tRNAj^y gene can recognize AGA as
wel1 as AGY codons.
INTRODUCTION
Within nucleotide sequences of the mitochondrial DNA (mtDNA) molecule of
Drosophila yakuba we have identified and reported the genes for two ribosomal
RNAs, twelve tRNAs and eight polypeptides (cytochrome c oxidase (CO) subunits
I, II and III, ATPase subunit 6, Unidentified Reading Frames (URF) 1, 2 and 3
and A6L) (1-4). Nucleotide sequences of sections of the JJ. melanogaster mtDNA
molecule, corresponding to some of the sequenced sections of the JJ_. yakuba
mtDNA molecule have also been determined (1,2,5). While the genes found 1n
Drosophila mtDNAs are the same as the genes found 1n a number of mammalian
mtDNAs (6-8), their arrangement within the DrosophUa and mammalian mtDNA
molecules differs considerably (1-5). Also, evidence obtained from
comparisons of sequences of Drosophila and mammalian polypeptide genes
suggests that 1n the Drosophila mitochondrial genetic code the triplet AGA,
which specifies arginine in the standard genetic code, and is used only as a
rare termination codon 1n some mammalian mtDNAs (6-8), may specify serine (3-
© IRL Press Limited, Oxford, England.
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Nucleic Acids Research
5). Further, the COI gene of Drosophila mtDNA appears to be unique in that it
may contain a four nucleotide initiation codon (4,5).
In this paper we report a sequence of the _D_. yakuba mtDNA molecule that
contains a cluster of six tRNA genes (tRNA a l a , tRNA a r 9, tRNA a s n , tRNAJjj^,
tRNA9 lu and tRNAP he ) bounded by the genes for URF3 and URF5. As shown
previously, the URF3 gene of JL yakuba mtDNA is one of a series of polypeptide
genes (URF2, COI, COI I, URFA6L, ATPase6, COIII and URF3) which occur in the
same relative order, and are transcribed in the same direction, in D_. yakuba
and mammalian mtDNAs (2-4). However, relative to the URF3 gene, all of the
six presently described 0_. yakuba mt-tRNA genes, as well as the URF5 gene, are
found 1n different locations in the mammalian mtDNA molecule. Further, the
structure of the tRNA predicted from the tRNAJ^y gene contains some unusual
features both in comparison to other Drosophila mt-tRNAs, and to its mammalian
counterpart.
MATERIALS AND METHODS
Experimental d e t a i l s regarding i s o l a t i o n of mtDNA from D_. yakuba (Stock
2371.6, Ivory C o a s t ) , preparation and i d e n t i f i c a t i o n of pBR322 and pBR325
clones of £ . yakuba mtDNAs, r e s t r i c t i o n enzyme d i g e s t i o n s , e l e c t r o p h o r e s i s ,
c l o n i n g of fragments i n t o M13mp8 or M13mp9 DNA, p u r i f i c a t i o n of s i n g l e stranded and double-stranded Ml3 DNAs and preparation of v i r a l DNAs c o n t a i n i n g
partial
d e l e t i o n s of cloned r e s t r i c t i o n fragments of mtDNA are given or
r e f e r r e d to i n ( 1 , 3 ) .
A l l DNA sequences were obtained from M13mp8- or M13mp9-cloned fragments
by the e x t e n s i o n - d i d e o x y r i b o n u c l e o t i de t e r m i n a t i o n procedure (9) using [ a dATP (600 C1/mmol; New England Nuclear) as described ( 1 ) .
P]
The sequencing
s t r a t e g i e s used are given 1n F i g . 1 .
Computer analyses of DNA sequences were c a r r i e d out using the programs
r e f e r r e d t o in
(3).
RESULTS AND DISCUSSION
The sequence of a continuous 1629 n u c l e o t i d e section of the £ . yakuba
mtDNA molecule, determined using the s t r a t e g i e s i n d i c a t e d 1n F1g. 1 , i s shown
i n F1g. 2.
Within the sequence are f i v e regions each of which can f o l d I n t o
the c h a r a c t e r i s t i c secondary s t r u c t u r e of a tRNA, w i t h anticodons
them to be the genes f o r tRNA
3).
a1a
ar
, tRNA 9, tRNA
asn
A s i x t h r e g i o n , which l i e s between the tRNA
, tRNA9
asn
lu
indicating
and tRNAPhe (F1g.
and tRNA g1u genes, can
also f o l d i n t o a s t r u c t u r e which resembles a tRNA, but t h i s s t r u c t u r e lacks a
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O-R-*
EcoRI | C
H/ndlll | C |
D
B
A
IE
|D|E|
—'
1
H/ndlll
J IECORI
loo be
A
B
^
~—
B
-J
H/ndlll
Figure 1. A map of the _D_. yakuba mtDNA molecule showing the relative
locations of the A+T-rich region (crosshatched) the two rRNA genes (dotted),
the origin (0) and direction (R) of replication, and EcoRI and Hindlll sites
and fragments (A-E and A-F respectively), (see (1) for details and
references). The bar under the map Indicates the segment sequenced. This
segment is expanded below and the restriction sites and strategies employed to
obtain the entire nucleotide sequence are shown. The origin of each sequence
1s as follows: a_, the EcoRI-E fragment. b_ and c_, the two ends of the 1.5 kb
Hindi II-EcoRI subfragment of the pBR325-c!oned EcoRI-A fragment. d_, the
Hindlll-E fragment which was subcloned from the cloned EcoRI-A fragment.
Unlabelled sequences with arrows pointing to the right are DNasel-EcoRI
deletions (cloned 1n M13mp9) of the 1.5 kb Hindi II-EcoRI subfragment of the
EcoRI-A fragment. Unlabelled sequences with arrows pointing to the l e f t are
TJNasel-BamHI deletions (cloned in M13mp9) of the H1ndIII-D fragment. The
small vertical terminal arrows on the lower bar indicate the extent of the
sequence shown in Fig. 2, and the solid arrowhead shows the 5'-3' direction of
the sequence.
standard dihydrouridine arm. Because 5'GCT 1s found in the anticodon position
of this structure, and for other reasons discussed below, this sequence is
Interpreted as being a gene for tRNA|gy. The tRNAP"e gene Is transcribed in
the same direction as that in which replication proceeds around the molecule,
while the remaining tRNA genes are transcribed in the opposite direction (F1g.
4).
The sequence to the l e f t of the tRNAa^a gene contains an open reading
frame of 351 nucleotides which would be transcribed in the same direction as
the tRNAa1a gene and which has been Identified as the URF3 gene; this
nucleotide sequence and the amino acid sequence predicted from i t are 52.4%
and 41.9% homologous, respectively, to the corresponding sequences of the
mouse URF3 gene (8). The 5' end of the D. yakuba URF3 gene is located
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Nucleic Acids Research
.
URF3 — •
AK y »—»• I F S I
I
I
I
AGTATAGATAlATTTTTTCTATTATTATTATTCCTTCAGTAATCTTATTAATCACAACTCTTGTTATATTTTTAGC
75
S
I
L
S
K
K
A
L
I
D
R
E
K
s
S
P
F
E
C
G
F
D
P
K
S
TTCAATTTTATCAAAAAAAGCTTTAATTGATCGAGAAAAAAGATCACCTTTTGAATGTGGATTTGACCCTAAATC
150
S
S
R
L
P
F
S
L
R
F
F
L
I
T
t
l
F
L
I
F
D
V
E
I
A
TTCTTCTCGATTACCATTTTCATTACGATTTTTTTTAATCACTATTATCTTTTTAATTTTTGATGTAGAAATTGC
22 5
L
I
L
P
M
I
I
I
L
K
Y
S
N
I
M
I
U
T
I
T
S
I
L
F
I
TTTAATTCTTCCTATAATTATTATTTTAAAATATTCTAATATTATAATTTGAACAATTACTTCGATTATTTTTAT
300
L I L L I G L Y H E W N Q G M L N U S N
••*
TTTAATTTTATTAATTGGGCTATACCATGAATGAAATCAAGGTATATTAAATTGATCAAATTAATAAATATTTAA
37 5
|AGGCTTGTAGTTAATTATAACATTTGATTTCCATTCAAAAACTATTGAATATTCAATCTACCTTA|TATATATATA
450
tRNAar8—•
'_
[
'_
'
TATATATATATATAATTfcAATATGAAGCGATTAATTGCAGTTAGTTTCGACCTAACCTTAGCTATTATATACCCT
tlWA a8n — » .
'
[
'
[
525
^
g
§
TATTTTtrTAATTGAAGCCAAAAAGAGGCGTATCACTGTTAATGATATAATTCAGTATAAACTCCAATTAAGlGAAG
600
[
TATGGTGATCAAGTAAAAGCTGCTAACTTTTTTCTTTTAATGGTTAAATTCCATTTATACTTCTUTTTATATAGT
67 5
TTAAAATAAAACCTTACATTTTCATTGTAATAATAAAATAATTTATTTTTATAAATTJACTATAATTAATTCACTA
750
|
|
'_
'_
'_
- * — "tRNAPhe
^rATTCAAAGATTAATTAATCTCCATAACATCTTCAGTCTCATACTCTAAATATAAGCTATTTGAAllATAAAAATA
1=1
* L F L
825
ATAAAAAACTAAATAAAATTATAATTCAAAATACAAATAATATTAAATAAATTTTTAAACTATTATTATGTATCA
L F S F L I M I W F V F L M L Y
I K L S N N H M L
F
L
T
K
S
T
F
S
K
V
V
f
L
N
Q
S
G
L
Y
K
N
Y
L
Y
P
L
Y
H
F
Q
I
G
M
G
G
F
Y
Y
E
S
W
G
Q
900
D
T
ATCATATTGAACCTAAAAATAATGTTAAATTATAATTTAATAGAGATTTATTCAAAGAATATAAATTTCTCATAG
W M S G L F L T L N Y N L L S K N L S Y L N s I S
1125
TTATATAAGGAAAAGCAAAAATTAACCAATTTAACATTCTACCTCCAATAATTCTTATAAATAATAAGCCTAGTA
1275
H
Y
P
F
P
F
I
L
G
R
L
M
V
W
S
E
Y
V
L
R
P
S
Y
S
W
H
L
M
S
G
C
I
I
J
D
N
L
M
N
L
S
C
C
N
V
T
L
G
T
S
F
F
Y
L
H
F
L
L
G
L
H
L
D
G
T
M
S
Y
F
F
S
F
M
N
I
TATTTCTAATTCTAACAATTTCTAAAATTATATCCTTAGAATAAAATCCAGCTAAAAATGGTATTCCACATAAAG
H
l
I
l
V
I
E
L
I
M
D
K
S
Y
F
G
A
L
F
P
M
G
C
L
A
15 0 0
CCAAATTAGAAACATTAAAACAAGCTGAAGTTAAAGGTATATGAATTCTCAACCCTCCTATTAACCGAATATCTT
L N S V N F C A S T L P M H I s L G C M L R I D Q
157 5
CACAATTATTTATATTATGAATAATAGCTCCTGCACATATAAATAATAAAGCTT
S N N M N H I I A G A C H F L L A
-•-"URF5
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Nucleic Acids Research
adjacent to the tRNA 9ly gene, as shown previously (Fig. 2; (2)). Alignment of
the nucleotide and amino acid sequences of the URF3 genes of d_. yakuba and
mouse indicate that three codons (nine nucleotides; 68-70; 104-106; 113-115,
Fig. 2) of the D_. yakuba gene are absent from the mouse gene. The URF3 genes
of both D_. yakuba and mouse have a TAA termination codon. In mouse this codon
is separated by a single A from the 5' terminus of the tRNA ar 9 gene, but 1n 0_.
yakuba 1t is separated by eleven nucleotides from the 51 terminus of the
tRNA ala gene.
The sequence of 811 nucleotides to the right of the tRNAP he gene contains
only one open reading frame which proceeds 1n the same direction as the sense
strand of the tRNAP he gene. The 270 amino acid sequence predicted from this
open reading frame 1s 25.7% homologous to the 271 amino acid sequence which
precedes the carboxyl terminus of the mouse URF5 gene (8). This alignment
(not shown) Includes insertion/deletions of only three internal codons between
the Drosophila and mouse genes, all within 108 nucleotides of the carboxyl
terminus. Interpretation of this D_. yakuba sequence as the URF5 gene is
strengthened by the finding that the amino a d d sequences corresponding to the
first 67 codons (nucleotides 1427-1627, F1g. 2) of the d_. yakuba and mouse
sequences have a homology of 55.2%. In mouse mtDNA the URF5 and URF6 genes
are transcribed from opposite strands of the molecule, the carboxyl termini of
the two genes overlap and each gene terminates with a standard TAA codon
(8). In D_. yakuba mtDNA the last codon of the URF5 gene is separated by a
single T from the 5' terminus of the tRNAP^ e gene. Presumably, as has been
argued for other genes with similar arrangements in both mammalian (6,10) and
DrosophUa (2-5) mtDNAs, a translation termination codon is created at the end
of the D_. yakuba URF5 transcript by polyadenylation when this transcript is
Figure 2. Nucleotide sequence of the segment of the 0_. yakuba mtDNA molecule
Identified in Fig. 1. The sequence contains a cluster of six tRNA genes
(boxed) the anticodons of which are underlined. From considerations of
nucleotide and predicted amino a d d sequence homologies to mouse mtDNA (8) the
sequence to the left of the tRNA genes 1s the entire URF3 gene, and the
sequence to the right of the tRNA genes is the carboxyl terminal segment of
the URF5 gene. The nucleotide sequence shown is the (5'-3') sense strand of
all of the genes except tRNApfie and URF5, and the arrows Indicate the
direction of transcription of each gene. Asterisks indicate partial or
complete termination codons. In the amino a d d sequences a lower case letter
s indicates the tentative assignment of serine to an AGA codon (see text).
The line above nucleotides 463 and 464 indicates the lack of a TA dinucleotide
1n one of the cloned fragments of this region of the sequence. The sequence
containing
the amino terminal 88 nucleotides of the URF3 gene and the entire
tRNA g l y gene are published 1n (2).
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Nucleic Acids Research
5'
A • I
G • T
G - C
G - C
T - A
r
T
G - C
T
A
T A A C T
A
T T G
A I
T G A
A
G C G
I
T G C
C
C A T A
G
G T A T
C
C G
C
T C A
G
A G T
G G C
• T - A T «
T -
A • T
T - A
C-G
t M A "
A - T
T
G - C
A - T
A - 7
G - C
T - A
T-
A
[j!
C
I
[
I
C
I
I
T G
A
A A C
T
T
A
A A A T
T I A
C
I
T C C
G
A G I
T A A
G A T T
'
T - A •
i
I
A - T
C - G
A - T
A - I
W* - T
A -
I
A - T
S-
C
Figure 3. Six tRNA genes of U_. yakuba mtDNA shown i n the presumed secondary
s t r u c t u r e of the corresponding" tRNAs. Also I n d i c a t e d 1n the diagram of the
tRNAJigY gene are possible secondary i n t e r a c t i o n s between nucleotides 1n the
d1hydrouridine replacement loop and the v a r i a b l e loop (dotted l i n e s ) , and
other possible t e r t i a r y I n t e r a c t i o n s ( s o l i d l i n e s ) proposed f o r tRNA^gy from
bovine and other mammalian mitochondria (23; see t e x t ) . The numbering shown
i s s p e c i f i c f o r t h i s tRNA gene.
excised from a multicistronic primary transcript.
Intervening nucleotides are not found between the central four tRNA genes
(arg, asn, ser(AGY) and glu) of the six tRNA gene cluster (Fig. 2 ) , nor do any
of these genes overlap. However, between the tRNA a l a and tRNA a s n genes are 27
nucleotides which include a twelve unit TA repeat (F1g. 2 ) . In a second
sequence of this region which was derived from a different mtDNA molecule, the
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Nucleic Acids Research
Figure 4. The arrangement of genes
determined to date in the circular
mtDNA molecule of £. yakuba, from the
results of various studies (for
references see 1-4) and the present
data. Each tRNA gene is Identified by
the one letter ami no acid code. Arrows
within and outside the molecule
indicate the direction of transcription
of each gene. Wavy lines indicate
uncertain gene boundaries. 0 and R
indicate the origin and direction of
replication respectively.
sequence between tRNA a and tRNA a s n genes included only an eleven unit TA
repeat. Whether this difference reflects heterogeneity 1n the size of the
Intervening sequence between the tRNA a 1 a and tRNA a s n genes 1n different mtDNA
molecules, either within or between flies, or 1s the product of a replication
error or of recombination during clone amplification, remains unknown.
Between tRNA9 l u and tRNAP n e there are 18 nucleotides, the only notable feature
of which is a direct tetranucleotide repeat at each end (F1g. 2 ) .
We have shown previously that the genes for URF2, COI, COII, URFA6L,
ATPase6, CO III and URF3 are found in the same relative order in D. yakuba and
mammalian mtDNAs, but that the tRNA genes which separate some of these
polypeptide genes differ between the two mtDNAs. All of the six tRNA genes
reported here as well as URF5 are in different relative locations in the d_.
yakuba and mammalian mtDNA molecules. In mammalian mtDNA the tRNA ar 9 gene is
adjacent to the URF3 gene; the tRNA a l a and tRNA a s n genes are adjacent to each
other and located between the tRNA tr P and tRNA c > s genes; the tRNAJj^ gene lies
between the tRNA n 1 s and tRNA^gJJ genes (which are located between the URF4 and
URF5 genes); the tRNA p h e gene 1s located between the replication origincontaining region and the large rRNA gene; and the URF5 gene Is located
between the tRNA^Jj and URF6 genes. Also, of these six tRNA genes and the
URF5 gene, only the tRNA a r g and tRNAj^f genes are transcribed 1n the same
direction in D_. yakuba and mammalian mtDNAs, relative to both the direction of
replication and to the seven polypeptide genes listed above.
The relative locations, and directions of transcription of all of the JD.
yakuba mitochondrial genes which have been determined to date are shown in
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Nucleic Acids Research
F1g. 4.
The d i r e c t i o n s of t r a n s c r i p t i o n of the newly mapped s i x tRNA genes
and of the URF5 gene add t o the trend which we have noted e a r l i e r ( 3 ) ; genes
In the approximate h a l f of the n_. yakuba mtPNA molecule which l i e s to the
left
of the A+T-r1ch region (F1g. 4) are t r a n s c r i b e d from one strand of the
molecule, w h i l e genes in the other h a l f of the molecule are t r a n s c r i b e d from
the other strand (F1g. 4 ) .
The only exceptions t o t h i s g e n e r a l i z a t i o n so f a r
are three tRNA genes ( g i n , cys, t y r ) to the l e f t of the A+T-rich r e g i o n , and
one tRNA gene ( s e r , UCN) t o the r i g h t .
This c o n t r a s t s w i t h what i s found i n
mammalian mtDNA, where a l l genes except the URF6 gene and e i g h t tRNA genes are
t r a n s c r i b e d from one strand (the H strand) of the molecule ( 6 - 8 ) .
Transfer RNA genes.
The t R N A a l a , tRNA a r 9, tRNA a s n , tRNA9 l u and tRNAPne
genes of D_. yakuba mtDNA are 62%, 32%, 56%, 54% and 56% homologous,
r e s p e c t i v e l y , t o the corresponding tRNA genes of mouse mtDNA ( 8 ) .
The value
f o r tRNA ar 9 1s the lowest f o r any of the 18 JJ_. yakuba-mouse tRNA gene homology
comparisons made so f a r .
However, the £ . yakuba mt-tRNA a r 9 gene 1s 84%
homologous t o the r e c e n t l y published sequence of tRNA ar 9 from mitochondria of
mosquito, Aedes a l b o p i c t u s (11).
The presently reported f i v e 0_. yakuba mt-
tRNA genes are s i m i l a r t o the other twelve D_. yakuba mt-tRNA genes we have
described p r e v i o u s l y (1-4) in regard t o t h e i r major secondary
characteristics (Fig. 3).
structural
A l s o , each of these tRNA genes lacks some of the
nucleotides which are constant i n p r o k a r y o t i c and non-organel le eukaryotic
tRNAs.
However, i n each of the t R N A a l a , tRNA a r 9, tRNA a s n and tRNAPne genes of
_D_. yakuba mtDNA, as 1n a l l other D. yakuba mt-tRNA genes, the constant Pu£g,
T33 and PU37 n u c l e o t i d e s are maintained (numbering system 1n 12).
In the
tRNA9' u gene T33 and PU37 are a l s o found, but n u c l e o t i d e 26 i s a p y r i m i d i n e
(C) r a t h e r than a p u r i n e .
The tRNA^
gene 1s again unusual i n t h a t I t s G+C
content 1s only 9%, compared to a range of 24% t o 31% f o r the remaining f i v e
newly described mt-tRNA genes.
Among the other 0_. yakuba tRNA genes
sequenced, only the tRNAasP gene has a s i m i l a r low (9%) G+C c o n t e n t .
In both
the tRNA9 1u and tRNA as P genes both the am1no-acyl and the T^C stems lack G-C
pairs ( 3 ) .
Among stems of other £ . yakuba mt-tRNA genes G-C p a i r s are absent
only 1n the am1no-acyl stem of tRNA9 l n .
Within the tRNA9 l u gene s t a b i l i t y of
secondary s t r u c t u r e may be aided by the absence of unusual nucleotide pairs
w i t h i n stems ( 2 ) , and by the stacking e f f e c t expected from runs of A's and T's
( F i g . 3 ) , as has been noted f o r the amino-acyl stems of the tRNAasP and
tRNA91n genes ( 3 ) .
As mentioned above, we have I n t e r p r e t e d the sequence between the tRNA asn
and tRNA9' u genes as a tRNAj^y gene.
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This sequence can be folded i n t o a
Nucleic Acids Research
structure (F1g. 3) which has an am1no-acyl stem and an anticodon stem with the
same number of nucleotide pairs (seven and f i v e , respectively), and an
anticodon loop with the same number of nucleotides (seven) as are found in a l l
other D_. yakuba mt-tRNA genes, and almost a l l other known tRNAs (12,13). The
T'^C arm could comprise either a stem of five nucleotide pairs (other 0_. yakuba
mt-tRNA genes contain four or five) and a loop of nine nucleotides (other V_.
yakuba mt-tRNA genes contain between three and e i g h t ) , or a six nucleotide
pair stem and a seven nucleotide loop. The variable loop of six nucleotides
1s larger than the variable loops (four or five nucleotides) of other d_.
yakuba, other insect, and mammalian mt-tRNAs (2,5,11-14), but is quite small
compared to the variable loops of some fungal mt-tRNAs (15,16), prokaryotic
and non-organelle eukaryotic tRNAs (12,13). Eleven nucleotides occur between
the last nucleotide of the amino-acyl stem (nucleotide 7, numbering system
shown in Fig. 3) and the f i r s t nucleotide of the anticodon stem (nucleotide
19), but these nucleotides cannot be arranged to form a structure resembling
the usual dihydrouridine arm of other tRNAs. Nucleotides T^g, GJJ and A ^ are
complementary to T ^ . C ^ and Ajg, as might be expected i f they were the
conserved dihydrouMdine stem; however, i f this is the case then two of the
A j 6 , Gj7 and T^g nucleotides must have been added during the evolution of this
gene from a gene coding a tRNA with a standard cloverleaf configuration. The
three nucleotides 1n the anticodon position, 5'GCT, would be expected to
recognize the t r i p l e t s AGU and AGC, which in a l l genetic codes known specify
serine. Recently, the sequence of an mt-tRNA isolated from mosquito tissue
culture cells has been obtained (D. T. Dubin, personal communcation) which is
91% homologous to the £ . yakuba t R N A ^ gene sequence, and can be folded Into
a structure similar to that shown 1n Fig. 3 for the d_. yakuba tRNA^y gene
sequence.
In each of the mt-tRNAJjgy genes of nine different mammals (hamster (17),
bovine (18,19), human (19), mouse (8,20), rat (21), g o r i l l a , chimpanzee,
orangutan and gibbon (22)) the d1 hydrouridine arm 1s replaced by a loop of
only five nucleotides. The variable loop has a maximum of five nucleotides,
the IK stem comprises five nucleotide pairs and the TK loop 1s always eight
or nine nucleotides. Based on information concerning t e r t i a r y structure
obtained from experiments Involving chemical probing, de Bruijn and Klug (23)
have derived a structural model for bovine tRNA^Qy. This model 1s similar 1n
overall shape to the crystal structure of yeast tRNAPhe (24-26) but is
smaller, and Involves a unique set of t e r t i a r y Interactions. The l a t t e r
include interactions of a l l five nucleotides in the d1hydrour1d1ne arm
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Nucleic Acids Research
replacement l o o p , two w i t h nucleotides i n the v a r i a b l e loop which e f f e c t i v e l y
extends the anticodon stem, and t h r e e w i t h nucleotides in the T^C l o o p .
The
s t r u c t u r a l model proposed f o r bovine tRNAjjjjy 1s also compatible w i t h the tRNAs
p r e d i c t e d from the other e i g h t described mammalian tRNA|gy genes, 1f some A-C
base p a i r i n g i s permitted ( 2 3 ) .
In the D_. yakuba tRNA p r e d i c t e d from the
tRNAJjgy gene (F1g. 3 ) , A^g, A^g, and G ^ i n the di hydrouridi ne arm replacement
loop could pair w i t h C33, l^g and T^Q i n the v a r i a b l e loop.
A l s o , the three
t e r t i a r y i n t e r a c t i o n s between nucleotides 1n the di hydrouridi ne arm
replacement loop and the 1>C loop thought t o occur in the mammalian tRNA|gy
(23) might be maintained (Gg-T 5 4 ; G g " 1 ^ '
i n t e r a c t i o n between T^g and Ag n .
T
10'A50'
F1
9-
3
'
as
wel 1
as
The s t r u c t u r a l model presented f o r mt-
tRNA|gy from bovine and other mammals includes an I n t r a l o o p U-A p a i r
found 1n yeast tRNAPne (24,25)) formed between the f i r s t and f i f t h
of the T+C loop.
(also
nucleotides
Such an I n t e r a c t i o n i n the n_. yakuba t R N A ^ 9 e n e ( F 1 9 - 3)
seems more l i k e l y between the second and s i x t h nucleotides (T48-A52) of the
T^C loop than between G47 and A g j .
Codon usage and the genetic code.
The frequency of codons ending i n A or
T i n the URF3 gene (93.2%) and i n the carboxyl terminal segment of the URF5
gene (91.9%) are w i t h i n the range of values (91.6% to 98.1%) found f o r the
other s i x polypeptide genes of 0_. yakuba mtDNA which have been analysed
(3,4).
The occurrence of the l e u d ne-sped f y i ng codon CTG (nucleotides 899-
9 0 1 , F1g. 2) 1n the URF5 gene leaves only four expected sense codons (CAG,
GAG, CGC, and AGG) unrecorded among the seven completed polypeptide gene
sequences and the p a r t i a l URF1 and URF5 sequences ( 1 - 4 ) .
The G+C content of the URF3 gene (20.6%) and the p a r t i a l
URF5 gene
(21.9%) are s l i g h t l y higher than the G+C contents of the URF2 and URFA6L genes
(18.6% and 17.2%, r e s p e c t i v e l y ) , but s t i l l
d i s t i n c t l y lower than the range of
G+C contents (24.2% to 30.1%) found f o r the D. yakuba COI, COM, COIII and
ATPase6 genes ( 3 , 4 ) .
As i n a l l other 0_. yakuba genes f o r which sequences have been o b t a i n e d ,
I n t e r n a l TGA and I n t e r n a l AGA codons are found 1n both the URF3 gene and the
p a r t i a l URF5 gene (F1g. 2 ) .
I t appears t h a t , as 1n fungal and mammalian
mtDNAs ( 8 , 2 7 - 2 9 ) , TGA codons specify tryptophan 1n the Drosophila
mltochondrial genetic code ( 1 , 3 - 5 ) .
While two out of three of the TGAs of the
JJ_. yakuba URF3 sequence correspond t o tryptophan-speci f y i n g codons (TGAs) i n
the mouse URF3 sequence, none o f the three TGA codons of the £ . yakuba URF5
sequence correspond to t r y p t o p h a n - s p e d f y i n g codons i n the mouse URF5
sequence.
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The d i f f e r e n c e 1n conservation of TGA codons between the URF3 and
Nucleic Acids Research
URF5 genes of D_. yakuba and mouse correlates with differences 1n overall
nucleotide sequence homology between these genes (52.4% and 39.7%,
respectively), as noted previously for other D_. yakuba-mouse mitochondrial
gene comparisons (4).
I t has been argued from comparisons of mtDNA sequences of Drosophila and
mammals (3-5), and from considerations of the characteristics of mitochondrial
genetic codes (3), that AGA may specify seMne rather than arginine in the
DrosophUa mitochondrial genetic code. The D. yakuba URF3 and URF5 genes
contain one and seven AGA codons respectively, which correspond in position to
codons specifying serine, asparagine (two), phenylalanine, alanine, threonine,
histidine and cysteine in the mouse URF3 and URF5 genes. A similar low
correspondence of serine-specifying codons to AGA codons has been found
previously for other mammal -DrosophUa comparisons Involving mitochondrial
genes with relatively low overall nucleotide sequence homologies (3-5).
As J). yakuba mtDNA contains a gene for a tRNA with a 5'GCU anticodon
which is expected to recognize only AGY codons, i t remains unclear as to how
AGA codons are decoded. Two p o s s i b i l i t i e s seem worth considering: The D_.
yakuba mtDNA molecule may contain one extra tRNA gene with a TCT anticodon (1)
which is specific for AGA (and possibly AGG). I f this 1s the case then D_.
yakuba mtDNA would contain three tRNAs to decode serine, a situation which is
Inconsistent with the otherwise overall economy of structure and organization
of this mtDNA molecule. The alternative possibility 1s that there is stable
pairing between the GCU anticodon and each of the three AGC, AGU and AGA
codons. This would necessitate either selective two-out-of-three nucleotide
pair recognition (30,31), or that the G In the wobble position of the
anticodon can effectively pair with C, U or A. As AGG codons have not been
found in DrosophUa gene sequences so f a r , there Is no need to include the
prediction of G-G pairing. Other cases of unusual base pairing of the
nucleotide 1n the wobble position of the anticodon have been Indicated for
mtDNAs. In mammalian mtDNAs the 5'CAU anticodon of tRNAf~met appears to be
able to recognize as methionine-specifying codons a l l four AUN codons when
they occur as I n i t i a t o r codons, and AUG and AUA when they occur Internally. A
similar situation 1s Indicated in DrosophUa mtDNA, except that evidence for
only AUG and AUU i n i t i a t i o n codons (and one possible special case of an AUA
I n i t i a t i o n codon (4,5)) has been found so far (1-5). Also, 1n DrosophUa
mtDNA, while genes contain both AAA and AAG codons, only a tRNA^ s gene with a
5'CTT anticodon has been located (3,5). I t seems reasonable, therefore, to
give serious consideration to the view that in Drosophila mitochondria, the
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GCU
anticodon can recognize AGA as w e l l as AGY codons.
ACKNOWLEDGEMENTS
We w i s h t o thank Donald T. Dubin f o r sending us data on mosquito tRNAs
p r i o r t o p u b l i c a t i o n , and F e l i x Machuca f o r e x c e l l e n t t e c h n i c a l a s s i s t a n c e .
T h i s work was supported by National I n s t i t u t e s of H e a l t h Grant No. GM 18375.
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