volume 8 Number 211880
Nucleic A c i d s Research
The nuckotide sequence of Scenedesmus obtiquus chloroplast
J.M.McCoy and D.S.Jones*
Department of Biochemistry, University of Liverpool, P.O. Box 147, Liverpool L69 3BX, UK
Received 19 September 1980
ABSTRACT
The chloroplast initiator tRNAfrom the green alga Scenedesmus
obliquua has been purified and its sequence shown to be p C-G-C-A-G-G-A-UA-G-A-G-C-A-G-U-C-U- Gm -G-D-A-G-C-U-C- m| G-y-G-G-G-G-C-U-C-A-U-A-A-'V-C-CC-A-A-U-rn^G-I>C>-G-C-A-G-G-T-V*-C-A-A-A-U-C-C-U-G-C-U-C-C-U-G-C-A-A-C-C-AOH.
This structure is prokaryotic in character and displays close
homologies with a blue green algal initiator tRNA
and bean chloroplast
initiator tRNA??8*.
INTRODUCTION
The protein biosynthetic machineries
of organelles have more features
in common with prokaryotic than with eukaryotic systems Cl, 2 ] .
chloroplast initiator tRNA
The
of a higher plant (bean) has been shown to be
highly homologous with prokaryotic initiator tRNAs [ 3 ] • However, the
mitochondrial initiator tRNAs of N.crassa [4] and yeast [5] are neither
prokaryotic nor eukaryotic in character.
initiator tRNA
We have isolated the chloroplast
of Scenedesmus obliquus, a unicellular green alga which
is an example of a lower plant.
The sequence of this tRNA possesses many
of the structural characteristics of prokaryotic initiator tRNAs,
notwithstanding the presence of the modified nucleoside mjG at position 26
which has not been found in any prokaryotic tRNA.
Met
tRNA
S.obliquus chloroplast
™
also has an anticodon stem which differs from all other initiator
tRNAs (both prokaryotic and eukaryotic) in that it lacks the G - C basepair immediately adjacent to the anticodon loop.
It has been suggested
that G - C base-pairing in the anticodon stem confers a unique anticodon
loop conformation on initiator tRNAs [6].
MATERIALS AND METHODS
Chemicals, enzymes, radiochemicals and chromatographic materials were
© IRL Press Limited, 1 Falconbarg Court, London W1V 5FG, U.K.
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obtained or prepared as described previously [ 7 ] .
Total crude tRNA was
i s o l a t e d from S.obllquus by the method of Osterman at_ al [8D. Chloroplast
tRNAMet was purified by successive s t e p s of column chromatography on BD c e l l u l o s e , arginine-agarose and two stages of RPC-5> followed by one
dimensional 15% polyacrylamide gel e l e c t r o p h o r e s i s .
The tRNA was
characterised by aminoacylation and formylation studies and a l s o by
hybridisation to S.obliquus chloroplast DNA (J.M.McCoy, J.A.Pryke and D.S.
Jones, manuscript in preparation).
The sequence was deduced using a combination of techniques, including;
mobility s h i f t a n a l y s i s [ 9 ] of formamide digested 5 ' - [ J t P ] - l a b e l l e d tRNA,
d i r e c t read-off g e l s [ 9 ] , and the limited hydrolysis method [ 1 0 ] as
described previously [ 7 3 .
Modified nucleosides were i d e n t i f i e d by the
t r i t i u m derivative method [ l l ] and by the r e s u l t s of the limited hydrolysis
procedure.
The 5 ' - and 3 1 - terminal nucleotides were characterised a f t e r
t o t a l d i g e s t i o n of 5' -C'*P]-labelled tRNA with nucleane P
and 3 ' - [ ' 8 P ] -
l a b e l l e d tRNA [12] with RNase Tn.
RESULTS
Nucleoaide Composition
Two dimensional t . l . c . of [ 3 H ] - l a b e l l e d nucleoside t r i a l c o h o l s showed
the presence of the following modified nucleosides in the molar r a t i o s
indicated:
D, 1.2; T, 0 . 8 ; V ,
1 . 2 | m G, 0 . 9 ; mjG, 0..8.
These, and a l s o
Gm, were the modified nucleosides detected during limited hydrolysis
sequential a n a l y s i s .
However,
2 residues of D and 3 residues of V were
found by t h i s l a t t e r method.
Nucleotlde Sequence
Direct read-off g e l s enabled most of the sequence from residues
t o be deduced. However in several regions i t was not p o s s i b l e t o
d i s t i n g u i s h C's from U ' s , and modifications were not i n d i c a t e d .
2-75
Mobility
s h i f t a n a l y s i s performed on 5" -["Pj-tRNA^J 0 * p a r t i a l l y digested with
formamide (100°C, 30 min) confirmed the sequence of residues 2 - 1 4 .
limited hydrolysis sequencing method gave the sequence of residues
except for residues 2, 3 and 19 (residue 18 i s Gm).
The
1-72,
The p o s i t i o n s and
i d e n t i t i e s of the modified nucleosides could a l s o be deduced by t h i s
nethod.
Total d i g e s t i o n of 5' -[ 3 I P]-tRNA with nuclease P, released
['*P]pC,
and t o t a l d i g e s t i o n using RNase T, of tRNA labelled at the 3 1 -end with
C'*P]pCp [12] released ["P]Ap. The nucleotide sequence of S.obllquua
Met
chloroplast tRNA
deduced from these data i s shown i n c l o v e r - l e a f f o r a .
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A-OH
C
C
A
p-C A
G»C
OG
A«U
G-C
U
-
rriir
U
Gni
A
A
A
GCAGG
m2G
CD
T
G«C
G«C
C
A
U
A
C AU
S c e n e d e s m u s o b l i a u u s CHLOROPLAST t R N A ^ e t
DISCUSS ION
Mot
S.obliquus chloroplast tlOIA
can be regarded as being generally
prokaryotic in character, although it does share some common features with
both prokaryotic and eukaryotic initiator tlS^As. Several characteristics
found in prokaryotic initiators, which are absent in their eukaryotic
counterparts, are found in S.obiIquus chloroplast tRNA
. These include
C! (not base-paired), the G,- - C,_ and JL^ - U_, base pairs, Don'
unmodified A^_i Ug^ and -GTT^C- in loop IV. These features are also found
in bean chloroplast tRNA.8
bean is A, - ^£7*
chloroplast tHNA™
tHNA™
tj2, except for the G, - C,- base pair which in
n
^ terms of general sequence homology S.obliquus
iis
s more homoloflou
homoloflous with E.coli tBNA™
(81%) than with
11
S.obliquus cytoplasnic tRNA ^* (64*).
Mat
The general
modification i n S.obliquus chloroplast tRNA
general level
level of
of base modifi
is low, there being just six types of modified nucleoside and only nine
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modifications in a l l . Gn'iq' D20 a n d T"i4 aro f o u n d * n prokaryotic, but not
eukaryotic i n i t i a t o r s , m Gig a n d V cr= ttre fo "nd in both classes and D at
position 47 has been found previously in eukaryotic, but not prokaryotic
i n i t i a t o r s . Many modifications common in eukaryotic i n i t i a t o r s are not
found i n S.obliquus chloroplast tRNA. These include; m1Gg, m*G10,
The modified mfG , i s interesting. This modification has been found
only at position 26 and only in eukaryotic tRNAs. Although i t i s present
in N.crassa mitochondrial tRNA1^ and yeast mitochondrial tRNA1^6 [4] i t
has not been found i n any other chloroplast tRNA, including S.obliquus
Met
chloroplast tRNA
(j.M.McCoy and D.S.Jones, unpublished r e s u l t s ) . The
m
Met
modification i s present in S.obliquus cytoplasmic tRNA
(J.M.McCoy and
—
•
m
D.S.Jones, unpublished results) but does not occur in S.obliquus
cytoplasmic tRNA . e , although it is commonly found in other eukaryotic
cytoplasmic initiator tRNAs. The apparently random selection of tRNAs for
this codified nucleoside makes it very difficult to suggest either a
functional role or a structural determinant which specifies the
modification.
Several tRNAs, including tRNA Met species, have y
m
at positions 2? and/
or 39, but t h i s i s the f i r s t time that these modifications have been found
i n an i n i t i a t o r tRNA.
It has been suggested [6] that i n i t i a t o r tRNAs have a unique anticodon
loop conformation, and that t h i s conformation may be due to the presence of
three G - C base-pairs found adjacent to the anticodon loops of a l l
i n i t i a t o r tRNAs.
Recently some supporting evidence for t h i s view has been
provided by an analysis of E.coli tRNA °
tertiary structure [133.
The
different from that of yeast
conformation of the anticodon loop of t h i s molecule
Met was found to be
G - y , q of S.obliquus chloroplast tRNA
l i e s immediately next to
the anticodon loop i n the position which would'be expected [ 6 ] to be
occupied by a G - C base-pair. The change of C to -y' would
weaken the base pairing i n the anticodon stem and therefore possibly
influence the anticodon loop conformation. Interestingly bean
chloroplast [ 3 ] , N.crassa mitochondrial [4] and yeast mitochondrial [ 5 ]
i n i t i a t o r tRNA species a l l have only two G - C base-pairs adjacent to their
anticodon loops, and not three as expected [63.
The sequence homology between Anacystis nidulans (blue green algal)
^6* and either S.obliquus chloroplast tRNA^6* (82%) or bean
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Met
chloroplast tRNA
(78%) is higher than the hoinology between the two
chloroplast tRNA^J6
divergence!
species (71%).
This could reflect evolutionary
According to the endosymbiont hypothesis [l4] blue green algae
are direct descendants of the organisms which formed the chloroplast
symbiosis in precambrian times. The sequences of blue green algal
initiator tRNA
species might therefore be expected to show homologies
with the initiator tRNAs of chloroplasts•
case.
This is now found to be the
Moreover it might be supposed that plants lower down the
evolutionary tree would have chloroplast tRNAs which would be more
homologous to A.nidulana.
The sequences of S.obliquua and bean chloroplast
initiator tRNAs support this view and suggest that S.obliquus might be
considered to be the more "primitive" of the two.
ACKNOWLEDGEMENTS
Ve thank Miss D.McDermott for her assistance in growing S.obliquus.
J.M.McCoy acknowledges the support of a S.R.C. research studentship.
*To w h o m correspondence should be addressed.
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