volume 9 Number 61981
Nucleic Acids Research
A putative precursor for the small ribosomal RNA from mitochondria of Saccharomyces cerevisiae
K.A.Osinga, R.F.Evers, J.C.Van der Laan and H.F.Tabak
Section for Medical Enzymology and Molecular Biology, Laboratory of Biochemistry, University
of Amsterdam, Jan Swammerdam Institute, P.O. Box 60.000, 1005 GA Amsterdam, The
Netherlands
Received 18 December 1980
ABSTRACT
We have characterized a putative precursor RNA (15.5S) for the 15S
ribosomal RNA in mitochpndria of Saccharomyces cerevisiae. Hybrids were
formed with mitochondrial RNA and mtDNA fragments terminally labelled at
restriction s i t e s located within the gene coding for 15S ribosomal RNA and
treated with S1 nuclease (Berk, A.J. and Sharp, J.A. (1977) 12, 721-732).
Sites of resistent hybrids were measured by agarose gel electrophoresis and
end points of RNAs determined. The 15.5S RNA is approximately 80 nucleotides
longer than the 15S ribosomal RNA, with the extra sequences being located at
the 5'-end. Both 15S ribosomal RNA and 15.5S RNA are fully localised within
a 2000 base pair HapII fragment.
This putative precursor and the mature 15S ribosomal RNA are also
found in petite mutants which retain the 15S ribosomal RNA gene. The petite
mutant with the smallest genetic complexity has i t s end point of deletion
(junction) just outside the HapII s i t e located in the 5' flank of the 15S
ribosomal RNA gene as determined by S nuclease analysis. This leaves a DNA
stretch approximately 300 base pairs long where an i n i t i a t i o n signal for
mitochondrial transcription may be present.
INTRODUCTION
In most e u k a r y o t e s
small r i b o s o m a l
the genes coding
RNAs (rRNAs)
are
transcribed
as a common p r e c u r s o r
rRNAs a r i s e
through p r o c e s s i n g
type of gene o r g a n i z a t i o n
amounts of
coding
are
for
far
physical
apart
mitochondrial
an e q u a l
the
and s e p a r a t e d
map [ 2 ] . We a r e
for
RNAs of
in
enzymes
t h e assembly of
This
equal
the
ribosome.
the mitochondrial
by 2 5 . 0 0 0 b a s e p a i r s
in
the
this
is
the mitochondrion
© IRL Press Limited, 1 Falconberg Court, London W1V 5FG, U.K.
(bp) on
to
the
the
the
establish
coordinated
of
genes
ribosome
s y n t h e s i s of
mechanisms o p e r a t e
and
individual
[1].
the mitochondrial
of b o t h rRNAs and i f
and import
and
t o each o t h e r
help to generate
interested
rRNAs t o s e e i f
output
synthesis
the large
RNA from which t h e
Saccharomyces c e r e v i s i a e
l a r g e and s m a l l
for
next
by s p e c i f i c
may be of
b o t h rRNAs r e q u i r e d
In t h e y e a s t
located
with
nuclear-
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Nucleic Acids Research
coded ribosomal proteins.
We have made a start with the study of these problems by investigating the structure of the rRNA genes and their transcripts. The gene for the large rRNA contains an optional intron
of 1143 bp in some strains and putative precursor RNAs still
containing the intervening sequence have been found [3-5].
Transcripts with extensions on the 3'-end of the gene have also
been described [5]. In the gene coding for the small rRNA we
have found no evidence for the presence of an intron [6] and
there are only limited indications for the existence of possible
precursor RNAs. Thus, Van Ommen et al. [7] identified a 19S RNA
mapping in the proximity of the 5'-end of the 15S rRNA gene and
Morimoto et al . [8] have shown that all petites retaining the
15S rRNA gene contain an RNA species slightly larger than the
15S rRNA itself. In this paper we have mapped this RNA species
and show that it may be a precursor of the 15S rRNA. The same
RNA was also characterized from wild-type ye,ast.
MATERIALS AND METHODS
Yeast strains. Saccharomyces cerevisiae KL14-4A (hisl,trp2,
+ R
R R
+
p ,omega , C.,.,. , 0,., P454 ) • The construction and isolation of the
petite strains LH25F2 (AMR24-11A, a,ile~,trp~,tsp25,PR) and
LH26D7-10, a secondary petite mutant from LH26D7 (AMR22-11C,
—
a,trp
—
,ilv
described
R
,tsp25,P
) was c a r r i e d out by L.A.M.Hensgens and i s
in Tabak et a l .
i s o l a t i o n of mitochondria
described previously
[ 6 ] . Methods for growth of c e l l s and
and mitochondrial
ribosomes have been
[9-11].
I s o l a t i o n of nucleic
a c i d s . mtDNA and RNA from S.
cerevisiae
KL14-4A and the p e t i t e mutants LH25F2 and LH26D7-10 were
from mitochondria by standard procedures
mitochondrial
[13] and
ribosomes was purified
isolated
[ 9 - 1 2 ] . RNA from
by hot phenol
extraction
alcohol-precipitation.
I s o l a t i o n of nucleic acids from agarose g e l s . RNA was
isolated
from agarose gels according
was i s o l a t e d
to Arnberg et
from agarose by a modification
Tabak and Flavell
[15]. The DNA was electrophoresed
Sepharose 4B instead of hydroxyapatite
After
1352
separation of
al.
[ 1 4 ] . DNA
of the procedure of
into
(suggestion of
lysine-
P.H.Boer).
DNA bands, holes were punched beside
the
Nucleic Acids Research
DNA band with a Pasteur pipette and filled with lysineSepharose 4B. The gel is then rotated 90° and electrophoresis
continued until all the ethidium fluorescence is bound to the
lysine-Sepharose 4B. This is then removed and rinsed into a
small Eppendorf pipette tip, plugged with glasswool and containing a thin layer of Sephadex G50. The column is topped by a
small layer of Sephadex G50 in order to avoid disturbance of
the lysine-Sepharose 4B layer during elution. The DNA is eluted
with 200-ul portions of 10 mM Tris-HCl, 0.1 mM sodium EDTA and
0.5 M NaCl (pH 8.0) and is usually present in the first two
fractions. Occasionally we have noticed that DNA recovered from
lysine-Sepharose 4B was broken down after subsequent incubation
in Mg-containing buffers, suggesting contamination with deoxyribonuclease. To avoid this, we routinely extract the eluted
fractions with phenol and concentrate the DNA by alcohol precipitation. The advantages over hydroxyapatite are 2-fold. The
procedure can be carried out with very small amounts of DNA
since there is no need to localize the DNA in the eluted fractions due to the absence of a dead volume in this very small
column. Contamination of DNA alcohol precipitates with EDTA or
phosphate used for elution of hydroxyapatite is prevented since
the DNA is eluted with NaCl from lysine-Sepharose 4B.
In vitro labelling of nucleic acids. RNA was labelled with
polynucleotide kinase and [iy-32P]ATP after alkaline fragmentation as described [16]. DNA was labelled by nick-translation
[17] as described [18]. DNA restriction fragments were labelled
at the 5'-ends with [y-32P]ATP and polynucleotide kinase [19]
or at the 3'-ends by filling in cohesive ends with DNA polymerase (Klenow fragment) and a-32P-labelled deoxyribonucleotides
[20].
Hybridization. Transfer of DNA to nitrocellulose filters
and filter hybridization were carried out as described [18].
Analysis of hybrids with S 1 nuclease. S.. nuclease mapping
was carried out according to Berk and Sharp [21] with minor
modifications as described [6], For DNA-DNA renaturation analysis the hybridization temperature was lowered to 31°C and S,.
nuclease digestion was carried out at 30°C. Electrophoresis in
neutral or alkaline flat-bed gels was carried out as described
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[6].
Restriction
10 mM Tris-HCl
dithiothreitol.
labelled
221,
Hinfl
220,
enzyme a n a l y s i s .
(pH 7 . 5 ) ,
For s i z e c a l i b r a t i o n we used a
digest
out
in
and 1 mM
terminally-
of pBR322 (1631, 517, 506, 396, 344, 298,
154 and 75 bp)
and an EcoRI d i g e s t of pBR322
bp) . For nomenclature of
Sanders e t
Digestion was c a r r i e d
8 mM MgCl2, 0.01% g e l a t i n
mtDNA r e s t r i c t i o n
al . [ 2 2 ] . T i s
Hindu ; D i s
fragments,
(4362
see
Hindlll.
RESULTS
Physical
mapping of
p e t i t e mutant
The DNA sequences r e t a i n e d
their
p o s i t i o n with r e s p e c t
indicated
in F i g .
1. P e t i t e
to the w i l d - t y p e p h y s i c a l
mutant LH26D7-10 i s of
g e n e t i c complexity with a r e p e a t
after
with
32
digestion
HapII
BamHI bp
a 2000 bp fragment
P-labelled
fragment
with
LH26D7-10
by LH25F2 are known [6]
unit
from w i l d - t y p e
therefore,
addition
to two small
(Fig. 2,
was found
lane e ) . After
that
with
corresponding
mtDNA ( F i g . 2 ) . The p e t i t e
derived
digestion
hybridizes
the complete gene coding
DNA fragments
map i s
much lower
l e n g t h of 2700 bp o b t a i n e d
15s rRNA and c o m i g r a t e s with the
contains,
and
for
mutant
15S rRNA in
from the 3 '
region
LH25F2
LH 2607-10
putative precursor/
15S rRNA
i
•"
•» *'
Fig. 1. Physical map of mtDNA in and around the 15S rRNA gene. The petite
mutants are positioned with respect to the physical map and the direction
of transcription is indicated.
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a b c d e
f g h i
Fig. 2. Characterization of petite mutant LH26D7-1O by restriction enzyme
analysis. DNA was digested with HapII and BamHI and analysed on a 1% agarose gel (lanes a-e). DNAs were blotted to nitrocellulose f i l t e r s and hybridized with in vitro 32 P-labelled 15S rRNA (lanes f - i ) . a) Marker pBR322 DNA
x Hinfl and pBR322 x EcoRI; b and f) wild-type mtDNA (KL14-4A) x HapII; c and
g) LH26D7-1O DNA (0.15 ug) x HapII; d and h) LH26D7-10 DNA (0.5 ug) x HapII;
e and i) LH26D710 DNA x BamHI. For marker DNA fragment lengths see Methods.
next to the gene
p o s i t i o n of
mapping
(see b e l o w ) . We have determined
t h e end p o i n t
of
the deletion
the
exact
with t h e S^ n u c l e a s e
technique.
mtDNA of
and the 3 '
Materials
t h e p e t i t e mutant
termini
labelled
with
LH26D7-10 was c u t with BamHI
32
P at
the 3'-ends
and Methods). DNA from the much l a r g e r
(see
petite
LH25F2 was c u t with MboII. The DNAs were mixed, melted
renatured.
nuclease,
After
treatment
of
the r e n a t u r a t i o n
t h e DNA was analysed by agarose g e l
p r o d u c t s with S^
electrophoresis.
When the l a b e l l e d
LH26D7-10 DNA r e n a t u r e s with i t s e l f
repeating
2700 bp i s e x p e c t e d .
unit
of
DNA of
petite
mutant
and
the
When t h e l a b e l l e d
10 DNA r e n a t u r e s
with
mutant
duplex r e s i s t a n t
to S^ n u c l e a s e can only s u r v i v e
LH26D7-
LH25F2, a h e t e r o from
the
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labelled
BamHI s i t e up to the junction or from the BamHI s i t e
to the i n t e r n a l
S.-resistant
MboII s i t e
(Fig. 3 ) . We find
three prominent
DNA bands on the gel under neutral
electrophoresis
(Fig. 4):
a 300 bp fragment
conditions of
corresponding
the distance between the MboII and the BamHI s i t e
fragments of
range of
1000 and 1300 bp f i t t i n g
1000-1700 bp predicted for
BamHI s i t e
within the expected
LH 26D7-10
size
the distance between the
and the position of the junction.
conditions of electrophoresis
to
and two
Under
alkaline
(Fig. 4) only the 1000 bp DNA
LH 25F2
junction
B M
B
M
Fig. 3. Outline for the mapping of the end point of deletion (junction) in
the petite mutant LH26D7-10. B, cleavage site for BamHI; M, cleavage site
for MboII (see also Fig. 1). Continuous arrows indicate the position where
the DNA duplex is broken, stippled arrows indicate the position of a nearby
restriction endonuclease site.
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Nucleic Acids Research
b e d
a
a b e d '
Fig. H. Mapping of the junction in the petite mutant LH26D7-1O. The BamHI
fragment of LH26D7-1O was labelled at the 3'-end with 3 2 P (lane a). After
addition of LH25F2 (0.3 ug) cut with MboII, the DNA mixture was denatured,
renatured, treated with S nuclease (lanes b-d, 100, 200 and 300 U/ml, respectively) and analysed on a neutral (lanes a-d) or alkaline agarose gel
(lanes a ' d') .
band can be seen. This f i t s
outlined
e x a c t l y with the e x p e c t a t i o n s
in Fig. 3 , where the 1000 bp fragment
DNA extending
bp fragment
from the BamHI s i t e
ary DNA. The end point of
t e l y upstream of
has f u r t h e r
the other
flanking
DNA from
reacted with complement-
the d e l e t i o n maps, t h e r e f o r e ,
the HapII s i t e
15S rRNA. Of the two small
the region
the length of
and the 1300
i s the one in which the s i n g l e - s t r a n d e d
the BamHI to the MboII s i t e
entirely,
is
to the junction
located
remaining
a t the 5'-end
of
immediathe
HapII fragments one maps
(the j u n c t i o n
fragment)
the 3'-end
the gene, as we have shown
of
almost e n t i r e l y
in
above.
mtRNAs from wild-type and p e t i t e
Morimoto et a l •
mutants
[8] have shown t h a t p e t i t e mutants
retain-
Nucleic Acids Research
ing the 15S rRNA gene have an extra RNA species (15.5S) migrating just behind the 15S rRNA on an agarose g e l . This same RNA .
species, with a length of 1680 nucleotides, is present in the
p e t i t e mutants LH25F2 and LH26D7-10 (Fig. 5 ) . When the agarose
gel is overloaded and electrophoresis is prolonged, an RNA
species with the same migration behaviour - but present in much
lower concentration r e l a t i v e to the 15S rRNA - can be seen in
the mtRNA derived from wild-type mitochondria (Fig. 5 ) .
Characterization of the 15.5S RNA
For the physical mapping of the RNA we have cut 15S rRNA
gene fragments with r e s t r i c t i o n enzymes t h a t cleave the gene
i n t e r n a l l y and end-labelled the codogenic strand e i t h e r at the
5'-end or the 3'-end (see Methods). The labelled fragments were
hybridized with the mtRNAs, the hybrids were treated with S,.
nuclease and analysed by agarose gel e l e c t r o p h o r e s i s . The
p e t i t e mutant RNAs both protect two s p e c i f i c DNA fragments
conserving the 3 2 P label at the 5'-end of the codogenic DNA
strand cut with BamHI (Fig. 6) and t h e i r r e l a t i v e concentrations
a
1358
b
c
Fig. 5. Agarose gel electrophoresis of mtRNA of wild-type
S. cerevisiae and two p e t i t e mutants s t i l l containing t h e
15S rRNA gene. Small RNA components have run off the gel due
to prolonged e l e c t r o p h o r e s i s . a) S. cerevisiae KL14-4A; b)
LH26D7-10; c) LH25F2. The bar indicates the position of the
15.5S RNA.
Nucleic Acids Research
are the same as the 15.5S RNA species and the 15S rRNA present
in the agarose gel (Fig. 5 ) . The difference in length is 70-90
nucleotides and compares well with the difference in migration
behaviour between the 15.5S RNA and 15S rRNA in the agarose gel
corresponding to 80 nucleotides difference in length. On the
basis of these experiments we conclude that the 15.5S RNA is a
15S rRNA molecule with at least a small extension at the 5'-end
of the gene. The same analysis on the wild-type mtRNA gives
only one band, because of the large excess of hybrid derived
a b c d e fg
Fig. 6. S nuclease analysis of hybrids between mtDNA fragments terminally
labelled with 3 2 P at the internal BamHI site and mtRNA. Hybrids were elc.ctrophoresed on an alkaline agarose gel of 2% DNA was blotted to nitrocellulose filters and radioactive bands visualized by autoradiography. a)
Marker DNA, pBR322 x Hinfl. S nuclease-treated hybrids formed with wild-type
mtRNA enriched for 15S rRNA: b) untreated; c) 100 U/ml; d) 50 U/ml. S
nuclease-treated hybrids formed with LH25F2 RNA: e) untreated; f), 100 U/ml;
g) 50 U/ml. For marker DNA fragment lengths, see Methods.
Fig. 7- Control on purification of 15.5S RNA component from wild-type S^_
cerevisiae KLI'l-'IA mtRNA. Agarose gel electrophoresis on 1.15% gel. a)
Petite mutant LH25F2 mtRNA; b) LH26D7-10 mtRNA; c) 15.5S mtRNA species
isolated from wild-type S. cerevisiae KL1i)-')A mtRNA. The bar indicates the
position of 15.5S RNA.
Nucleic Acids Research
from 15S rRNA. We t h e r e f o r e p r e p a r a t i v e l y
RNA from w i l d - t y p e
trates
the enrichment of
RNA was used for
ment,
RNA using gel
rRNA i t s e l f ,
is
the 15.5S RNA over
the S^ nuclease a n a l y s i s ,
70-90 n u c l e o t i d e s
isolated
electrophoresis.
larger
a l s o present
the
15.5S
Fig. 7 i l l u s -
15S rRNA. When t h i s
the p r o t e c t e d
(Fig. 6 ) .
This i n d i c a t e s
that
15.5S RNA species - more e a s i l y d e t e c t a b l e in the p e t i t e
strains
the
mutant
- but a l s o p r e s e n t in w i l d - t y p e mtRNA i s not due to
aberrant
transcription.
When the Mbol fragment
rRNA gene l a b e l l e d
with the p e t i t e
fragment
is
at
containing
the 3 1 terminus
nuclease mapping
a 2000 bp HapII fragment
e
its
the 3'-end
of
the gene or
the
as t o be u n d e t e c t a b l e by the S^
.
'
'
,
that
the 15S rRNA gene i s
[ 6 ] . Here we show t h a t
l o c a t e d on
the 15.5S RNA
e n t i r e t y on the same HapII fragment.
was i s o l a t e d
f _ ^
protected
15.5S RNA and 15S rRNA
We have shown e a r l i e r
bp fragment
the 15S
hybridization
technique.
P r e c i s e mapping of
in
of
( F i g . 8 ) . We conclude t h a t the 15.5S RNA
extension must be so small
is present
the 3'-end
i s used for
mutant and w i l d - t y p e mtRNAs, only one
found
species has no extension beyond
1360
frag-
than t h a t derived from the 15s
and l a b e l l e d
The 2000
at the 3 ' - e n d s with
32
P.
Fig. 8. S nuclease analysis of hybrids formed with
the 3' terminally-labelled Mbol DNA fragment originating from the 3' half of the 15S rRNA gene and mtRNA.
Hybrids were electrophoresed on alkaline agarose gels
of 2%. DNA was blotted to nitrocellulose f i l t e r s and
radioactive bands visualized by autoradiography. S
nuclease-treated hybrids formed with petite mutant
LH25F2 RNA: a) 100 U/ml; b) 50 U/ml; c) untreated.
S nuclease-treated hybrids formed with S. cerevisiae
KL1H-1A RNA: d) 100 U/ml; e) 50 U/ml; f) untreated.
g) As d) but with RNA and not treated with S nuclease;
h) 32 P-labelled DNA fragment; i) marker DNA, pBR32? x
Hinfl, see Methods.
Nucleic Acids Research
This fragment was hybridized with p e t i t e mutant mtRNA and the
hybrids were treated with S,. nuclease, electrophoresed on an
agarose gel and blotted onto a nitrocellulose f i l t e r . Fig. 9
shows that no 32 P end-label survives the S1 nuclease treatment,
but when the f i l t e r s are hybridized with in v i t r o 3 2 P-labelled
15S rRNA, the specific DNA fragments described e a r l i e r [6] are
found indicating that bona fide hybrids were made surviving S,.
nuclease a n a l y s i s . The experiment shows that the 15.5S RNA does
a b c d e f g h
a'b'c'd'e'f g'h'
a b e d
e
••
Fig. 9. S. nuclease analysis of hybrids between mtDNA fragments labelled
with 32 P a t the 3'-end of the HapII s i t e located a t the 5'-end of the 15S
rRNA gene and mtRNA. Hybrids were electrophoresed on a 2% agarose g e l . DNA
was blotted t o n i t r o c e l l u l o s e f i l t e r s and labelled DNA bands detected by
autoradiography (lanes a - h ) . Afterwards the n i t r o c e l l u l o s e sheet was
hybridized with in v i t r o 3 2 P - l a b e l l e d 15S rRNA (lanes a ' - h ' ) . a ) Marker
DNA, pBR322 x Hinfl. S nuclease-treated hybrids formed with LH25F2 RNA:
b) 100 U/ml; c) 50 U/ml; d) untreated, e) the HapII DNA fragment labelled
with 3 2 P a t the 3'ends.S nuclease-treated hybrids formed with S. c e r e v i s i a e
KLIt-tA mtRNA: f) 100 U/ml; g) 50 U/ml; h) untreated. Of the marker DNA
fragments in a ' only one i s s t i l l v i s i b l e due to decay of 32 P label
in the time elapsed between the f i r s t and the second experiment.
Fig. 10. S 1 nuclease analysis on hybrids formed with t h e MboII x HapII
double digestion DNA fragment and p e t i t e mutant LH25F2 RNA. Hybrids were
electrophoresed on a neutral 2% agarose g e l . DNA was blotted t o n i t r o c e l lulose f i l t e r s and hybridized with in v i t r o 3 2 P-labelled 15S rRNA. DNA
bands were detected by autoradiography. a ) DNA cut with MboII and HapII.
S nuclease-treated hybrids: b) 100 U/ml; c) 50 U/ml; d) untreated, e) Marker
DNA, pBR322 x Hinfl, see Methods.
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not overlap the HapII site located at the 5'-end of the 15S
rRNA gene.
The 15.5S RNA and the 15S rRNA also do not extend over the
HapII site flanking the 3' side of the gene. The isolated 2000
bp HapII fragment was cut in two halves with MboII and the
fragments were hybridized with petite mutant RNA. After S,.
nuclease treatment the hybrids were electrophoresed and blotted
on nitrocellulose filters. The protected DNA strands were
detected by hybridization with 32 P-labelled 15S rRNA. From the
5'-end of the fragment two protected pieces were found as
expected (Fig. 1 0 ) . From the 3'-end DNA fragment only one
protected piece is recovered and this migrates faster than the
original MboII x HapII fragment used for hybridization indicating that part of this hybrid fragment was not covered with RNA
in hybrid (Fig. 1 0 ) . The difference in migration suggests that
the 15S rRNA and the 15.5S RNA both end approximately 90 bp
short of the HapII site.
DISCUSSION
Yeast petite mutants retaining the gene for the 15S rRNA
contain a prominent mtRNA species (15.5S) migrating just behind
the 15S RNA on an agarose gel [8]. We have characterized and
mapped this RNA species on mtDNA, using the S- nuclease mapping
technique of Berk and Sharp [21]. It contains all RNA sequences
of the 15S rRNA plus approximately 80 nucleotides derived from
the 5' flanking region of the 15S rRNA gene. Both the 15.5S RNA
and the 15S rRNA map within the 2000 bp HapII fragment and their
3'-ends are the same within the limits of accuracy of the S^
nuclease mapping technique detected. An RNA species with the same
characteristics has been purified from wild-type mtRNA. This
shows that faithful transcription still occurs in petite mutant
mitochondria, but that the rate of processing of RNA species may
be altered leading to steady-state levels of RNA species very
different from those in wild-type. It is very tempting to
consider the 15.5S RNA as a precursor of 15S rRNA. In in vitro
RNA synthesis experiments with isolated mitochondria an RNA
species comparable with the 15.5S rRNA described here, shows
faster labeling kinetics than the mature 15S rRNA (Boerner, P.,
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Mason, T.L. and Fox, T.D., personal communication)
by Levens et a l .
[23] primary mitochondrial
In a study
transcripts
still
carrying
a diphosphate or triphosphate at the 5'-end were
detected
through specific
guanylyl
transferase
labelling with [<x-32P]GTP by the enzyme
from Vaccinia. An RNA species with the
same migration behaviour as the 15.5S RNA was capped with
[a-32P]GTP. We consider i t
very l i k e l y ,
therefore,
that
the
15.5S RNA is the primary transcript of the 15S rRNA gene.
I t i s of interest
precursor are s t i l l
that the 15S rRNA and i t s
synthesized
putative
in the low-complexity
petite
mutant LH26D7-10 for which we have shown that the end point of
the deletion i s located
5'
just
upstream of the HapII s i t e on the
flank of the gene. Considering the evidence of Levens et
[23],
who found a primary t r a n s c r i p t
ponding in length with the putative
described
signals
in this paper, we favour
for i n i t i a t i o n
al.
in wild-type mtRNA corres15S rRNA precursor we have
the idea that
specific
and termination of transcription
have
been conserved in the p e t i t e mutant LH26D7-10. This means that
in a DNA stretch of approximately 300 bp length extending
the HapII s i t e on the 5'
flank
of
the gene to the 5'-end
putative precursor RNA, we have located an i n i t i a t i o n
for mtRNA. Furthermore, this
contains a processing s i t e
signal
for
termination of
the
signal
low-complexity p e t i t e mutant
for
from
of
still
15S rRNA maturation and a stop
transcription.
ACKNOWLEDGEMENTS
We thank Profs P.Borst and L . A . G r i v e l l f o r h e l p f u l comments, Mr L.A.M.
Hensgens f o r p r o v i d i n g p e t i t e mutants and Mrs F.Fase-Fowler for g i f t s of
marker DNAs. This work was s u p p o r t e d i n p a r t by a g r a n t t o P . B o r s t / L . A . G r i v e l l
from The N e t h e r l a n d s Foundation f o r Chemical Research (SON) with f i n a n c i a l
aid from The Netherlands O r g a n i z a t i o n f o r t h e Advancement of Pure Research
(ZWO) .
Abbreviations:
rRNA, r i b o s o m a l
RNA; b p , b a s e
pair(s).
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