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Transcript
Reprint from
SYSTEM4TIC
AND
APPLIED
MICROBOOGY
formerly Zentralblatt für Bakteriologie,
Mikrobiologie und Hygiene
I. Abt. Originale C
Gropp e t a l . (1986)
H o m o l o g i e s o f compon e n t s o f DNA-dependent
RNA p o l y m e r a s e s o f
archaebacteria,
e u k a r y o t e s and e u b a c t .
Gustav Fischer Verlag
Stuttgart • New York
System. A p p l . M i c r o b i o l . 7, 95-101 (1986)
Homologies of Components of DNA-dependent RNA Polymerases of
Archaebacteria, Eukaryotes and Eubacteria*
F. G R O P P , W . D . R E I T E R , A . S E N T E N A C , W . Z I L L I G , R . S C H N A B E L , M . T H O M M ,
1
STETTER
1
1
2
1
3
4
and K . O .
4
Max-Planck-lnstitut für Biochemie, D-8033 Martinsried, Federal Republic of Germany
Centre d'Etudes Nucleaires de Saclay, F-91191 Gif-sur-Yvette Cedex, France
Present address: Medical Research Council, Cambridge CB2 2 Q H , Great Britain
Institut für Biochemie, Genetik und Mikrobiologie der Universität Regensburg, D-8400 Regensburg, Federal Republic of Germany
Summary
Using an immunochemical approach homologies between single components of DNA-dependent R N A
polymerases from eubacteria, archaebacteria and eukaryotes were investigated. The largest components of
all R N A polymerases included in this study are homologous to one another indicating a monophyletic
origin of these proteins.
Immunological crossreactions show that one of the large subunits present in the enzymes of sulfurdependent archaebacteria is split into two smaller components in methanogens and halophiles. One of
these smaller components roughly corresponds to the second largest subunit of the three eukaryotic
enzymes whereas the other one shares antigenic determinants with subunit ß of eubacterial R N A polymerases.
Semi-quantitative evaluation of the data suggests that the three nuclear R N A polymerases of eukaryotes
have evolved from an ancestral enzyme of the type that is found in sulfur-dependent archaebacteria.
K e y w o r d s : Archaebacteria - R N A polymerase - Immunological crossreaction - P h y l o g e n y - E v o l u t i o n
Introduction
Comparative cataloging of T R N a s e generated fragrnents of 1 6 S - r R N A s has led to the concept that all living
organisms can be classified into one of three major groups:
the " u r k i n g d o m s " of the eubacteria, the archaebacteria
n d the eukaryotes (Woese a n d Fox, 1977; Woese et a l . ,
1978; Fox et a l . , 1977). Since then m u c h additional evidence has been accumulated c o n f i r m i n g the idea that there
r e t w o groups of prokaryotes that are no more related to
°ne another than either of them is related to the eucyta
(i. e. the nuclear plus the cytoplasmic compartment of the
ukaryotes). This evidence includes comparison of differn t parts of the translation apparatus (Fox et a l . , 1982;
Matheson a n d Yaguchi, 1982; Yaguchi et a l . , 1982; Gup*, 1984; Kessel a n d Klink,
1982; Cammarano
et a l . ,
1985), of the enzymes involved i n replication (Prangishvilli a n d Zillig, 1984; Forterre et a l . , 1984; Nakayama et
al., 1985; Klimczak et a l . , 1985) and transcription (Zillig
r
a
a
e
e
tc
* Paper given at the E M B O Workshop on the Molecular Genetics of Archaebacteria, München-Martinsried, June 23 to 26,
1985.
et a l . , 1982; Schnabel et a l . , 1983; Huet et a l . , 1983;
Prangishvilli
et a l . , 1982), of cell w a l l composition (Kandier, 1982) a n d membrane structure (review by Langworthy et a l . , 1982).
In order to study phylogenetic relationships and evolution of different organisms it is desirable to compare cellular components that:
(1) are present i n all organisms under investigation,
(2) have already been present very early i n evolution,
and
(3) are sufficiently complex that statistically significant
data can be obtained.
These criteria are ideally met by some proteins a n d n u cleic acids involved i n transcription, translation a n d replication. Whereas ribosomal proteins a n d most D N A polymerases are too small or of too l o w complexity to be well
suited for investigating phylogenetic relationships, the
analysis of the large ribosomal R N A s a n d of R N A polymerases yields valuable i n f o r m a t i o n concerning phylogeny
and evolution.
The comparison of the antibiotic responses as well as
the component patterns a n d subunit homologies of R N A
strongly inhibit the R N A polymerases of eubacteria. A
typical i n h i b i t o r of eukaryotic R N A polymerases is the
m u s h r o o m poison a - a m a n i t i n that blocks transcription by
RNA
Polymerase II at very l o w concentrations. In most
eukaryotes the activity of polymerase III is affected by high
concentrations of this i n h i b i t o r whereas polymerase I is
insensitive. Examples can be f o u n d , however, where the
i n h i b i t i o n pattern is different. In yeast for instance R N A
Polymerase I is inhibited by a - a m a n i t i n but polymerase III
is not.
The component pattern of all three eukaryotic R N A
polymerases is very c o m p l e x . Analysis by SDS P o l y a c r y l amide gel electrophoresis shows the presence of 10 or
more different Polypeptide chains w i t h molecular weights
ranging f r o m 10 kilodaltons to more than 200 k i l o d a l t o n s .
W i t h the exception of polymerase III, where subunits of
intermediate size are f o u n d , each eukaryotic R N A polymerase is composed of t w o very large subunits (molecular
weight above 100 kilodaltons) and several smaller c o m p o nents (molecular weight below 50 kilodaltons). Usually,
designations have been given to single components that
include the type of enzyme as well as the molecular weight
of the subunit. T h u s the largest component of yeast R N A
Polymerase I is referred to as I , the second largest as I
etc. C o m p a r i s o n of single subunits of R N A polymerases
f r o m different organisms is facilitated, however, w h e n a
somewhat different nomenclature is employed. W e therefore decided to call the largest subunit of yeast R N A poly-
polymerases f r o m organisms belonging to different phylogenetic groups:
(1) confirms that there is a deep division w i t h i n the
prokaryotes separating eubacteria and archaebacteria,
(2) is independent evidence that w i t h i n the u r k i n g d o m
of archaebacteria there are t w o major branches, one
formed by the h a l o p h i l i c and methanogenic archaebacteria
and
the other formed by the thermophilic sulfur
metabolizing archaebacteria, and
(3) suggests that the three eukaryotic nuclear R N A polymerases have evolved f r o m an ancestral enzyme of the
archaebacterial type.
Properties of Different DNA-dependent R N A
Polymerases
nuclear RNA
polymerases
W i t h i n the nuclear compartment of eukaryotic cells
three R N A polymerases are involved in the transcription
of different "classes" of genes. Polymerase I (or A ) is responsible for transcription of r D N A (with the exception of
5 S - r R N A genes) whereas the f o r m a t i o n of h n R N A is
catalyzed by R N A Polymerase II (or B). T h e genes for
some small R N A species i n c l u d i n g 5 S - r R N A and t R N A s
are transcribed by R N A Polymerase III (or C ) .
A l l eukaryotic nuclear R N A polymerases are insensitive
the antibiotics r i f a m p i c i n and streptolydigin that
ARCHAEBACTERIA
Yeast
RNA
Pol
III
I
(A)amD 190 WD 220/185
VZ) 150
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(B)^
135
CD
CD
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CD
CD
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34
31
27
23
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10
19
14
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ann) cmp dmp
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, H = 0
CD
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21
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A CHHD
1 3 5
EUBACTERIA
3CCL
EUKARYOTES
Me
to
1 9 0
i c t e irium
Eukaryotic
CD
CD
D
cu
CD
CD
O
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10
Fig. 1: Schematic drawing of the component patterns of DNA-dependent R N A polymerases of eukaryotes, archaebacteria and
eubacteria obtained by Separation of Polypeptide chains by SDS Polyacrylamide gel electrophoresis.
The numbers give the apparent molecular weights in kilodaltons of the components of the yeast enzymes. Capital letters are used for
the designation of the components of archaebacterial R N A polymerases and of the two largest subunits of the yeast enzymes. Equal
design of components from different R N A polymerases indicates homology.
merases " A " a n d the second largest subunit " B " irrespective of the type of polymerase (I, II or III). A l l " A " subunits
of eukaryotic R N A polymerase are homologous and the
same is true for the " B " components (Huet et a l . , 1983
and data presented here). Some of the smaller components
are identical in all three R N A polymerases a n d others are
c o m m o n to t w o of the three enzymes. M o s t of the c o m p o nents, however, are unique and can only be f o u n d i n one
of the R N A polymerases (for a detailed review see Paule,
1981).
Eubacterial
RNA
polymerases
In contrast to the Situation found in eukaryotes all genes
of eubacteria are transcribed by one R N A polymerase.
The enzyme is strongly inhibited by the antibiotics rifampicin and streptolydigin but its activity is not affected by
a-amanitin.
A s i n the eukaryotic enzymes there are t w o large subunits (termed ß a n d ß') but very small components are
completely missing. T y p i c a l l y eubacterial enzymes contain
two copies of a subunit of intermediate size (a) as well as
an initiation factor termed o that is involved i n p r o m o t o r
recognition (cf. Zillig et a l . , 1976; Burgess, 1976). T h i s ofactor is a stoichiometric component of some eubacterial
R N A polymerases (Herzfeld and Kiper, 1976). In most
eubacteria, however, it is only temporarily associated w i t h
the enzyme.
Archaebacterial
RNA
polymerases
A s i n eubacteria only one R N A polymerase appears to
be responsible for transcription of all genes i n archaebacteria. Archaebacterial R N A polymerases are not inhibited
by rifampicin, streptolydigin or a - a m a n i t i n and no substance is k n o w n that specifically blocks R N A synthesis by
the archaebacterial enzyme. Its activity is blocked, h o w ever, by such general inhibitors of transcription as heparin
and actinomycin D . T w o different types of R N A polymerases are observed corresponding to the t w o major
branches of this u r k i n g d o m . T h e component pattern of the
enzymes f o u n d i n sulfur-dependent archaebacteria a n d
Thermoplasma
closely resembles that of the eukaryotic
R N A polymerases (especially polymerase I a n d II). In
halophilic and methanogenic archaebacteria a somewhat
different subunit composition is observed. T y p i c a l l y there
is one large subunit of a molecular weight greater than 100
kilodaltons and t w o components of about 6 0 - 9 0 k i l o d a l tons. A n a l o g o u s to the enzymes f r o m sulfur-dependent archaebacteria (including Thermoplasma)
a n d eukaryotes,
several smaller Polypeptide chains strictly copurify w i t h
the larger components. In the f o l l o w i n g , the term „sulfurdependent archaebacteria" is meant to include the thermoacidophilic genus Thermoplasma
if not noted otherwise.
"
System. A p p l . M i c r o h i o l .
Vol. " 1
Homologies Between Subunits of R N A Polymerases
from Eubacteria, Eukaryotes and Archaebacteria
RNA polymerases and antibodies
of homologies
used for
investigation
Antibodies were directed against single components of
the R N A polymerases f r o m Sulfolobus acidocaldarius and
Methanobacterium
thermoautotrophicum
(Schnabel et a l . ,
1983), against the subunits of the Escherichia coli polymerase and against those of the three nuclear enzymes of
Saccharomyces cerevisiae (A. Sentenac).
T h e yeast enzymes I and II (Buhler et a l . , 1980) were
used for i m m u n o b l o t t i n g as representatives of eukaryotic
R N A polymerases. Archaebacterial R N A polymerases
were f r o m Sulfolobus acidocaldarius
(Zillig et a l . , 1979).
Halobacterium
halobium (Zillig et a l . , 1978; Madon a n d
Zillig,
1983).
Methanococcus
thermolithotrophicus
(Thomm a n d Stetter, 1985) and Methanolobus
vulcanii
(M. Thomm,
unpublished). A s a representative of a
eubacterial R N A polymerase the enzyme f r o m E. coli was
used.
The technique
of
immunoblotting
The simplest w a y to detect homologies between different Polypeptide chains is to look for c o m m o n antigenic
determinants.
The antibodies used in such studies must meet t w o demands:
(1) they must be p o l y c l o n a l so that different antigenic
determinants of a Polypeptide chain are recognized;
(2) they must be directed against a denatured Polypeptide chain (so-called "sequence-specific antibodies");
otherwise homolgies of sequences that are hidden i n the
interior of a native protein can be overlooked.
After Separation of different Polypeptide chains by SDS
Polyacrylamide gel electrophoresis, the resulting c o m p o nent pattern is blotted to nitrocellulose sheets and challenged w i t h the antibody of interest. B o u n d antibody can
be visualized by employing labelled protein A f r o m S. aureus that is k n o w n to bind specifically to the F part of
i m m u n o g l o b u l i n s . A somewhat different approach uses a
second antibody that is directed against the F part of the
first one. This second antibody is usually covalently linked
to an enzyme capable of catalyzing a colour reaction (a
peroxidase is used i n most cases).
c
c
The most important disadvantage of the i m m u n o l o g i c a l
approach is the fact that similar amino acid sequences do
not necessarily result i n c o m m o n antigenic determinants.
Thus it must be borne in m i n d that the absence of an
i m m u n o l o g i c a l crossreaction does not necessarily mean
that t w o proteins are completely unrelated o n sequence
level. T h o u g h direct comparison of protein sequences
yields more Information than the i m m u n o c h e m i c a l
method, the latter is simple to perform a n d results can be
obtained w i t h very little experimental effort. Nevertheless
it js highly desirable to compare amino acid sequences of
those proteins for w h i c h an i m m u n o l o g i c a l crossreaction
has been demonstrated.
The "A" components
polymerases
of DNA-dependent
RNA
Immunological crossreactions show the f o l l o w i n g c o m ponents to be h o m o l o g o u s :
- T h e largest subunit of the three eukaryotic nuclear R N A
polymerases (i. e. the " A " subunits of these enzymes)
- T h e second largest subunit of the R N A polymerases
f r o m sulfur-dependent archaebacteria
— T h e largest subunit of the R N A polymerases f r o m
halophilic a n d methanogenic archaebacteria
— Subunit ß ' of eubacterial R N A polymerases
Those components of archaebacterial R N A polymerases that are homologous to the A subunits of the eukaryotic enzymes have also been given the designation " A " ,
though i n the case of sulfur-dependent archaebacteria this
subunit is not the largest one. F o r the homologies listed
above the f o l l o w i n g evidence has been obtained:
(1) Antibodies raised against the largest subunit of the
three eukaryotic R N A polymerases crossreact w i t h c o m ponent A of a l l archaebacterial enzymes (i.e. the largest
component i n the halophilic/methanogenic branch and the
second largest one of sulfur-dependent archaebacteria).
T y p i c a l l y this crossreaction is m u c h stronger w i t h the archaebacterial R N A polymerase than it is w i t h the corresponding subunits of the t w o other eukaryotic enzymes.
Antibodies directed against the A subunit of yeast R N A
Polymerase III for instance give a strong signal w i t h the
archaebacterial A components whereas the i m m u n o l o g i c a l
crossreaction w i t h the largest component of yeast polymerase I is weak a n d such a reaction is barely detectable
w i t h the A subunit of polymerase II (Fig. 2). Since antibodies directed against the largest components of eukaryotic R N A polymerases I a n d II strongly crossreact w i t h the
A component of the archaebacterial enzymes one must
conclude that all A components of the eukaryotic R N A
polymerases are homologous though direct crossreaction
cannot always be demonstrated.
(2) Antibodies directed against subunit ß ' of E. coli
R N A polymerase crossreact w i t h the A component of the
archaebacterial enzymes a n d w i t h the A component of
yeast R N A polymerase I (Fig. 2). T h i s crossreaction is
strongest w i t h the enzyme f r o m H. halobium suggesting
that the archaebacteria belonging to the halophilic/
methanogenic branch are more closely related to eubacteria than the sulfur-dependent archaebacteria.
The "B" components
polymerases
of DNA-dependent
RNA
Immunological crossreactions show the f o l l o w i n g c o m ponents to be h o m o l o g o u s :
— T h e second largest subunits of the three eukaryotic
R N A polymerases (i.e. the " B " subunits)
— T h e largest component (B) of the R N A polymerases of
sulfur-dependent archaebacteria
— T h e second largest (B') a n d the third largest ( B " )
component of the R N A polymerases of h a l o p h i l i c a n d
Anti - Pol l l l
(A)
methanogenic archaebacteria. B ' a n d B " w i t h i n one
Anti - ß' ( E . c o l i )
(S. c e r e v i s i a e )
R N A polymerase are i m m u n o l o g i c a l l y unrelated, but
both components crossreact w i t h the B subunit of the
R N A polymerases of sulfur-dependent archaebacteria.
— Subunit ß of eubacterial R N A polymerases
A
W h e n antibodies directed against the second largest
subunit (subunit B) of eukaryotic R N A polymerases are
used, a strong crossreaction w i t h component B of Sulfolobus R N A polymerase is observed (Fig. 4). Similar to
the S i t u a t i o n w i t h the A components, crossreactions between the B subunits of the three eukaryotic R N A polymerases themselves are mostly rather weak o r not detectable
at all whereas the crossreaction w i t h the corresponding
Sulfolobus component can easily be demonstrated.
Antibodies against component B of Sulfolobus R N A
Polymerase crossreact w i t h t w o components of the enzymes f r o m h a l o p h i l i c a n d methanogenic archaebacteria,
that have therefore been termed B ' a n d B " (Fig. 3).
H.H.
Antibodies directed against B ' a n d B ' f r o m
Methanobacterium thermoautotrophicum
both strongly crossreact
w i t h component B of Sulfolobus R N A polymerase, but no
Fig. 2: The homology group of " A " subunits.
crossreaction is observed between the components B ' a n d
After Separation of components of different R N A polymerases by
SDS Polyacrylamide gel electrophoresis and transfer to nitrocelB " themselves. T h i s strongly suggests that B ' a n d B "
lulose sheets, homologous subunits were detected by incubation can be viewed as fragments of a larger B component. Since
with the antibodies indicated. Bound immunoglobulin was vis- the sizes of B ' and B " of different R N A polymerases
ualized using peroxidase-coupled anti-antibody.
vary to some degree (in Methanolobus
B " is even someAbbreviations used:
what larger than B') the question arises whether the split
H . h.: Halobacterium halobium
of the large B subunit is i n the same position i n R N A
S.a.: Sulfolobus acidocaldarius
polymerases f r o m different halophilic a n d methanogenic
M c . th.: Methanococcus thermolithotrophicus
archaebacteria. Experimental data show that the distribuS.c: Saccharomyces cerevisiae
1 6 0
Anti — B
Anti - B
(S.acidocaldarius)
(Mb. tharmoautotrophicum)
Anti - B*
1
B
B' ~
2 <•
»
>
B"*
H.h.
i
(Mb. tharmoautotrophicum)
1
Mc.th.
S.a.
Ml.v.
H.h.
Mc.th.
Ml.v.
S.a.
H.h.
B-4
Mc.th.
B
'
Ml.v.
Fig. 3: The homology group of ' B " components of archaebacteria. For details of the method used to detect homologies see Fig. 2.
M c . t h . : Methanococcus thermolithotropbicus
Abbrevations used:
H . h . : Halobacterium halobium M l . v . : Methanolobus vulcanii
S.a.: Sulfolobus acidocaldarius
Anti - Pol l l l
Anti - ß
I
I
H.h.
Ml.v.
(B)
(S. cerevisiae)
(E. c o l i )
•
1 2 8
B'
S.a.
S.a.
H.h.
Ml.v.
Mc.th.
Fig. 4: " B " component homology group of R N A polymerases
from archaebacteria, eubacteria and eukaryotes.
For details of the method used to detect homologies see Fig. 2.
Abbrevations used:
S.a.: Sulfolobus acidocaldarius
H . h . : Halobacterium halobium
M l . v . : Methanolobus vulcanii
M c . t h . : Methanococcus thermolithotropbicus
tion of antigenic determinants to B ' a n d B " is not identical i n different enzymes of this type. T h u s antibodies
directed against B ' of Mb. thermoautotrophicum
crossreact w i t h the B ' subunits of Mc.
thermolithotropbicus,
Ml. vulcanii a n d H. halobium exclusively. Antibodies d i rected against B " of Mb. thermoautotrophicum,
however, show a crossreaction w i t h B ' of H. halobium a n d
Ml. vulcanii though the reaction is weaker than that w i t h
B " . In the case of Mc. thermolithotropbicus
only B "
crossreacts, indicating that antigenic determinants have
been separated i n the same ways i n
Methanobacterium
and Methanococcus
(Fig. 3).
Antibodies directed against subunit ß of E. coli R N A
Polymerase crossreact weakly yet significantly w i t h the B
components of Sulfolobus R N A polymerase a n d yeast
Polymerase II (Fig. 4). A s far as the enzymes f r o m
halophilic a n d methanogenic archaebacteria are c o n cerned, anti-ß shows the same pattern of crossreactions
that is observed w i t h antibodies directed against c o m p o nent B " of Mb. thermoautotrophicum
(Fig. 3 a n d 4).
T h e B " components of a l l enzymes of this branch are
recognized by anit-ß. In addition there is also a weaker
crossreaction of anti-ß w i t h B ' of Halobacterium
and
Methanolobus.
B ' of Methanococcus,
however, does not
react.
O n the other hand antibodies directed against the B
subunits of eukaryotic R N A polymerases show a pattern
of crossreactions w i t h enzymes f r o m halophilic a n d
methanogenic archaebacteria that is very similar to that
obtained using antibodies against component B ' of Mb.
thermoautotrophicum
(Fig. 3 a n d 4). Strong crossreaction
is observed w i t h B ' but not B " of these enzymes. O n e
exception is a weak crossreaction of anti-III 8 w i t h H.
halobium
B " though the reaction w i t h B ' is m u c h
stronger.
12
Homologies
between smaller
components
W i t h decreasing length of Polypeptide chains there is an
increasing probability that existing homologies between
two proteins cannot be demonstrated any more by the
i m m u n o c h e m i c a l approach. W i t h i n the u r k i n g d o m of archaebacteria, it is possible to identify the " C " components
Anti-C
Anti-Pol
(S. acidocaldarius)
l
folobus C is at the limit of detection (using anti-o) o r
cannot be observed at all (using a n t i - I ) .
The only significant i n t e r - u r k i n g d o m crossreaction i n volving an archaebacterial component smaller than C is
the recognition of component D of the Sulfolobus R N A
Polymerase by antibodies directed against the 4 0 k i l o d a l ton component of yeast R N A polymerase I (Fig. 6). Clearly one must expect that this is not the only h o m o l o g y
between smaller components of archaebacterial a n d
eukaryotic R N A polymerases. Other methods have to be
used, however, to clarify this point.
4 0
(S. cerevisiae)
49
Conclusions
Mc. th.
Pol I
(S.c.)
E.c.
Pol I
(S.c.)
S.a.
Fig. 5: Immunological crossreactions between smaller components of R N A polymerases from archaebacteria, eubacteria and
eukaryotes.
For details of the method used to detect homologies see Fig. 2.
Abbrevations used:
S . c : Saccharomyces cerevisiae
S.a.: Sulfolobus acidocaldarius
M c . t h . : Methanococcus thermolithotropbicus
E . c : Escherichia coli
of all R N A polymerases using antibodies directed against
component C of the Sulfolobus enzyme.
E m p l o y i n g antibodies raised against components D a n d
E of Sulfolobus R N A polymerase, homologous c o m p o nents of other archaebacterial R N A polymerases could o n ly be identified in some cases. Occasionally it is possible to
observe i m m u n o l o g i c a l crossreactions between smaller
components of R N A polymerases, even when the enzymes
belong to organisms f r o m different u r k i n g d o m s .
Antibodies directed against component C of Sulfolobus
R N A polymerase crossreact w i t h the o factor of E . coli as
well as w i t h the 49 k i l o d a l t o n component o f yeast R N A
Polymerase I (Fig. 6). By using antibodies against E. coli o
and S. cerevisiae I , however, a crossreaction w i t h Sul4 9
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B"
Sulfolobus
B
Halobacterium
B'*B"
Methanobacterium
B'* B"
i ^ m m m m ^ d E. CO I i
Saccharomyces
ß
B
Fig. 6: Schematic drawing of the distribution of conserved antigenic determinants in the component " B " homology group of
DNA-dependent R N A polymerases.
Corresponding antigenic determinants are indicated by equal
hatching.
(1) D N A - d e p e n d e n t R N A polymerases from organisms
belonging to the three different urkingdoms are h o m o l o gous to one another. T h i s can be clearly s h o w n for the
largest subunits present i n all of these enzymes a n d it is
probably also true for some of the smaller components.
(2) T h e largest subunits of the three different eukaryotic
nuclear R N A polymerases are more related to the corresponding components of the enzymes from archaebacteria
than to one another. This strongly suggests that these three
eukaryotic R N A polymerases have evolved f r o m a n ancestral enzyme that was very similar to the R N A polymerase as it is f o u n d today i n archaebacteria, especially the
sulfur-dependent branch.
(3) T h e large B subunit present i n sulfur-dependent archaebacteria seems to have been subject to different cleavage a n d t r i m m i n g events during evolution. W i t h i n the
halophilic/methanogenic branch of archaebacteria a split
of a larger " B " component into the smaller components
B ' a n d B " is observed. This split separates antigenic
determinants in different ways depending o n the organism
f r o m w h i c h the R N A polymerase has been isolated. T h i s
fact makes it highly unlikely that i n evolution there was a
fusion event between the B ' a n d B " components resulting i n the formation of the large B subunit of the R N A
polymerases of sulfur-dependent arachebacteria. T h e hypothesis of an ancestral " B " gene that is still present in the
R N A polymerases f r o m sulfur-dependent archaebacteria,
but has been split into t w o different components i n R N A
polymerases f r o m h a l o p h i l i c a n d methanogenic archaebacteria, appears much more attractive. T h e investigation of the Organization of the genes c o d i n g for B ' a n d
B " i n halophilic and methanogenic archaebacteria is the
obvious approach to obtain more detailed i n f o r m a t i o n .
Whereas i n a l l archaebacteria the w h o l e ancestral " B "
gene appears to be present (whether split or not), in eubacteria a n d eukaryotes obviously only part of it has been
conserved. Experimental data indicate that a part chiefly
corresponding to the B " component of h a l o p h i l i c a n d
methanogenic archaebacteria has been conserved in the ß
subunit of eubacteria whereas another, somewhat overlapping part of the putative ancestral " B " gene (mainly corresponding to B ' i n halophilic a n d methanogenic archaebacteria is f o u n d i n the B subunits of R N A polymerases of eukaryotes (Fig. 5). Since the ß subunit of E . coli
R N A polymerase is believed to harbour the catalytic sites
for the polymerization reaction, these findings are of special interest.
(4) Thermoplasma
acidophilum,
w h i c h o n the basis of
r R N A sequence data appears somewhat closer to the
methanogenic than to the sulfur-dependent archaebacteria, has a R N A Polymerase of the sulfur-dependent type
indicating an intermediate phylogenetic position.
Since molecular cloning techniques and methods for sequencing of D N A are well advanced, it appears w o r t h while to obtain more detailed Information about the
evolution of R N A polymerases by direct comparison of
amino acid sequences. Using such approaches it might also
be possible to gain some insight into the basic mechanisms
of transcription.
References
Buhler, ]. M., Huet, ]., Davies, K. E., Sentenac, A., Fromageot,
P.: Immunological studies of yeast nuclear R N A polymerases
at the subunit level. J. Biol. Chem. 255, 9949-9954 (1980)
Burgess, R. R.: Purification and Physical Properties of E. coli
R N A Polymerase. In: R N A Polymerase, P. Losick and M.
Chamberlin, eds., pp. 69-100. Cold Spring Harbor/New
York, Cold Spring Harbor Laboratory 1976
Cammarano, P., Teichner, A., Londei, P., Acca, M., Nicolaus, B.,
Sanz, J. L., Amils, R.: Insensitivity of archaebacterial ribosomes to protein synthesis inhibitors. Evolutionary implications. E M B O J. 4, 811-816 (1985)
Forterre, P., Elie, C , Kohiyama, M.: Amphidicolin inhibits
growth and D N A synthesis in halophilic archaebacteria. J.
Bact. 159, 800-802 (1984)
Fox, G. E., Pechmann, K.J., Woese, C. R.: Comparative cataloging of 16S ribosomal R N A : molecular approach to procaryotic
systematics. Int. J. System. Bact. 27, 44-57 (1977)
Fox, G. £., Luehrsen, K. R., Woese, C. R.: Archaebacterial 5S
ribosomal R N A . Z b l . Bakt. Hyg., I. Abt. Orig. C 3, 330-345
(1982)
Gupta, R.: Halobacterium volcanii tRNAs: Identification of 41
t R N A s covering all amino acids, and the sequences of 33 class
I tRNAs. J. Biol. Chem. 259, 9461-9471 (1984)
Herzfeld, F., Kiper, M.: The reconstitution of Anacystis nidulans
DNA-dependent R N A polymerase from its isolated subunits.
Europ. J. Biochem. 62, 189-192 (1976)
Huet, /., Schnabel, R., Sentenac, A., Zillig, W.: Archaebacteria
and eukaryotes possess DNA-dependent R N A polymerases of
a common type. E M B O J. 2,1291-1294 (1983)
Kandier, O.: Cell wall structures and their phylogenetic implications. Z b l . Bakt. Hyg., I. Abt. Orig. C 3, 149-160 (1982)
Kessel, M., Klink, F.: Identification and comparison of eighteen
archaebacteria by means of the diphtheria toxin reaction. Z b l .
Bakt. Hyg., I. Abt. Orig. C 3, 140-148 (1982)
Klimczak, L. ]., Grummt, L, Burger, K. ].: Archaebacterial D N A
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F. Gropp, Max-Planck-Institut für Biochemie, D-8033 Martinsried
Molecular Genetics of Archaebacteria", held in Martinsried,
June 23-26 (1985)
Langworthy, T. A., Tornabene, T. G . , Holzer, G.: Lipids of archaebacteria. Z b l . Bakt. Hyg., I. Abt. Orig. C 3, 228-244
(1982)
Madon, ]., Zillig, W.: A form of the DNA-dependent R N A Polymerase of Halobacterium halobium containing an additional
component, is able to transcribe native D N A . Europ. J.
Biochem. 133, 471-474 (1983)
Matheson, A. T., Yaguchi, M.: The evolution of the archaebacterial ribosome. Z b l . Bakt. Hyg., I. Abt. Orig. C 3, 192-199
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Nakayama, M., Mahrez, K. B., Kohiyama, M.: D N A polymerases and reverse transcriptase-primase from Halobacterium
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23-26 (1985)
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Schnabel, R., Thomm, M., Gerardy-Schahn, R., Zillig, W., Stetter, K. O . , Huet, ].: Structural homology between different
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Yaguchi, M., Visentin, L. P., Zuker, M., Matheson A. T., Roy,
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1976
Zillig, W., Stetter, K. O., Tobien, M.: DNA-dependent R N A
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System. A p p l . M i c r o b i o l . 7, 102-105 (1986)
Transfer RNAs of Halobacterium volcanii: Sequences of Five Leucine
and Three Serine tRNAs*
RAMESH
GUPTA
Department of Medical Biochemistry, and Department of Chemistry and Biochemistry, Southern Illinois University, Carbondale, IL
62901, U.S.A.
Summary
The sequences of eight class II transfer R N A s (those having the large extra arm) of Halobacterium
volcanii, five for leucine and three for serine are presented here. In principle, these tRNAs cover at Ieast 11
out of the possible 12 codons for these two amino acids. Although these tRNAs follow general patterns for
the class II tRNAs, in detail they are distinct from both eucaryotic and eubacterial tRNAs.
K e y w o r d s : t R N A - Halobacterium
volcanii
Archaebacteria - Extreme halophiles - R N A sequencing
Introduction
Transfer R N A plays a major role i n protein biosynthesis
and so this molecule has been extensively studied. W i t h i n
the last 20 years, sequences of more than 350 t R N A s f r o m
various sources have been determined (Sprinzl et a l . ,
1985). T h o u g h the sequences of these t R N A s differ significantly, nearly all fit the "cloverleaf" secondary structure (some m i t o c h o n d r i a l t R N A s are the exceptions).
W i t h i n a cell, the t R N A molecule interacts w i t h several
macromolecules. In some of these interactions, different
t R N A s associate w i t h the same macromolecule, e. g., a
ribosomal component, while in others, each t R N A (or one
group of isoacceptors) interacts w i t h a specific cognate
member of a set of macromolecules, e. g., a m i n o a c y l t R N A synthetases. Therefore, i n any t R N A molecule,
there are some features w h i c h are c o m m o n to all t R N A s ,
while there are other characteristics w h i c h are specific to
that particular t R N A (or that particular group of isoaccep-
* Paper given at the E M B O Workshop on the Molecular Genetics of Archaebacteria, München-Martinsried, June 23 to 26,
1985.
tors). Furthermore, a comparative study of t R N A sequences has suggested several eubacteria- and eukaryotespecific features both in the sequences, as well as i n the
modification pattern of the t R N A s {Singhai and Falls,
1979; Gupta, 1985).
A m o n g archaebacteria, the sequences of 37 t R N A s f r o m
the extreme halophiles, 3 f r o m the thermoacidophiles and
2 f r o m the methanogens are so far reported
(Kilpatrick
and Walker, 1981; Kuchino et a l . , 1982; Gu et a l . , 1983,
1984; Gupta, 1984). These archaebacterial t R N A s show
the general cloverleaf structure c o m m o n to all t R N A s .
H o w e v e r , they have several unique characteristics, and
show similarity to the eubacterial t R N A s i n some features,
while to the eukaryotic t R N A s in other [Kuchino et a l . ,
1982; Gupta, 1984, 1985). The same holds for the overall
modification patterns of the archaebacterial t R N A s (Gupta and Woese, 1980). A detailed review of the archaebacterial t R N A s has recently been published (Gupta,
1985).
The sequences of five t R N A s for leucine and three
t R N A s for serine, f r o m an archaebacterium,
Halobacterium volcanii, are presented here. These, along w i t h the
previously reported 33 t R N A sequences (Gupta, 1984) of
H . volcanii can be compared w i t h the available sets of
t R N A sequences for eubacteria and eucaryotes (Sprinzl et
a l . , 1985).
Abbreviations:
p, 5'-phosphate. The modified residues are referred to as: t A ,
N-[9-ß-D-ribofuranosylpurin-6-yl)carbamoyl]threonine;
m^C,
5-methyl C ; ac C, N -acetyl C ; C m , 2'O-methyl C ; m G , 1methyl G ; m G , N -methyl G ; m^G, N ,N -dimethyl G ; m o U ,
Materials and Methods
5-methoxy U ; U, a specific unidentified modified U ; \p,
pseudouridine; m'ij), 1-methyl \p; D, dihydrouridine; T, ribothymidine; m I, 1-methyl Inosine; R, purine; Y, pyrimidine; X , a
All the procedures for Separation and sequencing of these
specific unidentified modified G ; N , any nucleoside.
t R N A s have been described previously (Gupta, 1984).
6
4
2
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