Elements of an archaeal promoter defined by mutational analysis

Abstract

The sequence requirements for specific and efficient transcription from the 16S/23S rRNA promoter of Sulfolobus shibatae were analysed by point mutations and by cassette mutations using an in vitro transcription system. The examination of the box A-containing distal promoter element (DPE) showed the great importance of
the TA sequence in the center of box A for transcription efficiency and the influence of the sequence upstream of box A on
determining the distance between the DPE and the start site. In most positions of box A, replacement of the wild type bases
by adenines or thymlnes are less detrimental than replacements by cytosines or guanines. The effectiveness of the proximal
promoter element (PPE) was not merely determined by its high A + T content but appeared to be directly related to its nucleotide
sequence. At the start site a pyrimldlne/purine (py/pu) sequence was necessary for unambiguous initiation as shown by analysis
of mutants where the wild type start base was replaced. The sequence of box A optimal for promoter function in vitro is identical
to the consensus of 84 mapped archaeal promoter sequences.

Full text

Nucleic
Acids
Research,
Vol.
20,
No.
20
5423-5428
Elements
of
an
archaeal
promoter
defined
by
mutational
analysis
Johannes
Hain*,
Wolf-Dieter
Reiter+,
Uwe
Hudepohl
and
Wolfram
Zillig
Max-Planck-lnstitut
fOr
Biochemie,
8033
Martinsried,
Germany
Received
June
24,
1992;
Revised
and
Accepted
September
25,
1992
ABSTRACT
The
sequence
requirements
for
specific
and
efficient
transcription
from
the
16S/23S
rRNA
promoter
of
Sulfolobus
shibatae
were
analysed
by
point
mutations
and
by
cassette
mutations
using
an
in vitro
transcription
system.
The
examination
of
the
box
A-
containing
distal
promoter
element
(DPE)
showed
the
great
importance
of
the
TA
sequence
in
the
center
of
box
A
for
transcription
efficiency
and
the
influence
of
the
sequence
upstream
of
box
A
on
determining
the
distance
between
the
DPE
and
the
start
site.
In
most
positions
of
box
A,
replacement
of
the
wild
type
bases
by
adenines
or
thymines
are
less
detrimental
than
replacements
by
cytosines
or
guanines.
The
effectiveness
of
the
proximal
promoter
element
(PPE)
was
not
merely
determined
by
its
high
A+
T
content
but
appeared
to
be
directly
related
to
its
nucleotide
sequence.
At
the
start
site
a
pyrimidine/purine
(py/pu)
sequence
was
necessary
for
unambiguous
initiation
as
shown
by
analysis
of
mutants
where
the
wild
type
start
base
was
replaced.
The
sequence
of
box
A
optimal
for
promoter
function
in
vitro
is
identical
to
the
consensus
of
84
mapped
archaeal
promoter
sequences.
INTRODUCTION
Two
consensus
regions
have
been
defined
by
comparison
of
promoter
sequences
of
Archaea
[1]
(Archaebacteria):
the
box
A
centered
about
27
bases
upstream
of
the
transcription
start
site
and
the
box
B
at
the
start
site
[2,
3].
Mutational
analysis
of
the
16S/23S
rRNA
promoter
of
Sulfolobus
shibatae
using
an
in
vitro
transcription
system
[4]
has
identified
the
position
and
function
of
three
essential
promoter
elements:
(i)
a
distal
promoter
element
(DPE)
encompassing
box
A,
which
is
important
for
transcription
efficiency
and
start
site
selection;
(ii)
a
proximal
promoter
element
(PPE)
between
position
-11
and
-2
which
also
contributes
to
transcription
efficiency;
and
(iii)
a
pyrimidine/purine-sequence
which,
at
the
proper
distance
from
the
DPE,
serves
as
start
site
[5].
Utilisation
of
heterologous
promoters
in
the
S.
shibatae
transcription
system
showed
that
constitutive
promoters
are
functionally
conserved
between
distantly
related
archaea
[6].
An
analysis
of
the
Methanococcus
vannielii
tRNAVaI
gene
promoter
[7]
is
in
fair
agreement
with
these
data
but
extends
the
determination
of
sequence
requirements
by
point
mutations
in
which
certain
bases
between
position
-35
to
+2
were
replaced
with
guanine.
In
this
study
we
analyze
the
functional
importance
of
positions
in
promoter
elements
in
more
detail,
especially
within
box
A
where
each
base
was
replaced
with
the
other
three.
We
also
examined
the
effect
of
all
possible
base
exchanges
at
the
start
site
in
order
to
check
the
requirement
for
a
pyrimidine/purine
sequence.
Moreover,
we
replaced
the
proximal
promoter
element
by
streches
of
adenines
or
thymines,
or
the
complement
of
the
wild
type
PPE-sequence,
to
test
the
hypothesis
that
a
high
A+T
content
is
sufficient
for
its
function
in
the
16S/23S
rRNA
promoter
[5].
Promoter
strength
and
transcript
start
sites
of
all
mutant
promoters
were
determined
by
Si
nuclease
mapping
of
the
in
vitro
transcripts.
The
box
A
sequence
optimal
for
promoter
function
in
vitro
was
compared
to
the
consensus
of
mapped
archaeal
promoters.
MATERIALS
AND
METHODS
Materials
Restriction
enzymes,
RNase-free
DNase
and
T4
ligase
were
obtained
from
Boehringer
Mannheim,
T4
polynucleotide
kinase,
Sl-nuclease
and
Klenow-fragment
of
E.coli
DNA
polymerase
I
from
Pharmacia,
radiochemicals
from
Amersham.
The
soluble
cell-free
extract
of
S.
shibatae
was
prepared
as
described
[4].
Methods
Construction
of
the
vector
and
mutants.
A
sequence
identical
to
the
-39
to
+6
promoter
region
of
the
S.
shibatae
16S/23S
rRNA
operon
[14]
was
cloned
into
the
phagemid
pBluescript
II
KS'
(Stratagene)
between
the
Xho
I
and
Bam
HI
cleavage
sites,
using
synthetic
oligonucleotides.
The
Pst
I-site
in
the
middle
of
the
promoter
region
was
generated
by
exchange
of
a
thymidine
against
guanosine
at
position
-19
and
against
cytidine
at
position
-21
in
the
oligonucleotide
sequence
(Fig.
1).
This
difference
in
the
promoter
sequence
had
no
negative
effect
on
transcription
efficiency
and
start
site
selection.
This
vector
construct
was
named
pSP2
and
used
as
standard.
The
Pst
I-site
together
with
either
the
XhO
I-
or
the
Bam
HI-site
were
used
to
generate
mutants
by
introduction
of
synthetic
oligonucleotides
comprising
distinct
mutations.
These
oligonucleotides
were
designed
with
ends
*
To
whom
correspondence
should
be
addressed
+
Present
address:
MSU-DOE
Plant
Research
Laboratory,
Michigan
State
University,
East
Lansing,
MI
48824-1312,
USA

5424
Nucleic
Acids
Research,
Vol.
20,
No.
20
complementary
to
the
sticky
ends
of
either
the
Xho
IVPst
I-or
Pst
I/Bam
HI-vector
fragment
generated
by
digestion
of
pSP2
with
the
respective
enzymes.
Both
double
digests
always
led
to
a
vector
fragment
with
overhanging
ends
on
the
same
strand
(Fig.
1).
Ligation
[8]
of
the
oligonucleotides
with
these
vector
fragments
led
to
a
circular
vector
with
a
short
single
stranded
region.
Upon
transformation
[9]
the
DNA-repair
system
of
the
recipient-cell
strain
E.coli
XL1-blue
converted
the
construct
into
double-stranded
DNA.
After
selection
on
LB
agar
containing
tetracyclin
(25
pg/ml)
and
ampicillin
(75
itg/ml)
the
recombinant
DNA
was
isolated
[10]
and
the
mutation
verified
by
DNA-sequencing
[11].
Mutated
phagemids
obtained
through
this
procedure
were
cleaved
with
Bgl
I
yielding
two
fragments
and
used
as
linear
templates
for
in
vitro
transcription.
In
vitro
transcription.
100
ng
template-DNA
were
incubated
for
10
min
at
60°C
in
50
/d
of
a
reaction
mixture
containing
50
mM
Tris-HCl
(pH
8.0),
25
mM
MgCI2,
1
mM
EDTA,
1
mM
Dithiothreitol,
2
mM
AT?,
1
mM
CTP,
1
mM
GTP,
1
mM
UTP
and
8
pl
of
the
cell-free
extract
from
S.
shibatae
[4].
The
mixture
was
chilled
on
ice,
50
/l
ddH20
were
added
and
the
reaction
mixture
was
extracted
three
times
with
100
pl
of
phenol/chloroform/isoamylalcohol
25:24:1
(vol/vol/vol).
The
nucleic
acids
were
precipitated
with
ethanol
and
the
template-
DNA
was
removed
by
digesting
with
25
U
RNase-free
DNase
I
(Boehringer,
Mannheim)
in
50
1d
reaction
buffer
for
30
min
at
room
temperature.
After
addition
of
50
pl
ddH20
the
reaction
mixture
was
extracted
twice
with
phenol/chloroform/
isoamylalcohol
and
with
chloroform/isoamylalcohol
24:1
(vol/vol).
The
aqueous
phase
containing
the
in
vitro
RNA
was
stored
at
-700C.
SI
analysis.
DNA
probes
for
SI
nuclease
analysis
[12,
13]
were
prepared
by
extension
of
the
5'-32P-labeled
M13
universal
sequencing
primer
hybridized
to
single
stranded
pSP2
or
its
respecfive
mutant
derivative
single
strand.
Identical
volumes
(two
microliters)
of
the
in
vitro
RNA
solutions
were
hybridized
to
an
at
least
fivefold
molar
excess
of
DNA
probe.
Hence
only
the
quantity
of
the
in
vitro
RNA
determined
the
strengit
of
the
signal
and
therefore
the
transcription
efficiency.
The
condition
for
hybridization,
SI-nuclease
digestion
and
the
electrophoresis
through
denaturing
polyacrylamide
gels
were
as
described
[4].
The
transcription
efficiencies
were
quantified
by
densitometry
of
the
autoradiographs.
A
calibration
curve
was
used
to
correct
for
nonlinearity.
Primer
extension.
After
in
vitro
transcription
and
DNase
I
digestion
the
reaction
mixtures
were
extracted
twice
with
phenol/chloroform/isoamylalcohol
and
were
ethanol
coprecipitated
with
30,000
cpm
of
the
5'-32P-labeled
M13
universal
sequencing
primer.
Reverse
transcription
was
performed
as
described
[14]
yielding
a
fragment
of
99
bp
(in
the
case
of
initiation
at the
wild
type
start
site).
The
cDNA
was
analyzed
on
a
6%
polyacrylamide
sequencing
gel.
RESULTS
In
order
to
define
the
sequence
requirements
within
the
archaeal
promoter
elements
responsible
for
transcription
efficiency
and
start
site
selection,
an
extensive
mutational
analysis
of
the
promoter
region
of
the
16S/23S
rRNA
operon
of
S.
shibate
was
performed.
For
this
purpose
a
cassette
with
a
sequence
almost
A
5,
-38
DPE
-25
1
-11
PPE
-2
(Start)
I
I
.
cccctcga
ggactgaaqaa&tAaOgqtcc
______
Xho
I
B
box
A
Pat
I
box
B.
Bar
HI
3,
5'
tcgag
.ctgc
3'
(oligo
with
a
miutation)
5'
ccc
S
I
cccc
3'
ggggagct
Xho
I
5,
3,1
3,
5S
Pat
I
Pat
I
cccctcgagttagatttatatgqgactgca
qgggagctcaatctaaatataccctg,
Bam
HI
gatcc
3'
5,
(oligo
with
a
mutation)
3'
acgtc
.................
cctag
5'
Figure
1:
(A).
Sequence
of
the
promoter
region
of
the
16S/23S
rRNA
operon
of
S.
shibata
containing
a
Pst
I
site
introduced
by
two
nucleotide
exchanges
at
posiions
-19
(F
to
G)
and
-21
(F
to
C).
Oligonueooides
with
this
promoter
sequence
from
position
-39
to
+6
(tnscription
stat
site
poiion
+1)
have
been
inserted
into
pBluescript
II
KS+
between
the
Xo
I
ad
te
Bwn
HI
sites
yielding
the
constrct
pSP2.
The
sequence
shows
the
disl
promoter
element
(DPE,
underined)
iluding
box
A
(uppercase
and
bod),
the
proximal
pmoter
ekment
(PPE,
undrlned)
and
box
B
(u
e
and
bold).
Th
M13-20
univra
primer
used
for
sequencing,
generation
of
the
SI
probes
and
for
prie
extension
binds
at
position
+83
to
+99.
(B).
Strategy
of
the
insrtion
of
synthetic
oligonuclides
containing
certain
mutai
into
pSP2.
The
pSP2
cosrct
was
cleaved
with
either
Xho
IIPst
I
(upper
part)
or
with
Pst
I/M
HI
(lower
part).
The
sythtc
doigoleotides
were
c-eny
at
both
ends
to
these
restiction
sites.
After
ligaton
and
transfonnation
the
single
straned
region
was
filled
in
by
the
DNA
repair
system
of
the
host
cell.
identical
to
that
in
the
natural
promoter
from
position
-39
to
+6
(transcription
start
site
defined
as
+
1)
was
assembled
from
overlapping
oligonucleotides
and
cloned
into
pBlescript
II
KS+
yielding
pSP2
(Fig.
1).
To
permit
the
facile
and
efficient
manipulation
of
all
parts
of
the
promoter,
the
pSP2
promoter
cassette
contained
a
Pst
I
site
between
the
distal
and
the
proximal
promoter
element
leading
to
two
nucleotide
exchanges
compared
to
the
wild
tpe
promoter.
In
vitro
transcription
of
pSP2
and
a
similar
construct
containing
an
entirely
wild
type
promoter
sequence
indicated
that
the
introduction
of
the
Pst
I
site
did
not
alter
the
initiation
site
or
negatively
affect
tnription
efficiency
(data
not
shown).
Mutations
were
focussed
on
three
regions:
(i)
box
A
(position
-32
to
-26),
where
each
of
the
seven
bases
of
the
wild
type
sequence
was
substituted
by
each
of
the
other
ihree
possibilities
(figure
2,
upper
part),
(ii)
the
PPE,
which
was
examined
by
three
cassette
mutants
(figure
2,
middle),
and
(iii)
box
B
with
the
start
site,
where
the
wild
type
guanine
at
position
+
1
was
substituted
by
each of
the
three
other
bases
(figure
2,
bottom).
The
mutations
in
the
promoter
region
were
introduced
by
ligation
of
synthetic
oligonucleotides
and
verified
by
DNA
sequence
detm
ination.
The
various
constru
acted
as
template
for
in
vtro
tnscription
with
a
cell
free
extract
of
S.
shibatae.
The
transcription
efficiency
of each
mutant
promoter
was
determind
by
Sl-nuclease
mapping
of
fte
resulting
transcription
products
followed
by
densitometric
analysis
of
the
respective
signals
on
the
autoradiogram.
To
check
the
reproducibility
for
each
individual
construct,
at
least
three
independent
repetitions
of
these
experiments
were
performed.
In
the
case
of
the
box
A
mutants,
a
pSP2-derived
probe
was

Nucleic
Acids
Research,
Vol.
20,
No.
20
5425
-4U
-30
-20
-10
-
1
.-
Start)
pSP2:
5'
tcgagttagatttatatgggactgcagaacaatatgtataatgcgga
3'
tcgagttaga2ttatatqqggactgcagaacaatatgtataatgcgqa
tcgagttagatgtatatgggactgcagaacaatatgtataatgcgga
tcgagttagatt.&atatgggactgcagaacaatatqtataatgcgga
tcgagttagatLt&atatgggactgcagaacaatatgtataatgcgga
tcgagttagattta&atgggactgcagaacaatatgtataatgcgga
tcgaqttagattta5atgggactgcagaacaatatgtataatgcgga
tcgagttagatttatStgggactgcagaacaatatgtataatgcgga
tcgagttagatttataCgggactgcagaacaatatgtataatgcgga
tcgagttagatttata2gggactgcagaacaatatgtataatgcgga
tcgagttagatAtaAatgggactgcagaacaatatgtataatgcgga
:gagtt&Alttatatgggactgcagaacaatatgtataatqcgga
tcgagttagatttatatgggactgcagaac
flTfltqcgga
tcgagttagatttatatgggactgcagaacaaA3
a&aatgcgga
tcgagttagatttatatgggactgcagaacaatatgtataatZcgga
tcgagttagatttatatgggactgcagaacaatatgtataatScgga
tcgagttagatttatatgggactgcagaacaatatgtataat&cgga
Si
Transcription
Si
(rmutants)
efficiency[%]
(wild
type,
100
reference)
3'
11
i
2
3'
69
±11
3'
45tS
\
3'
67
9
3'
5±
1
_
3'
3
63±5
3'
7±23t
/
3'
20i4
3'
18
4
3'
9
a8±2
3'
4±
1
3'
3±
1
3'
116±18
\
3'
5±1
3'
b
39
3
3
'
28
6
3'
=
27±
9
'3'
*
96±10
3'
20
±
4
3'
89
±13
3'
19±
5
3'
:m
83
±14
*.1
3'
MU
3'
3'
3'
9:
31
9
3'
J
23±6
3
115
±20/
Fue
2:
Transcription
efficiencies
of
all
mutant
constructs
mapped
by
Sl-analysis.
The
data
were
derived
from
at
least
three
independent
repetitions
of
the
SI-analysis.
Box
A-mutations
upper
part,
PPE-mutations
in
the
middle
and
box
B-mutations
at
the
bottom
of
the
list.
Mutations
are
shown
by
bold
underlined
uppercase
letters,
wild
type
nucleotides
in
lowercase
letters.
The
box
A
motif
defined
by
functional
analysis
is
double
underlined.
The
autoradiographs
from
SI
experiments
and
the
deduced
transcription
efficiencies
(4+
standard
deviation)
are
shown
to
the
right
of
the
template
sequences.
Autoradiographs
shown
were
assembled
from
several
gels;
the
corresponding
wild
type
(=pSP2)
controls
are
shown
to
the
right
of
the
transcription
efficiency
data.
Autoradiographs
from
the
start
site
determinations
by
primer
extension
(PE)
are
shown
to
the
extreme
right
of
the
figure;
arrows
indicate
the
fragment
corresponding
initiation
at
the
wild
type
+
1
position.
Mapped
start
sites
are
also
indicated
on
the
template
sequences,
if
deviating
from
the
wild
type
start
site
position:
major
initiation
site:
black
dot;
niinor
initiation
site(s):
open
circle.
used
in
S1
analysis
for
the
examination
of
the
transcription
efficiency
and
the
start
site.
Since
the
probe
was
complementary
to
the
template
DNAs
up
to
box
A,
the
start
sites
of
all
these
mutants
could
be
monitored
without
difficulty.
In
case
of
mutations
in
the
PPE
and
in
box
B
promoter
mutants,
specific
DNA
probes
were
prepared
for
each
construct
to
exclude
SI
nuclease
digestion
at
mismatch
positions.
DPE
mutants
In
the
following
part,
the
influence
of
mutations
in
box
A
of
the
16S/23S
rRNA
promoter
shall
be
described
in
the
order
of
decreasing
effects
on
transcription
efficiency.
All
nucleotide
exchanges
at
positions
-30
and
-29
reduced
transcription
efficiency
dramatically
(Fig.
2),
indicating
the
importance
of
the
TA-sequence
at
these
positions
for
the
function
of
box
A;
only
the
adenine
at
position
-30
maintained
a
high
transcription
efficiency.
Every
exchange
at
position
-27
also led
to
a
reduction,
though
to
a
lesser
extent
than
at
the
positions
-30
and
-29.
The
thymine
at
position
-32
could
only
be
replaced
by
cytosine,
the
thymines
at
positions
-31
and
-28
only
by
adenines,
in
each
case
leaving
the
transcription
efficiency
higher
than
67%
of
that
of
the
wild
type
promoter
(Fig.
2).
Guanine
at
position
-32
was
tolerated,
but
in
a
lesser
extent
(45%
transcription
efficiency).
The
other
possible
exchanges
at
each
of
these
positions
left,
at
most,
11%
of
the
transcription
efficiency.
The
tolerance
of
the
transcription
system
especially
towards
cytosine,
but
not
adenine
or
guanine,
at
position
-32
shows
that
at
this
position
occupation
by
a
pyrimidine
rather
than
a
high
A+T
content
is
necessary
for
promoter
function.
In
contrast,
the
tolerance
towards
transversion
at
positions
-31
and
-28
suggests
that
a
weak
base
pairing
rather
than
a
certain
base
is
required
there.
At
position
-26,
only
the
replacement
of
the
wild
tpe
thymine
by
cytosine
led
to
a
strong
reduction
in
transcription
efficiency:
the
other
two
possible
exchanges
were
tolerated
(Fig.
2;
see
also
figure
3
for
an
overview).
All
of
these
box
A
mutations
influenced
the
transcription
efficiency
without
leading
to
a
shift
of
the
transcription
start
site.
One
mutant
('5S
DPE',
Fig.
2)
contained
the
DPE
of
the
5S
rRNA
gene
promoter
of
S.
shibatae.
This
alteration
in
the
region
immediately
upstream
of
the
box
A,
which
itself
was
the
same
as
in
the
16S/23S
rRNA
promoter,
left
the
transcription
efficiency
at
83%
but
introduced
ambiguity
of
transcription
initiation.
Starts
were
mapped
at
positions
-7
(guanine),
-5,
-3
(adenines)
and
+1
(guanine)
with
the
major
start
site
at
position
-5.
The
mutation
'Euka'
transfonned
the
wild
type
box
A
sequence
'TTTl-lATAT'
to
'TATAAAT'
which
represents
the
'TATA-box'
consensus
of
eukaryotic
pol2
promoters.
The
transcription
efficiency
from
this
promoter
mutant
was
19%
and
thus
M32
A:
M32
C:
M32
G:
M31
A:
M31
C:
M31
G:
M30
A:
130
C:
M30
G:
M29
T:
M29
C:
M29
G:
M28
A:
M28
C:
M28
G:
M27
T:
M27
C:
M27
G:
M26
A:
M26
C:
M26
G:
Euka:
5S
OPE:
5'
5
.
5'
5'
5'
5'
5
'
5'
5'
5'
5'
5'
5'
5.
5'
5'
5'
5'
5'
5.-
5'
5'
5'
Trans
AT:5'
oligo
T:
5'
oligo
A:
5'
Start
T
5'
Start
C
5'
Start
A
5'
Sf
I
I.
a
21
±
6
5
±
1
7
±
3

5426
Nucleic
Acids
Research,
Vol.
20,
No.
20
pSP
2:
T
T
T
A
T
A
T
ACG
ACG
ACG
TCG
ACG
TCG
ACG
N
C)
C
8
a a
C
o
X
2
2
2 2
2 2
2~~~~~~~~~~~2
Figure
3:
Transcription
efficiencies
of
box
A
mutants
obtned
by
quantitative
Si-mapping.
The
names
of
the
mutants
are
given
below
the
x-wis
at
their
corresponding
column
and
are
in
accordance
with
those
at
figure
2.
The
black
column
at
the
right
represents
the
pSP2
(the
16S/23S
rRNA
operon
promoter
introduced
into
pBluescript
II
KS+).
significant
though
weak.
Transcription
was
initiated
at
the
same
site
as
in
the
wild
type
promoter.
PPE
mutants
The
three
cassette
mutations
in
the
PPE,
an
oligo
adenine
stretch,
an
oligo
thymine
stretch
and
the
complement
of
the
wild
type
PPE-sequence,
reduced
the
tnscription
efficiency
to
7%,
5%
and
21%,
respectively.
The
start
site
of
each
of
these
ffiree
mutants
was
the
same
as
that
of
the
wild
type
promoter
(Fig.
2).
Box
B
mutants
In
box
B
the
guanine
at
the
trnscription
start
site
was
substituted
by
each
of
the
three
other
bases.
The
replacement
of
the
wild
type
guanine
by
adenine
did not
shift
the
transcription
start
site
but
increased
the
transcription
efficiency
slightly.
The
replacement
by
thymine
shifted
transcription
initiation
to
the
positions
-3
(adenine)
and
+2
(cytidine)
accompanied
by
a
reduction
of
transcription
efficiency
to
31%
overall.
The
replacement
by
cytidine
led
to
an
ambiguous
initiaton
in
the
vicinity
of
the
wild
type
start
site
but
mainly
at
the
position
+
1
(cytidine)
with
an
overall
htanscrition
efficiency
of
23%
(Fig.
2).
DISCUSSION
Using
an
in
vitro
transcription
system
from
Sulfolobus
shibatae
[4],
the
functional
significance
of
single
positions
widtin
archaeal
promoter
elements
was
examined
by
detennining
the
streng
of
altered
promoters
and
their
start
site
selecdon
in
comparison
to
the
wild
type
promoter
of
the
16S/23S
rRNA-operon
of
S.
shibatae.
The
latter
was
chosen
because
of
its
strength
in
vivo
organism
and
gene
Hc.
rRNA
P1
H.c.
rRNA
P2
H.c.
rRNA
P3
H.c.
LlI,l
H.c.
LIe
B.c.
NA
H.c.
SOD
N.h.
RIAP
N.h.
512
N.h.
HOP
H.h.
DOP
N.h.
Glycopr
.h.
flgA
Rh.
flgS
N.h.
Mn-SOD
N.h.
p-vac
N.h.
p-gvpO
N.h.
TS1,2,3
N.h.
NR
T4
N.h.
N9
TN
N.h.
N8
T7
N.h.
*b
TN
N.h.
N8
T9/10
N.h.
b
TINSSI.
N.h.
0
TLXI
N.h.
*N
Tant
N.m.
168/235
rRIA
P1
.mm.
168/238
rlUA
P2
N.m.
16S/238
rRNA
P3
Nt.
mvhDCA8
NM.
tcr
Mt.
purl
Mv.
165/238
eDNA
NMv.
tRIA
G
5
reUNA
M.v.
tRyArg
Mv.
ORFa
M.v.
S17
Mv.
hisA
M.v.
mcr
N.V.
ORrl
Ta.
165
rRNA
T.a.
23S
rRIA
Ta.
SS
rRNA
T.a.
tRNAOt
D.&.
Ligase
D.a.
SOR
(ar)
D.e.
sor-OF2
(konst.)
Da.
sor-ORN3
(aer)
D.a.
sorODF4
(anaer)
DG..
55
rRNA
D.-.
rRNA-Pl
D.m.
rRNA-P2
S..
S12
S.&.
ONFNS
(rpoN)
Se.
rpoC
S.a.
ORF-X
S.a.
SOD
S.a.
S7
Na.
tRNAMSr
S.a.
ef
la
S.a.
Ef
2
Ss.
16S/23S
rRNA
Ss.
5S
rRNA
S.s.
tRNAArg
S.s.
SSVI
T1,2
S.s.
SSV1
T3
S.9.
SSV1
T4
S.s.
SSV1
T9
S.s.
SSV1
T5
S.s.
SSV1
T6
T.c.
5S
rRNA
Tc.
rpoHIl)
T.c.
rpoH(2)
Tc.
rpl30
Tc.
rpsi2
Tc.
tRNAThr
Tc.
tRNAPro
T.p.
16S/23S
rRNA
T.p
tRNAMnt
T.p.
ORF1
T.p.
ORF2
T.t.
16S/23S
rRNA
Tt.
tRNAAla
T.t.
tRNAMet
consensus:
H.
c.
ORF
H.
h.
BRP
N.
h.
ON
TLX3
H.me.
mc-gvpA
S.s.
SSV1
Tind
sequence
TCC"=TcC
SAACCOCA
CCTCs
OAOa
M
CCA
OTCCOATOCCC
A
OCCTACTTC
CTGCAATC
AAC
ATTCOTOCCATAATM
OOOTTTCO
ATOMASO
SAC
MMMOG
n8WCO00
COO=zOOTTTC
TOACM~
OCT
CTTC
S-C-CTCCA
TCMCSOTG
T
ACA
GTTTCOACW
2SZc
MRO
GTGOAMO
OCWA?M~
TOG
OTTQACAOOAOAMTOCTOTO
OSADCARMY
G8OCPT6--
ATC
GTTGOOOOA
A
C
M
TOCTC
CTTOCAC
CAT
_GGTCSTAOMZACATAT
CCTCO!S
TACTSYWA
TOT
OCTAGAAACIMSOAOTOOCC
OOOTCATSACTC
RM
Cm
Tax=AT
CMC
cTCMTC
=
c
CAC
CATACACATCOO
WATOCC
CGSTATAM
TAGMISS
TTA
GAOOOTCAATC1ATACS
Gm0Cal
ACDAC
GCA
TAOATAGAZ*ZWAGSO
CCTCITAM
JOIO
MSA
ATOGGAACUCSTOOOOC
AAMOWT
a
I
a
SI-?
CCA
CTOCTAIW
C
CCASAOCA
CAT
ATCAAACAGAMTAOOTAO
GOGTCA!
OTIOW
GTSA
TGOCACOAA
AOCCAT
CcTAWC
?ATICMCA
TOA
CTCACAAhA
G5WOAA9Y=T=
GAGCORACOCM
C
CS=
IS
Im
CE
COCC
1IC
--AAAATSONGA
MTCA
GAO
CTACCTTT5_SOCOA
T0TOA£S?YS
C
AYCT
GOC
CTT~OA0O~5WG
AACR
CAOAU
SIA
AAG
C~~~~~~~~~~~CS
TCCO
R
lCAMGTM
OM:lCASTTB
AAUSt
MO
ATAACCZZZ
STTACCA
COCTA
ASA
TAC
OOOCACYIM&ATO
TcOAT
5C_T
CSM
TASCCTAAACS=AZMlATCA
ACSIIS
2AAIWA
T
TACCGAAAACSCTATTATA
ACACTATAT
TCMATAT
AaM
AACCGAAATAZZT
ACTA
ATMCCTC
TATATAC
?CT
TAAOCATAAT
ATCAT
AMCCAOTATS
TCTAT6c5T
MAC
CTATATTAAACZZUATCTOT
ATTcTATS
AMCTMA
CT
TAOTACCAMSZLTOTAM
ACCTAMTIA
ACATsWT
AAT
TGAAAACTTOMS&=TCTT~CT
TIMYMYTAT
TAYMTOTAT
ATA
TATOCAAAMAG
AANTAMcS
TAA
cTTATT
AAMACMT
TAT
GCCTTCGAAMZSIATM
ATTTOCTATT
CYTTACTS
CM
GATCAAAAT
CTCT
TMAATRTA
TCCA2SM
OCT
TCACGAAAATCZATTMATOT
CTTCTATATA
GTOIOCAcM
CCCTACAGCCMTSLSTC=C
TATOCTOIA
TGOCO
TO
CATCATCAAZAZSATATCT
TTCMACT
...A.Ta
GaO
AAMAAA_ASIS
TAO
AOAAATOTAT
ATITACAA
AMA
MTTSCACTTAAZATTTGT
TAAMSAOS
ITATATTATS
CAT
GAMATAOA1RZEZTCCTOAA
TAAAOTAOT
YMATTATO
TCG
CTCAACTATT1CAS8kCTAGT
OCLAAAAM
ATRAPAT
MCT
CCTAACCACAATATATT
GATOCTCOGA
TSTOM
TCC
TATATAAACQAj
CATMsTAGA
OTAAT
SMOYM
P
AccGTcATqaUAZMCTTO
OAGCMAAM
AcTCcIOAY
a
CM
CTAMGTTMTIZZMLMAGUT
TIOGATATI
mAITOTOW
TOT
CTAMCATMATmSLZAMCTA
AATOMYA
MSAW
ITO
cTGTGMaAMA3zzzSSAOaT
ATTOTICAMB
TM
O
COGTAAACCT2A=ATCT
TTATTASA
TAVSI*
AGM
AMTCAAAATAZCMZZAWM0T
TOOCAYTT
TT20TSM
MCA
CACATCTAM
ACCCTTCA
TMATATAWT
ASACM
TM
TTTAT
CATC
OTATAM
ATTM&SSC
C0o
TMCTAAACTZS&WCTMTC
TACBTATAaA
AATO
M
OTA
TCCACACACZAACTMAA
MYTATTAA
CTAFSa
csC
AGAAGTTAGAmSaZOTA
TT
TCAMAUCAMT
ATOTAMIC
TAGTTAATTTZSZMTOTGTTA
TGACTTTA
ATT
mA
CCC
TGTCTAACAGIZZM&ATATCCG
ATGAGOCAYA
TTTATOTT
ACC
ACTGGAGGGGZTAAAACGTAA
GCGGGAAOCC
ATAT"ACC
AAG
TTAGGCTCTTUTSA&GTCTACC
TTCTTTTTO
CTTAllTOA
OGA
GATAGCCCTTAZMGCCATAA
ATTTTTTATC
GCTTCATAA
GTG
AAGTAGGCCCZZAZAGTCAT
ATTCTTTTTC
TTTCCCTGAT
GAG
TAGAGTAAGAGIIAMTACTAAT
TTATACATAG
AGTATAGAT&
GAG
TAGAGTAAAGZZM&TACTTAT
ATAGATAGAG
TATAGAT&GA
OGG
GACCGTAACCTTTATAAACCCAG
GCCGGGAGTT
CCGTACCOT
ACG
TCGTAACCGAIAIAMCCCCTCT
GAGCGAGTTG
TCACfCAA
AGC
ACTCGAAAAGCTAUTACCGAA
ACCCOCAOOG
OOMTAOTG
ATA
ACCCGAAAAGGZTTAAAAGCACC
GCCTTAAGOG
TCTTTCQC
GTC
CTAGGAACAT=IUS&GA=CT
CGGGGTTAAM
GTTAAAMO
TCC
GAGGGAAAAATTTATAAATGCCA
ATCTGCATOC
TATCATCAC
C
CC
GAGGGAAAAGCTTAATAAGGAAC
CCTTTAACCC
ACTACCOCCC
CATO
GCATAATATTCAZAIACCCCC
GTTTACTAMC
TACATTSCC=
CCA
ATGCTAAAGGZTIAIIACCCAGG
AAGTATTCCO
GTCATW)GC
GTT
GCATTCGACAAA,TTAACCT
CGTTCGACGA
GTAT5MAGT
OCT
TAAATAGCTTZIACGTAGGAG
GTGCTACCTC
CAqWATCG
TAA
GCGAAAAATT*ATTAGGGT
GTTTTAGGAT
GGTOCCCT
TAA
AGCGAAAAAAflIAMTCGGTGA
GTMGTACOC
TCBGGCCGG
TAG
ACAAAAGCTTTZAIMTTCGCGC
AAAGCTTAGA
CCTIGCGGGG
TAG
TTA
A
C
A
GGAAAGCGCTTTTCOGCGCTTGCTGTCTACGQCCACGTATG
GGTCTTTTTTGATGCTCGGTAGTGACGTGTGTATTCATGCA
CGATGGGTTCAAGAACCTCGCTGCCCGTCTATTTCAATATGCCC
CACGAATGATTTTGTTACTTGCCAACACGTTTTCAGATOPGTA
Fure
4:
The
list
(first
part
euyarchaeotal,
second
part
c
al
si)
shows
the
in
start
sites
of
mapped
archaal
gensa
d
teir
pro
er
(box
A-seqences
are
underlined
and
the
Ipton
start
sites
bold
and
underlined.
The
sequences
are
aligned
for
the
box
A
elmet,
except
the
last
five
which
are
aligned
for
the
st
sits
since
no
consensus
box
A
could
be
found.
Abbreviations
usd
for
r
s:
H.c.:
Halobwntrim
ibrum
H.h.:
Halcrimj
halobium,,
H.mm.:
iu
marismortui,
H.me.:
Halobacterium
medierrani,
M.t.:
Me
bterium
thermoautoar
icum,
M.v.:
Methanococcus
vannielii,
T.a.:
acidophilum,
D.a.:
Desulfurolobus
ambivalens,
D.m.:
mobilis,S.a.:
Sulfolobus
acidocaldarius,
S.s.:
Sul
lobs
shibat,
T.c.:
Thermococcus
celer,
T.p.:
Thermofilum
pendens,
T.t.:
Throproeu
tenax.
reference
1161
(161
(161
1171
(171
(17)
(181
1201
121)
1221
1231
1231
1241
(25)
(261
(271
(271
(271
(271
1291
(291
1301
1311
(291
1321
(321
(321
(331
(341
(35)
(15)
(15)
1151
(36)
(36)
1371
(391
(39)
(401
(401
1401
(411
(421
1431
(43)
(43)
143)
144)
(44)
(441
(45)
(45)
(45)
1451
1461
1471
(47)
1481
(481
121
121
1491
12)
121
121
121
(2)
121
150)
1511
1511
1521
1521
(461
1461
1531
153)
(531
1531
154)
1541
1541
171
1551
311
1561
121

Nucleic
Acids
Research,
Vol.
20,
No.
20
5427
and
in
vitro
and
its
identity
to
the
promoter
consensus
defined
by
sequence
comparison
[2,
3, 15].
Promoter
efficiency
The
box
A.
All
box
A
mutations
replacing
the
thymine
at
position
-30
and
the
adenine
at
position
-29
in
the
center
of
box
A
showed
strong
reductions
of
transcription
activity
indicating
the
importance
of
the
TA-sequence
at
these
positions.
The
only
exception,
a
tolerance
towards
the
exchange
of
thymine
-30
against
adenine,
can
be
interpreted
as
a
shift
of
the
TA-sequence
one
position
upstream.
These
data
are
in
accordance
with
a
high
conservation
of
the
TA-sequence
in
the
center
of
box
A
in
archaeal
promoters
(Fig.
4).
The
distance
between
the
TA-
sequence
and
the
start
site
is
29
bases
in
the
16S/23S
promoter
but
between
25
to
28
bases
on
average.
These
results,
and
the
sensitivity
of
the
promoter
to
certain
base
exchanges
at
single
positions
of
box
A,
defined
an
optimal
box
A
sequence
for
promoter
function.
For
the
determination
of
this
optimal
box
A
sequence,
only
mutants
with
a
transcription
efficiency
higher
than
66%
of
the
wild
type
were
considered.
Position
-26
of
the
16S/23S
promoter
was
not
considered
important
for
promoter
function
since
only
one
of
the
three
possible
base
exchanges,
the
introduction
of
cytosine
instead
of
the
wild
type
thymine,
led
to
a
strong
reduction
of
promoter
strength.
Applying
the
above
criteria,
an
optimal
box
A
sequence
5'
T/CTTAT/AA
3'
(positions
-32
to
-27)
was
derived.
This
functionally-determined
sequence
was
in
good
agreement
with
the
consensus
of
archaeal
box
A
sequences
5'
TTTAT/AA
3'
[2,
3,
15].
Inspection
of
84
mapped
archaeal
promoters
(Fig.
4)
showed
that
cytosine
did
indeed
sometimes
replace
thymine
in
the
first
position
of
the
consensus
box
A.
The
last
position,
-27,
in
the
consensus
occupied
by
adenine
proved
very
sensitive
against
base
exchange
in
accordance
with
its
conservation
in
all
but
the
halobacterial
promoters.
The
PPE.
Box
A
is
not
the
only
element
determining
the
efficiency
of
transcription.
Exchange
of
a
second
essential
promoter
element,
the
A
+T
rich
PPE
at
positions
-11
to
-2,
by
stretches
of
adenines
or
thymines
(Fig.
2)
led
to
a
20
fold
reduction
in
promoter
efficiency
and
replacement
of
this
element
by
its
complementary
sequence
(Fig.
2)
resulted
in
a
5
fold
reduction
of
promoter
strength.
Thus,
the
element
is
not
merely
an
A+T-rich
region
facilitating
strand
separation
during
the
initiation
of
transcription,
as
previously
discussed
[5].
A
certain
sequence,
the
alternating
purine/pyrimidine
sequence
5'
ATATGTATA
3'
in
the
case
of
the
strong
16S/23S
rRNA
promoter
of
S.
shibatae,
appears
to
be
required
for
maximal
promoter
strength.
The
strong
reduction
of
transcription
efficiency
upon
replacement
of
the
element
by
its
complementary
sequence
indicates
that
the
sequence
must
be
correctly
positioned
with
respect
to
the
start
site.
The
PPE
sequence
is
not
generally
conserved
between
archaeal
promoters
(Fig.
4)
and
may
therefore
represent
a
particular
feature
of
the
16S/23S
rRNA
promoter.
The
start
site.
Promoter
strength
appears
to
depend
on
the
distance
between
the
DPE
and
the
start
site
[5]
as
well
as
on
the
occupation
of
this
site.
Apart
from
the
elements
already
discussed,
the
core
promoter
region
thus includes
the
start
site
itself.
When
an
unfavourable
start
site
was
introduced,
e.
g.
a
pyrimidine
instead
of
the
wild
type
purine,
the
start
shifted
to
nearby
sites
at
less
favourable
distances
concomitant
with
a
reduction
of
total
transcription
efficiency.
In
these
cases,
it
is
difficult
to
estimate
the contributions
of
the
changes
of
distance
and
start
site
context
to
the
overall
effect.
But
it
appears
that
promoter
strength
declines
sharply
whenever
a
dinucleotide
different
from
a
pyrimidine/purine
(py/pu)
occupies
the
optimal
initiation
region
defined
by
its
distance
from
the
DPE.
Start
site
selection
Analysis
of
promoter
mutants
with
regard
to
start
site
selection
indicated
the
necessity
of
a
start
motif
as
well
as
a
distance
measurement
in
defining
a
start
region
in
which
this
motif
serves
its
role.
We
suggested
previously
[5]
that
a
purine
preceeded
by
a
pyrimidine
acted
as
minimal
start
signal
since
most
initiations
on
a
number
of
mutant
constructs
occured
at
a
purine
following
a
pyrimidine.
Results
from
a
methanogene
transcription
system
[7]
and
those
of
our
current
work
corroborate
this
assumption.
Furthermore
a
comparision
of
mapped
archaeal
transcription
start
sites
showed
that
79
of
89
transcripts
initiate
at
a
purine
after
a
pyrimidine
(Fig.
4).
Since
such
a
dinucleotide
sometimes
occurs
more
than
once
at
an
appropriate
distance
from
box
A
and
initiation
nevertheless
remained
specific
there
must
be
additional
information
to
provide
specific
initiation.
The
formerly
proposed
box
B
consensus
T/ATGC/A
found
around
the
start
site
[2]
does
not
appear
to
play
this
role
since
initiation
occurs
either
upstream,
or
within,
or
downstream,
and
thus
not
at
a
defined
site
in
this
sequence.
Moreover
many
of
the
mapped
promoters
in
figure
4
do
not
show
this
consensus.
Aligning
promoter
sequences
for
the
start
site
yielded
a
different,
rather
weak
consensus
A/TT/CG/A
with
the
initiation
at
the
last
position
and
in
crenarchaeotal
promoters
a
weakly
conserved
(py/pu)4
pattern
(see
above).
The
DPE
as
well
as
the
py/pu
dinucleotide
determines
the
start
site.
This
had
already
been
shown
with
the
insertion
and
deletion
mutants
[5]
and
was
reconfirmed
by
some
of
our
promoter
mutants.
Alteration
of
the
distance
between
the
DPE
and
the
wild
type
start
site
led
to
a
shift
of
the
start
site
and
concomitantly
sometimes
to
ambiguity
of
initiation.
This
ambiguity
was
also
observed
when
the
wild
type
start
guanine
was
substituted
with
thymine
or
cytosine.
The
mutant
containing
the
5S
rRNA
promoter
DPE
instead
of
the
16S/23S
rRNA
promoter
DPE
showed
both
an
upstream
shift
of
the
start
site
and
ambiguity
in
start
site
selection.
The
three
different
types
of
mutants
share
one
common
feature:
all
of
the
new
start
sites
were
positioned
within
a
region
of
eight
bases.
This
indicates
that
the
DPE
delimits
(by
some
sort
of
distance
measurement)
a
certain
window
of
about
eight
bases
in
which
initiation
occurs.
Furthermore,
the
mutant
with
the
5S
DPE
shows
that
a
stretch
of
more
than
four
thymines
upstream
of
box
A
altered
the
structure
of
the
promoter
in
such
a
way
that
the
transcription
system
used
a
start
point
nearer
to
box
A
than
with
the
16S/23S
DPE-sequence.
Several
other
archaeal
promoters
carrying
stretches
of
thymines
in
this
region
also
showed
a
reduction
in
the
distance
between
box
A
and
the
start
site.
A
stretch
of
more
than
three
thymines
causes
bending
of
the
DNA
which
therefore
could
be
the
reason
for
the
reduction
of
this
distance.
ACKNOWLEDGEMENTS
We
wish
to
thank
Felicitas
Pfeifer
and
Melissa
Holmes
for
critical
reading
of
the
manuscript.

5428
Nucleic
Acids
Research,
Vol.
20,
No.
20
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