Interchangeable RNA polymerase I and II enhancers

Abstract

The RNA polymerase I (pol I) enhancer of Saccharomyces cerevisiae contains at least three elements commonly associated with RNA polymerase II (pol II) enhancers, binding sites for the transcriptional activators general regulatory factor 2 and autonomously replicating sequence-binding factor I, and a thymidine-rich element. When the particular form of the thymidine-rich element found in the pol I enhancer was placed in front of a pol II promoter, transcription was stimulated 43-fold, comparable to the effect of a powerful pol II activator such as Gal4. Conversely, when two copies of a thymidine-rich element from a pol II enhancer were placed upstream of a pol I promoter, transcription was stimulated 38-fold. This functional reciprocity of pol I and II enhancers may reflect similarities in the mechanisms of transcriptional activation. The pol I enhancer also contains an element that appears to be pol I-specific and prevent the activation of pol II.

Full text

Proc.
Nati.
Acad.
Sci.
USA
Vol.
87,
pp.
8202-8206,
November
1990
Biochemistry
Interchangeable
RNA
polymerase
I
and
II
enhancers
(Saccharomyces
cerevisiae/transcription/upstream
activation
sequence/thymidine-rich
element)
YAHLI
LORCH*,
NEAL
F.
LUE,
AND
ROGER
D.
KORNBERG
Department
of
Cell
Biology,
Fairchild
Center,
Stanford
University
School
of
Medicine,
Stanford,
CA
94305
Communicated
by
E.
Peter
Geiduschek,
August
6,
1990
(received
for
review
May
18,
1990)
ABSTRACT
The
RNA
polymerase
I
(pol
I)
enhancer
of
Saccharomyces
cerevisiae
contains
at
least
three
elements
com-
monly
associated
with
RNA
polymerase
H
(pol
II)
enhancers,
binding
sites
for
the
transcriptional
activators
general
regula-
tory
factor
2
and
autonomously
replicating
sequence-binding
factor
I,
and
a
thymidine-rich
element.
When
the
particular
form
of
the
thymidine-rich
element
found
in
the
pol
I
enhancer
was
placed
in
front
of
a
pol
U
promoter,
transcription
was
stimulated
43-fold,
comparable
to
the
effect
of
a
powerful
pol
II
activator
such
as
Gal4.
Conversely,
when
two
copies
of
a
thymidine-rich
element
from
a
pol
U
enhancer
were
placed
upstream
of
a
pol
I
promoter,
transcription
was
stimulated
38-fold.
This
functional
reciprocity
of
pol
I
and
U
enhancers
may
reflect
similarities
in
the
mechanisms
of
transcriptional
activation.
The
pol
I
enhancer
also
contains
an
element
that
appears
to
be
pol
I-specific
and
prevent
the
activation
of
pol
H.
Eukaryotic
RNA
polymerases
I
and
II
(pol
I
and
II)
have
much
in
common,
including
a
high
degree
of
sequence
homology
of
the
two
largest
subunits
(1, 2),
three
subunits
identical
in
the
two
enzymes
(3,
4),
and
similarities
in
promoter
structure.
Both
enzymes
are
capable
of
initiation
at
a
minimal
promoter,
consisting
of
sequences
in
the
immediate
vicinity
of
the
transcription
start
site;
in
both
cases
the
frequency
of
initiation
is
greatly
increased
by
the
presence
of
additional
sequence
elements,
termed
enhancers,
which
may
be
located
a
variable
distance
from
the
promoter
and
in
either
orientation
(5-11).
The
question
arises
whether
enhancers
are
functionally
interchangeable
between
pol
I
and
pot
II
promoters.
Others
have
found
(12)
that
a
number
of
pol
II
enhancers
fail
to
stimulate
initiation
by
mammalian
pol
I,
suggesting
that
the
two
enzymes
are
activated
by
different
mechanisms.
We
report
here
on
findings
that
lead
to
the
opposite
conclusion.
RNA
polymerase
I
is
responsible
for
the
synthesis
of
a
large
precursor
of
ribosomal
RNAs,
35S
in
yeast
and
40S
in
higher
organisms.
The
35S
precursor
is
encoded
by
-=120
tandem
repeats
of
a
gene
and
2.5-kilobase
(kb)
spacer.
Two
parts
of
the
spacer
are
involved
in
initiation
of
the
35S
precursor:
""210
base
pairs
(bp)
immediately
upstream
of
the
initiation
site
and
"'190
bp
located
2.2
kb
further
upstream
(10,
11,
13).
The
190-bp
segment
stimulates
initiation
17-fold
and
functions
at
a
variable
distance
from
the
initiation
site
in
either
orientation,
leading
to
its
designation
as
an
enhancer.
Progressive
deletions
of
the
190-bp
segment
gradually
dimin-
ish
enhancer
activity
(14),
suggesting that
the
enhancer
is
made
up
of
multiple
DNA
elements.
Similarities
between
some
of
these
elements
and
previously
identified
pol
II
enhancer
elements
prompted
us
to
investigate
the
question
of
functional
reciprocity.
One
pol
I
enhancer
element
proved
highly
effective
in
the
activation
of
pol
II
transcription,
whereas
other
pol
I
enhancer
elements
may
play
accessory
roles
at
both
pol
I
and
pol
II
promoters.
MATERIALS
AND
METHODS
Plasmid
DNAs.
Plasmids
for
transcriptional
activation
of
pol
II
were
members
of
the
pCZ
family
of
Escherichia
col-Saccharomyces
cerevisiae
shuttle
vectors,
containing
a
polylinker
upstream
of
a
yeast
CYCJ-E.
coli
lacZ
fusion
gene
(15).
The
synthetic
oligonucleotides
listed
in
Table
1
were
ligated
with
the
1-kbp
EcoRI-Cla
I
and
7.8-kbp
Cla
I-BamHI
fragments
of
pCZ.
The
control
with
no
oligonucleotide
(des-
ignated
A)
inserted
in
the
polylinker
was
as
described
(15).
For
construction
of
a
plasmid
with
a
Gal4-binding
site
and
residues
157-180
of
the
pol
I
enhancer
in
pCZ
(plasmid
designated
pCZGALpolI157-180),
the
synthetic
oligonucleo-
tide
had
the
sequence
CGGGTGACAGCCCTCCGAAGGC-
AAAGATGGGTTGAAAGAGAAGG,
with
termini
as
de-
scribed
in
Table
1.
Synthetic
oligonucleotides
containing
the
minimal
en-
hancer
with
sequences
downstream
or
upstream
of
the
thy-
midine-rich
(T-rich)
element
deleted
(Fig.
3
downstream
del.,
upstream
del.)
were
also
ligated
with
the
1-kbp
EcoRI-Cla
I
and
7.8-kbp
Cla
I-BamHI
fragments
of
pCZ.
The
entire
and
minimal
enhancers
[EcoRI-Xba
I
and
EcoRI-HindIII
frag-
ments
of
pSES5,
respectively
(ref.
18;
gift
of
S.
Roeder,
Yale
University)]
depicted
in
Fig.
3
were
ligated
with
the
2.2-kbp
EcoRI-Bgl
II
and
6.6-kbp
BgI
II-Xho
I
fragments
of
pCZ
and
the
following
Xho
I-Xba
I
or
Xho
1-HindIII
adapter
oligo-
nucleotides:
5'-TCGAGGAAGGGGTTCCCTTCCCCAAGGTCA-5'
and
5'-TCGAGGAAGGGGTTCCCTTCCCCAAGTCGA-5'
(Xmn
I
sites
in
the
adapters
are
underlined).
Orientation
of
the
enhancer
fragments
was
reversed
in
constructions
des-
ignated
entire.,,
and
minimalrey
by
ligation
of
the
same
fragments
of
pSES5
with
the
6.6-kbp
EcoRI-Bgl
II
and
2.2-kbp
BgI
II-Xba
I
or
2.2-kbp
BgI
II-HindIII
fragments
of
pCZ.
The
region
adjacent
to
the
minimal
enhancer
(HindIII-
Xba
I
fragment
of
pSES5)
was
ligated
with
the
6.6-kbp
Xba
I-Bgl
II
and
2.2-kbp
Bgl
II-HindIII
fragments
of
pCZ.
Plasmids
for
transcriptional
activation
of
pol
I
were
deriv-
atives
of
pCZ;
the
pol
I
promoter
was
supplied
by
a
640-bp
Xba
I
(end-filled)-BamHI
fragment
of
pSES5,
extending
from
212
bp
upstream
of
the
initiation
site
of
the
35S
rRNA
precursor
into
vector
sequences
428
bp
downstream.
Inser-
tion
of
the
640-bp
pol
I
promoter
fragment
between
the
EcoRI
(end-filled)
and
BamHI
sites
of
pCZ(DED48)2
(17)
gave
pCpolIZA.
Insertion
of
the
pot
I
promoter
fragment
between
the
Xho
I
(end-filled)
and
BamHI
sites
of
pCZ(DED48)2
gave
pCpolIZ-(DED48)2.
For
construction
of
pCpolIZ-ACT,
the
pol
I
promoter
fragment
and
large
EcoRI-BamHI
fragment
of
Abbreviations:
pol
I
and
II,
RNA
polymerase
I
and
II,
respectively;
GRF2,
general
regulatory
factor
2;
ABFI,
autonomously
replicating
sequence-binding
factor
I;
T-rich,
thymidine-rich.
*To
whom
reprint
requests
should
be
addressed.
8202
The
publication
costs
of
this
article
were
defrayed
in
part
by
page
charge
payment.
This
article
must
therefore
be
hereby
marked
"advertisement"
in
accordance
with
18
U.S.C.
§1734
solely
to
indicate
this
fact.

Proc.
Natl.
Acad.
Sci.
USA
87
(1990)
8203
Table
1.
RNA
pol
I
enhancer
elements
activate
RNA
pol
II
transcription
Oligonucleotide
A
GRF2
binding
35SRNAl
35SRNA2
RAP1
X40
ABFI
binding
rABFI
SPT2
pol
I
enhancer
residues
8-29
40-56
Relative
transcription
1.0
3.7
2.8
1.3
2.5
1.8
10.7
T-rich
r(dAdT)
116-156
43
r(dAdT)rev
156-116
3.2
The
synthetic
oligonucleotides
listed
were
inserted
in
the
poly-
linker
of
pCZ.
Sequences
of
the
35SRNA1,
35SRNA2,
RAPI,
and
X40
oligonucleotides
are
given
in
ref.
16
and
that
of
SPT2
in
ref.
17.
The
rABFl
and
T-rich
oligonucleotides
contained
the
residues
listed
5'-GATCC
G-5'
(Fig.
1),
with
before
the
first
residue
and
3'-G
CAATfPG-3'
after
the
last
residue.
The
resulting
plasmids
were
introduced
into
yeast
strain
15C
(a
leu2-31,
12,
ura3-52,
trpi,
his4-580,
pep4-3),
and
,/-galactosidase
activities
in
cell
extracts
(expressed
as
units
per
mg
of
protein)
were
determined.
Relative
transcription
refers
to
units
of
,8-galactosidase
activity
per
mg
of
protein
divided
by
the
result
obtained
for
the
plasmid
with
no
oligonucleotide
(A)
inserted
in
the
polylinker
(0.6
unit
per
mg
of
protein).
Each
value
listed
is
an
average
of
at
least
two
determinations
on
independent
transformants.
pCZ(DED48)2
were
ligated
with
the
following
double-
stranded
oligonucleotide:
AATTCTCTGTCACCCGGCCTCTATTTTCCATTTTCTTCTTTACCCGCCAC
GAGACAGTGGG
CGGAGATAAAAGGTAAAAGAAGAAAIGGG
GGTG
[general
regulatory
factor
2
(GRF2)-binding
sites
are
under-
lined;
ref.
16].
The
pol
I
promoter
fragment
was
inserted
between
the
Xho
I
(end-filled)
and
BamHI
sites
of
pCZ-3GAL
(19)
to
give
pCpolIZ-3GAL
with
three
Gal4-binding
sites
in
front
of
the
promoter.
Assays
of
Transcriptional
Activation.
Plasmid
DNAs
were
introduced
into
yeast
by
the
lithium
acetate-transformation
procedure
(20).
Transformants
were
picked,
grown,
har-
vested,
and
disrupted,
and
B-galactosidase
assays
were
done
as
described
(16).
Values
of
relative
transcription
or
-fold
activation
represent
3-galactosidase
units
per
mg
of
protein
for
the
fragment
or
oligonucleotide
in
question
divided
by
that
for
no
insert
(A,
0.6
unit
per
mg).
GRF2
RESULTS
The
pol
I
Enhancer
Contains
Three
Sequence
Elements
Commonly
Associated
with
pol
II
Promoters.
Residues
14-23
of
the
pol
I
enhancer
(Fig.
1)
match
the
consensus
sequence
YNNYYACCCG
(Y
=
C
or
T;
N
=
A,
G,
C,
or
T)
for
recognition
by
the
pol
II
activator
protein
GRF2
(16)
and
bind
the
protein
in
vitro
(16).
A
protein
termed
REB1
that
binds
this
region
of
the
pol
I
enhancer
has
been
identified
(21)
and
appears
identical
with
GRF2
(16).
Binding
sites
for
GRF2
occur
upstream
of
many
pol
II
promoters
and
function
synergistically
with
other
upstream
activation
sequence
ele-
ments,
possibly
by
excluding
nucleosomes
from
these
ele-
ments
(16,
22,
23).
A
gene
for
GRF2
has
been
isolated
from
yeast
and
is
essential
for
viability;
the
deduced
amino
acid
sequence
of
the
protein
exhibits
some
homology
to
that
of
human
c-MYB
(39).
Residues
39-58
of
the
pol
I
enhancer
match
the
consensus
sequence
RDCNYNNNNNACGAD
(R
=
A
or
G,
D
=
A,
G,
or
T)
for
recognition
by
the
pol
II
activator
protein
autono-
mously
replicating
sequence-binding
factor
I
(ABFI)
(17),
except
for
the
substitution
of
thymidine
for
cytidine
at
the
third
position.
Substitution
of
adenosine
for
cytidine
at
this
position
was
previously
shown
to
reduce,
but
not
abolish,
ABFI
binding
(17),
and
an
oligonucleotide
with
the
sequence
of
residues
39-58
(Table
1,
rABFI)
competes
with
other
ABFI
sites
for
ABFI-binding
in
vitro
(16).
A
protein
termed
REB2
that
binds
this
region
of
the
pol
I
enhancer
has
been
identified
(21)
and
is
probably
identical
with
ABFI.
Like
GRF2,
ABFI
binds
to
many
pol
II
promoters
and
exerts
a
modest
effect
on
transcription
on
its
own
but
functions
synergistically
with
other
activators
(17).
The
third
region
of
the
poI
I
enhancer
that
resembles
previously
described
pol
II
activation
sequences
is
the
thy-
midine-rich
(T-rich)
stretch
of
residues
116-155.
Although
the
sequence
of
this
region
varies
somewhat
among
the
many
copies
of
the
pol
I
enhancer
in
the
yeast
genome,
the
first
part
(Fig.
1,
residues
116-131),
containing
three
stretches
of
2-5
thymidines,
and
the
last
part
(Fig.
1,
residues
143-153),
containing
nine
contiguous
thymidine
residues,
are
con-
served
among
all
sequences
so
far
determined
(10,
24,
25).
Similar
T-rich
sequences
occur
upstream
of
many
yeast
pol
II
promoters
and
contain
runs
of
3-11
thymidine
residues,
with
as
few
as
15
thymidines
sufficing
for
activation
of
transcription
(26-30).
From
the
previous
deletion
analysis
of
the
pol
I
enhancer
(14),
it
is
apparent
that
all
three
regions,
the
GRF2
and
ABFI
sites
and
the
T-rich
element,
contribute
to
activating
pol
I
transcription.
Deletion
of
residues
7-21,
invading
the
GRF2
site
(Fig.
1),
reduced
transcription
2.7-fold.
Extending
the
deletion
of
residue
32,
removing
the
rest
of
the
GRF2
site,
had
ABFI
GAATTCTATGATCCGGGTAAAAACATGTATTGTATATATCTATTATAATATACGAT
GGAGGATGATAGTGTGTAAGAGTGTACCAT
II I
20
30
40
50
60 70
80
T-rich
TTACTAATGTATGTAAGTTACTATTTACTATTTGGTCTTTTTATTTTTTATTTTTTTTTTTTTTTTCGTTGCAAAGATGGGTTGAAA
I
90
100
110
120
130
140
150
160
170
Hind
avj=
GAGAAGGGCTTTCACAAAGCTTCCCGAGCGTGAAAGGATTTGCCCGGACAGTTTGCTTCATGGAGCAGTTTTTTCCGC
-
7/
A
-
II
1a0
190
200
210
220
230
240
250
336
FIG.1.
Nucleotide
sequence
of
an
RNA
polymerase
I
enhancer
from
S. cerevisiae,
as
determined
by
Stewart
and
Roeder
(18).
GRF2-binding,
ABFI-binding,
and
T-rich
sequences
analyzed
as
synthetic
oligonucleotides
in
the
present
study
are
shaded
and
in
boldface
type.
Biochemistry:
Lorch
et
al.

Proc.
Natl.
Acad.
Sci.
USA
87
(1990)
no
greater
effect,
but
deletion
of
residues
7-46,
encroaching
on
the
ABFI
site,
reduced
transcription
a
further
2.4-fold.
An
even
larger
deletion,
removing
the
entire
T-rich
element,
caused
an
additional
2.3-fold
drop
in
transcription.
The
T-Rich
Region
of
the
pot
I
Enhancer
Activates
pot
II
Transcription.
In
view
of
the
similarity
of
the
GRF2,
ABFI,
and
T-rich
regions
of
the
pot
I
enhancer
to
pol
II
activation
sequences,
we
tested
the
capacity
of
the
regions
to
stimulate
pol
II
transcription
in
vivo.
Synthetic
oligonucleotides
with
sequences
of
all
three
regions
were
inserted
in
a
polylinker
16
bp
upstream
of
the
major
TATA
element
of
the
yeast
CYCI
promoter
fused
to
the
E.
coli
lacZ
gene
in
a
yeast
centromeric
plasmid.
Levels
of
8-galactosidase
activity
were
determined
in
extracts
of
yeast
harboring
the
various
constructions
(Table
1).
The
T-rich
sequence
from
the
pol
I
enhancer
activated
pol
II
transcription
to
a
remarkable
extent
[r(dAdT)
oligonucleotide,
43-fold
stimulation
above
background
with
no
oligonucleotide],
more
than
a
T-rich
sequence
from
a
pol
II
promoter
(the
DED)
T-rich
element,
in
the
form
of
the
DED48
oligonucleotide,
7.4-fold
stimulation;
ref.
15).
Acti-
vation
by
the
pol
I
T-rich
sequence
was
strongly
orientation
dependent
[reduced
from
43-
to
3.2-fold
stimulation
on
re-
versing
orientation
in
the
r(dAdT)re,
oligonucleotide],
as
observed
previously
for
the
DEDI
T-rich
element
(15).
The
effect
of
the
pot
I
T-rich
sequence
was
comparable
to
those
of
the
most
potent
pol
II
activation
sequences
known,
such
as
a
single
Gal4-binding
site
(-100-fold;
ref.
31).
Two
controls
were
done
to
confirm
that
transcription
from
the
CYCI
promoter
constructs
was
from
pot
II
and
not
from
pol
I
recruited
to
the
promoter
by
the
pol
I
T-rich
sequence.
(i)
A
form
of
the
CYCJ
promoter
was
used
from
which
the
major
TATA
element
had
been
deleted
(pCThal,
ref.
32).
This
deletion
abolishes
pol
II
transcription
from
the
promoter
in
vitro
and
in
vivo
(32)
and
reduced
transcription
stimulated
by
the
pol
I
T-rich
element
5.8-fold.
The
low
level
of
transcrip-
tion
remaining
may
be
attributable
to
two
TATA-like
se-
quences
(TATTTT)
within
the
pol
I
T-rich
element.
(it)
The
second
control
was
to
compare
the
transcription
start
sites
obtained
with
a
known
pot
II
activation
sequence
to
those
found
in
the
presence
of
the
pol
I
T-rich
element.
For
this
purpose,
RNA
was
isolated
from
cells
bearing
plasmids
with
either
a
Gal4-binding
site
or
the
pot
I
T-rich
element
in
front
of
the
CYCI
promoter.
The
5'
ends
of
CYCI
transcripts,
revealed
by
RNase
protection
mapping
with
32P-labeled
RNA
probe,
were
the
same
in
the
two
cases
(Fig.
2),
with
a
larger
amount
of
RNA
probe
protected
for
the
Gal4binding
plas-
mid,
congruent
with
the
somewhat
greater
enhancer
activity
of
the
Gal
element
than
the
T-rich
sequence.
The
pattern
of
CYCI
transcription
start
sites
was
the
same
as
reported
(32).
Despite
the
capacity
of
the
pot
I
T-rich
sequence
to
activate
pot
II
transcription,
the
entire
pot
I
enhancer
failed
to
do
so.
The
enhancer
was
initially
tested
in
the
form
of
a
larger
fragment,
including
the
190-bp
minimal
enhancer
region
described
above
and
sequences
immediately
adjacent
to
this
region
because
the
adjacent
sequences
also
contribute
to
enhancer
function
(18).
The
larger
fragment
failed
to
activate
pol
II
transcription
in
either
orientation
(Fig.
3,
entire
and
entirerey),
so
the
minimal
enhancer
region
and
the
adjacent
sequences
were
tested
separately,
but
again
there
was
no
effect
(Fig.
3,
minimal,
minimalre,,
and
adjacent).
Appar-
ently,
sequences
flanking
the
T-rich
element
within
the
minimal
enhancer
not
only
fail
to
contribute
to
activation
of
pol
II
transcription
but
are
actually
inhibitory.
Location
of
the
inhibitory
component
was
determined
by
deleting
the
flanking
sequences.
Removal
of
the
region
down-
stream
from
the
T-rich
element
restored
function
to
nearly
the
level
obtained
with
the
element
alone
(Fig.
3,
downstream
del.).
On
the
other
hand,
addition
of
only
the
downstream
residues
157-180
to
the
T-rich
element
abolished
function
(Fig.
3,
upstream
del.).
We
conclude
that
residues
157-180
1
2
3
603-
310-
281
-
271-
$
234-
sf
194-
FIG.
2.
Patterns
of
transcription
start
sites
with
pol
I
and
pol
II
enhancer
ele-
ments
upstream
of
the
CYCI
promoter.
Synthetic
oligonucleotides
containing
a
Gal4-binding
site
(same
as
G4-1
oligonu-
cleotide
of
ref.
22,
here
designated
GOiP)
(lane
2)
or
the
pol
I
T-rich
element
[same
as
r(dAdT)
oligonucleotide
of
Table
1,
here
designated
HOT1]
(lane
3)
were
inserted
in
the
polylinker
upstream
of
the
CYCI
promoter
in
pCZ.
The
resulting
plasmids
were
introduced
into
yeast
strain
15C
(see
Table
1),
and
RNA
was
isolated
and
subjected
to
RNase
protec-
tion
mapping
with
RNA
probe
from
pSPCTB
as
described
(32).
Markers run
in
lane
1
were
from
an
Hae
III
digest
of
4X174
restriction
fragment
DNA;
sizes
in
nucleotides
are
indicated
at
left.
prevent
activation
of
pot
II
transcription.
This
conclusion
is
supported
by
the
effect
of
inserting
residues
157-180
between
a
Gal4-binding
site
and
the
CYCI
promoter
(in
pCZGAL
polI157-180,
see
Materials
and
Methods).
Activation
by
Gal4
protein
was
reduced
from
>100-
to
only
7-fold.
The
GRF2-
and
ABFI-binding
sequences
associated
with
pot
II
promoters
exert
only
modest
effects
on
transcription
(2-
to
10-fold;
refs.
16
and
17),
so
it
was
not
surprising
to
find
that
the
GRF2-
and
ABFI-binding
sequences
from
the
pol
I
enhancer
barely
stimulated
pot
II
transcription
at
all.
The
GRF2-binding
sequence
(35SRNA2
oligonucleotide)
stimu-
lated
slightly
(2.8-fold),
as
did
a
second
GRF2-binding
se-
quence
from
immediately
upstream
of
the
pot
I
transcription
start
site
(35SRNA1
oligonucleotide,
oppositely
oriented
to
35SRNA2,
3.7-fold
stimulation).
The
ABFI-binding
se-
quence
from
the
pol
I
enhancer
(rABFI
oligonucleotide)
also
activated
pot
II
transcription
slightly
(1.8-fold).
The
DEDI
T-Rich
Element
Activates
pot
I
Transcription.
Identification
of
pot
I
enhancer
elements
that
stimulate
pol
II
transcription
led
us
to
investigate
the
possibility
that
some
pol
II
enhancers
might
activate
pol
I
transcription.
Various
pol
II
enhancers
were
placed
in
front
of
the
35S
rRNA
initiation
region
(212
bp
upstream
of
the
start
site
of
pot
I
transcription),
and
levels
of
transcription
in
yeast
harboring
the
constructions
were
determined
by
RNase
protection
mapping
of
cellular
RNA
(Table
2).
A
pair
of
T-rich
elements
from
upstream
of
the
DED)
gene
(two
copies
of
the
DED48
oligonucleotide)
was
a
potent
activator
of
pot
I
transcription
(38-fold
stimulation),
more
so
than
the
pol
I
enhancer
itself
(17-fold
stimulation).
RNase
protection
mapping
also
showed
that
transcription
stimulated
by
the
DEDI
T-rich
element
was
initiated
at
the
correct
pot
I
start
site
(34),
so
transcription
was
in
all
likelihood
due
to
pol
I.
A
pair
of
GRF2-binding
sites
upstream
of
the
ACT
gene
stimulated
pot
I
transcription
less
(3.8-fold)
when
compared
with
the
effects
of
these
sites
on
pol
II
transcription
(16).
Other
pot
II
enhancers,
such
as
a
Gal4-dependent
activation
sequence,
were
also
less
effec-
tive.
The
capacity
of
both
the
pot
I
and
DED)
T-rich
elements
to
activate
pol
I
and
pol
II
transcription
raised
the
question
whether
the
effects
of
these
elements
are
mediated
by
a
common
protein
factor.
Insertion
of
the
DEDI
T-rich
element
upstream
of
a
pot
II
promoter
activates
transcription
10-
to
8204
Biochemistry:
Lorch
et
al.

Proc.
Natl.
Acad.
Sci.
USA
87
(1990)
8205
fragment
entire
entirerev
minimal
minimalrev
adjacent
downstream
del.
upstream
del.
enhancer
residues
4-336
336-4
4-191
191-4
336-192
4-156
116-180
GRF2
ABFI
T-rich
-
-
-
GRF2
ABFI
T-rich
-
-
-
GRF2
ABFI
T-rich
-AffmW-
-
--ric
T-rich
FIG.
3.
Identification
of
a
region
of
the
pol
I
enhancer
that
prevents
activation
of
pol
1I
transcription.
The
fragments
indicated
(residue
numbers
as
in
Fig.
1)
were
incorporated
in
the
polylinker
of
pCZ, and
their
effects
on
transcription
were
analyzed
as
in
Table
1.
Fragments
are
designated
entire
and
minimal
for
entire
and
minimal
enhancers,
entirer,
and
minimalrv
for
the
same
fragments
in
the
opposite
orientation,
adjacent
for
the
region
immediately
downstream
of
the
minimal
enhancer,
and
downstream
del.
and
upstream
del.
for
synthetic
oligonucleotides
containing
the
minimal
enhancer
with
sequences
downstream
or
upstream
of
the
T-rich
element
deleted.
30-fold
in
vitro,
and
activation
is
inhibited
by
addition
of
free
DEDI
T-rich
element
(DED48
oligonucleotide)
to
the
reac-
tion
(Fig.
4;
ref.
15),
indicating
involvement
of
a
DEDI
T-rich-binding
factor.
By
contrast,
the
pol
I
T-rich
element
caused
no
activation
of
transcription
in
vitro,
and
the
low
level
of
transcription
observed
was
unaffected
by
addition
of
the
free
element
[r(dAdT)
oligonucleotide,
Fig.
4].
Thus,
action
of
the
pol
I
fragment
is
probably
mediated
by
a
distinct
factor,
as
appears
so
for
other
T-rich
elements
that
have
proved
ineffective
in
vitro
(15).
Consistent
with
this
inter-
pretation,
the
pol
I
element
was
a
poor
competitor
of
tran-
scription
from
a
DEDJ
T-rich
template,
and
the
inhibition
that
did
occur
(25%
of
that
with
the
DEDI
T-rich
element;
data
not
shown)
probably
reflects
low
affinity
of
the
DEDI
T-rich-binding
factor
for
the
pol
I
element.
DISCUSSION
Dissection
of
the
pol
I
enhancer
was
essential
for
revealing
the
capacity
of
one
sequence,
the T-rich
element,
to
activate
pol
II
transcription
because
another
sequence,
between
res-
idues
157-180,
was
inhibitory.
These
residues
probably
con-
tain
a
factor-binding
site,
as
deletion
of
residue
166
and
insertion
of
a
linker
greatly
diminishes
activity
of
the
en-
hancer
(18).
It
has
also
been
noted
that
residues
166-173
nearly
match
the
simian
virus
40
enhancer
core
consensus
Table
2.
Effects
of
RNA
pol
II
enhancers
on
RNA
pol
I
transcription
Relative
Sequence
transcription
A
1.0
(DED48)2
38
ACT
4.0
3GAL
3.8
The
sequences
listed
(A,
no
insertion;
DED48,
48-bp
DEDI
T-rich
element;
ACT,
two
GRF2-binding
sites;
3GAL,
three
GALA-binding
sites)
were
placed
in
front
of
a
pol
I
promoter
in
derivatives
of
pCZ
designated
pCpolIZA,
pCpolIZ-(DED48)2,
pCpolIZ-ACT,
and
pCpolIZ-3GAL.
Plasmids
were
introduced
into
yeast
strain
5C
(a
his3A200,
ura3-52);
transformants
were
isolated
and
grown
as
de-
scribed
(15)
to
an
A6w
value
of
0.5
and
RNA
was
isolated
(33);
transcripts
initiated
at
the
35S
precursor
start
site
in
the
plasmids
were
quantitated
by
hybridizing
with
an
RNA
probe,
RNase
diges-
tion,
and
gel
electrophoresis
as
described
(34),
and
then
counted
with
an
AMBIS
(San
Diego)
radioanalytic
imaging
system.
(11).
Factor-binding
between
the
T-rich
element
and
pro-
moter
may
block
activation
of
pol
II,
much
as
was
shown
for
protein-binding
sites
interposed
between
a
Gal4-dependent
activation
sequence
and
promoter
(37).
The
minimal
pol
I
enhancer,
containing
GRF2,
ABFI,
and
T-rich
elements,
but
lacking
residues
157-180,
gave
no
in-
crease
in
activation
of
pol
II
transcription
above
the
level
obtained
with
the
T-rich
element
alone
(Fig.
2).
Both
GRF2
and
ABFI
sites
exert
synergistic
effects
when
located
adja-
cent
to
T-rich
elements
in
other
constructs
(16,
17),
but
in
the
case
of
a
GRF2
site,
the
effect
has
been
shown
to
be
strongly
distance-dependent,
declining
by
85%
when
spaced
from
a
T-rich
element
only
half
that
in
the
pol
I
enhancer.
The
distance-dependence
of
the
ABFI
effect
as
well
as
possible
synergism
between
GRF2
and
ABFI
elements
have
not
been
investigated.
100
n
DED48
c
80-
r(dAdT)
00
fi60-
2
40-
20
.
0
200
400 600
800
oligonucleotide
(ng)
FIG.
4.
Effects
of
T-rich
elements
from
upstream
of
the
DEDI
gene
(DED48
oligonucleotide)
and
from
the
pol
I
enhancer
[r(dAdT)
oligonucleotide,
Fig.
1]
on
pol
II
transcription
in
vitro.
Templates
for
transcription
were
p(DED48)2CG-,
with
two
copies
of
the
DED48
oligonucleotide
(48-bp
DEDI
T-rich
element)
in
front
of
the
yeast
CYCI
promoter
(35),
and
pr(dAdT)CG-,
with
the
r(dAdT)
oligonu-
cleotide.
pr(dAdT)CG-
was
constructed
by
insertion
of
r(dAdT)
between
the
Xho
I
and
HindI.1
sites
of
pGAL4CG-
(35).
Transcrip-
tion
reactions
with
p(DED48)2CG-
(E)
contained
200
ng
of
template
and
the
amounts
of
DED48
oligonucleotide
indicated,
whereas
reactions
with
pr(dAdT)CG-
(*)
contained
380
ng
of
template
and
r(dAdT)
oligonucleotide
[levels
of
transcription
from
pr(dAdT)CG-
were
divided
by
1.8
to
correct
for
the
greater
quantity
of
DNA
used].
Procedures
were
as
described
(36).
relative
transcription
1.7
0.5
0.8
0.7
0.3
38
2.3
Biochemistry:
Lorch
et
al.

Proc.
Natl.
Acad.
Sci.
USA
87
(1990)
The
evidence
presented
here
for
reciprocal
actions
of
pol
I
and
pol
II
enhancers
can
be
interpreted
in
two
ways.
(i)
pol
I
and
pol
II
enhancers
may
function
by
similar
mechanisms.
Stimulatory
proteins
that
bind
to
these
enhancers,
such
as
GRF2,
ABFI,
and
T-rich
binding
factors,
may
interact
with
common
components
of
the
pol
I
and
pol
II
transcription
machineries.
The
selection
of
one
or
the
other
type
of
polymerase
at
a
particular
promoter
would
be
determined
by
additional
sequences.
For
example,
the pol
II-inhibitory
component
of
the
pol
I
enhancer
described
above,
or
the
TATA
element
specific
to
pol
II
promoters,
might
fulfill
this
role.
(ii)
An
alternative
interpretation
would
be
that
different
proteins
bind
to
the
same
enhancer
sequence,
depending
on
whether
it
is
located
in
front
of
a
pol
I
or
pol
II
promoter.
The
detailed
mechanisms
of
transcriptional
activation
might
then
be
different
at
the
two
types
of
promoter.
Although
there
is
precedent
for
multiple
transcription
factors
recognizing
the
same
DNA
sequence
in
higher
cells
(38),
a
single
factor
appears
to
bind
a
particular
sequence
in
most
cases.
The
question
of
whether
one
or
multiple
factors
are
involved
in
the
present
examples
could
be
addressed
with
the
use
of
mutant
binding
sequences,
such
as
those
described
for
ABFI
(17)
and
GRF1
(31).
We
thank
Dr.
S.
Roeder
for
providing
pSES5.
This
research
was
supported
by
National
Institutes
of
Health
Grant
GM36659
to
R.D.K.
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