J. Mol. Biol. (1990) 211. l-4
COMMUNICATIONS
Transcription
from the rha Operon p,, Promoter
J. F. Tobin? and R. F. Schleifl
Department of Biochemistry
Brandeis University
Waltham, MA 02254, U.S.A.
(Received 25 April
1989, and in revised form 13 July
1989)
S, nuclease mapping
experiments
performed
with RNA extracted
from cell lines that were
unable to metabolize
t-rhamnose
demonstrated
that L-rhamnose
and not a metabolite
was
the inducer
of the L-rhamnose
operons
of Escherichia
coli. In vitro transcription
studies
showed that purified
RhaR activates transcription
from the pSr promoter
in the presence of
r,-rhamnose.
In the absence of L-rhamnose,
RhaR binds to the pSr promoter
but does not
activate
transcription
until L-rhamnose
is added.
We have shown (Tobin & Schleif, 1987) that the
product
of the rhaK gene is required
for efbcient’
transcription
of the L-rhamnose
operons of Escherichia coli. The experiments
in the accompanying
paper
(Tobin
& Schleif.
1990) demonstrate
that
RhaR is an r,-rhamnose-dependent,
DNA-binding
protein that binds specifically
to an inverted repeat
DNA sequence
located
upstream
from the RNA
polymrrase
binding
site within
the pSr promoter.
Here, we show that purified
RhaR can stimulate
transcription
from
the pSr promoter,
and that
L-rhamnose
is required
for its activit’y.
LVe used
in vitro transcription
to determine
if
purified
RhaR was capable of activating
transcription from the psr promoter.
The system necessitated
the use of supercoiled
templates,
since we detected
no
RhaR-specific
transcription
from
linear
tjemplates.
RhaR-p,,
D?U’A complexes
were formed
to which a sat,urating
concentration
of RNA polymerase was added and allowed
to react for four
minutes.
At this point,
heparin
and nucleotide
triphosphates
were added and RNA polymerase
was
allowed to elongate for ten minutes. The addition
of
heparin
and triphosphates
allows only those RNA
polymerase
molecules
that
have
formed
open
complexes
to elongate.
Incubation
of the RhaR-p,,
DNA
complex
with
RNA
polymerase
for times
longer
than four minutes
does not result in an
increased
level of transcription.
Figure
1 shows results from a typical
transcription
experiment.
RhaR-dependent
transcription
from psr gives rise to a 170 base-pair
transcript.
There is a detectable
background
of synthesis from
the pSr promoter
but. upon the inclusion
of RhaR
and r,-rhamnose,
transcription
is stimulated
fivefold. In t’he absence of L-rhamnose,
there is no
increase in the level of pSr transcription
as the RhaR
concentration
is increased.
The result that L-rhamnose
is required
for in vitro
transcription
from the pSr promoter
could be an
artifact,. Suppose RhaR can activate
transcription
but, is unstable
in vitro in the absence of I,-rhamnose. If this were the case, then r,-rhamnose
would
appear to be the inducer because its addition
would
stabilize
RhaR.
We excluded
this possibility
by
showing that RhaR is stable in vitro in the absence
of L-rhamnose
(Fig. 1). RhaR-p,,
DNA complexes
were formed in the absence L-rhamnose
and allowed
to incubate
for the appropriate
time. r,-Rhamnose
was added
with
RNA
polymerase
followed
by
heparin
and nucleotide
triphosphates.
Under the
conditions
of this experiment
with
L-rhamnose
added late, the level of transcription
was increased
threefold,
and in the tubes where 1,.rhamnose
was
present
for the entire
preincubation
period,
the
stimulation
was fivefold.
This experiment
demonstrates t,hat the lack of transcription
in the absence
of L-rhamnose
cannot be attributed
to irreversible
denaturation
of the protein.
Therefore,
RhaR
requires r,-rhamnose
to stimulate
transcription
from
PSV
The affinity of RhaR for its pSr binding
site in the
absence of L-rhamnose
is decreased
approximately
20.fold
to 7.5x 10~‘2 M (Tobin
& Schleif,
1990).
However,
since the RhaR concentration
is 50-fold
greater
than the Kapp measured
in the absence of
r,-rhamnose,
the pSr site should be fully occupied
under t’he conditions
of the in vitro transcription
experiment.
Therefore,
the lack of transcription
in
the absence of L-rhamnose
is not due to failure to
bind to DNA. To examine this question more care-
‘r %‘resent address: Department of Tropical Public Health.
Harvard
School of Public Health,
Boston, MA 02115, U.S.A.
f Present address: Hiology Department,
Johns Hopkins
I’niversity.
31th and (Iharles St., Baltimore.
MD 21218. 1J.S.A.
c~2~~2x3ci/~o~oloool~~4
$03.00/0
I
0
1990 Academic
Press Limited
L-Rhamnase
f=lO
+
+
+
+
+
-
-
-
-
-
+
+
+
+
L-Rhamnose
t; 0
RhaR
+
-
+
+
+
+
+
t
+
+
-t+++tt+t
-
-
-
-
-
-
-
PSR
ANA1
I
le-IO
I
2e-IO
[RhaRl
I
3e-IO
4
4e-i0
(M )
(b)
Figure 1. Rhalt is reyuired
for
transcription
from psr. (a) The 170 Ljasr-pair
ljSr t ransc:ript
anti
ttrt.
IO(I I~~~w-~~~~II~
RNA- 1 transcript are marked with arrows. A minus sign indicates the absrnc.e and a plus sign indicatr>s thts presents 01’
RhaK and/or 50 mM-L-rhamnose.
r,-Rhamnose at t = 0 indicates t,hat r)-rhamnose LV~N present tluriny the IO rnin
incubation
of RhaR wit,h the pSr DNA4 template. r,-Rhamnose at, t = 10 indicates that I.-rhamnose was not atldrd until
aft,er the 10 min incubation of pSI template DNA with RhnR. (b) 4 plot of transcription
level OPTSU.S
c,c)nc.t,trtration of
RhaR. The unit.s are arbitrary.
The level of RhaR.-spec+fic transcript
was determined
by densitometr~
ot’ thf,
;tutoradiograph
and normalization
to the const.itutively expressed Rh’A-I transcript. (+) With r.-rhamnose: (0) without
I.-rha,mnose. To detect t.ranscription
from supercoiled templates. a rho-independent
transcription
terminator.
q~)f‘
(Squires rt ~1.. 1981), was cloned downstream
from t,hr prr transc~ription
start sit,<,. Transcriptiou
rctac*tions w~r’(.
performed using a modification
of the procedure described by Hahn rt 4. (IYHB). Puritied RhaK was added to
I4 nM-supercoiled
t.emplate in transcription
buffer (20 m#-Tris-acetate
(pfl 34). IO0 rn~-KC’]. 50 1~1M~~.--rlI~~rnt~oS~
10 tIIM-~bfgC1,,
0.5 mmEDTA.
1 mr\l-dithioerpthritol
and 50 pg boGne serum albumin,ml)
and incubat,ed for 5 to IO mill
at 37°C:. Purified fC”.coli RX;A polgmerase (Burgess
& Jendrisak. 1975: I,owe ct ccl.. 1979). kindly provided t)J, S. Hahn.
was added (final cone I.5 ny) and incubated for 4 min at 37°C. after which 100 pg heparini ml. zX)O ~M-~~‘IT’.
“00 PM-GTP. 100 PM-CTP and 10 PM-I’TP (15 &i [a-32P]I’TP/nmol)
was added. Tncubati&
was contirlurd for 10 min
at 37°C’ and the reaction stopped with d0 ,uI of 8096 (v/v) f ormamide. 50 mM-EDTA. and OM”i, (w/v) bromphnol
hluc.
The entire react.ion mix was loaded onto a 7.5 M-urea/s
*& polyacrytamidr
gel and electrophoresed
in Tris-boratr
hutGt
(Schleif & Wensink, 1981). The amount of RhaR-specific transcript was determined by densit,ometry of’ thta atltoradiograph and normalized to the constitutively
expressed RNA-I transcript (Lrvk
& Rupp: 1!478; Morita & Oka. 19~:
Rosenberg et al.. 1983).
Communications
I.0
nu-PSR
I-D nu-puc19
Rho R
3
Rha B
t
f
-
t
+
+
Rha A
t
t
t
+
-
t
t
RhaD
Rha
L-Rhomnose
Rha A-DNA
R
t
t
t
+
t
-
+
t
t
t
-
t
t
+
+
+
-
DNA 4
Figure 2. RhaR is bound to supercoiled ps, DNA in the
absence ot’ I,-rhamnose. A plus sign indicates the presence
and a minus sign indicates the absence of RhaR. supercoiled psr competitor
DNA. or supercoiled pUCIS competitor I>K,L\. The band above t#he RhaR-DKA
complex is
labeled vect’or I)?iA.
fully, we performed
a competition
gel shift assay in
parallel with t’he in vitro transcription
assay. RhaR
was mixed with unlabeled
supercoiled
psr template
or supercoiled
pUCI9
template
in transcription
buffer lacking
I,-rhamnose.
The reaction
was incuhated for an appropriate
time, after which
endlabeled
psr DNA was added. The incubation
was
continued
and the mixture
was loaded onto a 60/b
non-denaturing
gel (Tobin
& Schleif,
1990). The
results, Figure 2. indicate
that all of the RhaR is
bound to the supercoiled
psr template
in the absence
of I,-rhamnose.
Therefore,
even though
the psr
binding
site is fully occupied
by RhaR
in the
absence of I,-rhamnose.
RhaR
does not’ activate
transcription.
Therefore,
RhaR must exist, in two or
more states or conformations,
one, in the presence of
L-rhamnose.
activates
transcription,
and the other,
in the absence of L-rhamnose,
does not appreciably
activate
transcription.
The trancriptionally
active
form of RhaR could interact
directly
with RNA
polymerase
/bin a protein-protein
interaction
or
indirectly
through
the Dh’A.
U’e used the SI nuclease
protection
assay to
det.ermine
if r.-rhamnose
was the inducer
of the
rhamnose operons in ~izro. Total RNA was extracted
from strains of E. coli grown
in the presence
of
L-rhamnose
that contained
point mutations
in the
Figure 3. I,-Rhamnose is the inducer of the rha operon.
S, nucalease mapping
analysis of (a) the p1 and p2
promotjers, (b) the p3 promoter, and (c) the psr promoter
in rell lines incapable of metabolizing
L-rhamnose. Cells
were grown in minimal salt media in the absence ( -) or
presence ( + ) of @%?‘O (w/v) L-rhamnose.
The hybridization conditions
this
experiment
(1987).
and the end-labeled
are as described
by
DNA
fragments
in
Tobin
& Schleif
Rha
E
t
+
-
+
Rha
A
+
t
t
-
t
+
t
+
+
-
-
t
+
+
+
RhaD
L.-Rhamnose
+
(b)
Rha B
Rha A
Rho
RhaR
L-Rhomnose
0
t
+
t
+
-
+
t
t
t
t
t
+
t
t
+
t
t
t
+
t
t
t
t
t
t
t
4
J. k’. Tobin
and R. k’. Schleif
r,-rhamnose isomerasegene rhaA502.
t’he r,-rhamnuwe surmise that t,he lower levels of mKSt1 in the
lose kinase gene rhaRl07,
the 1,.rhamnulose-l-phosRhaH
and rhaD mutants may bfs due to a similar
phate aldolase gene rhaD701
and the regulator!
phenomenon.
gene rhaR702 (Chen et al., 1987). The RNA was used
in S, nuclease mapping experiments t’o measure the
References
level of mRNA produced from each of the four rhrr
promoters. Mutations in rhaA or rhaR do not allow
Al-%arba,n.S., He&reman, L., Chitani. .J., Ransonr. I,.
r,-rhamnose to be metabolized: while mutat’ions in
& Wilrox. (:. (1984).J. Racteriol. 158. 603WW3
ISuraeas.R. & ,Jendrisak. ,J. (1975). Hiochumistry. 14.
rhaB and rhaD allow conversion of L-rhamnose to
4634-4638.
1-phosphate,
I,-rhamnulose and I,-rhamnulosr
(‘hen. Y.-,X, Tobin. J. F.. Zhu. I’.. Srhlrait’.R. F‘. & Lirl,
respectively. The results are shown in Figure 3.
E. t’. (‘. (1987)..J. Bockrid.
169, 3712~-3719.
Yutations in the st’runtural genes do not signifi(‘ozzarelli, I\‘. R.. Koch, ,J. P.. Hayashi. S. & Lin. ELi’. (‘.
cantly interfere with t,ranscription from the st)ruch(I 96.5). J. Bnctwiol. 90, 1325-1329.
tural gene promoters, pl. pZ and p3. while a
Hahn. S.. Hendrickson.W. Br Schleif. It. (19%). ./. .I/ol.
mutation in RhaR abolishes transcription as
Hid. 188. X2X67.
expected. The data for the pSr promoter (Fig. X(C)) ,Jiuks-Robertson.S.. (:oursr, R. I,. & Notr~~~ra.
JI. ( I!#:{).
gives the same results but is of poor quality due to
(‘ell. 33. 866-876.
ineficient, digestion by nuclease S,. However. t)htA I,evirre. A4. & Rupp. 12’. (IBSH). 111 .Vi?rohio/oyy
(Schlessinger. I).. ~1.). pp. 16%166. i\rnrric:an
results of t,he %n>vitro transcription experiments
Society for Microbiology. Washin@n. I)( ‘.
demons&ate that r,-rhamnose is t’he inducer at thrx
Lowe. t’., Hager. 1). & Hurgrss. R. (1979). Hiochwr~risfr!/.
pS, promoter. Taken toget)her. these result’s indic~atr
18. 1.X44-13.52.
t.hat, I,-rhamnose and not a met,aholit’e is the inducer
Morita. M. k Oka. :I. (I97!)). Eu~. .I, /liochrr,/ 97
of the rhn operons.
435--443.
Strains containing mutations in the ‘&al) and
Xorris. 1’. E. B tioc*h. .I. I,. (1972). ,i. .Iloi. I<io/. 64.
rhal3
genessynthesize somewhat lessthan the wiltl6X-64!J.
type level of rha mRNA. The lower mRlVA levels
K,osenbert_r.
bl.. (‘he@insky. .A. & >lcKrnnr~-. K. (I!W).
may be due tjo accumulation of I,-rhamnose metakw
AVirnce. 222. 734-739.
lites to physiologically harmful levels. Growth of
R,vals, ,J.. Little. K. bi Krernrbr.H. (I 9X2). ./. /~mcf~,~/~/.
151, 142.5-1432.
rhaB mutants is slightly inhibited by I,-rhamnose.
while growth of rhaD mutants is inhibited severely
(Al-Zarban et al., 1984), presumably due to t,he
accumulation of phosphorylated mt’ermediates,
which generally are growth inhibit’ory (C’ozzardli
et al., 1965). RNA levels vary greatly depending on
the growth rate of cells (Norris & Koch. 1972: Ryals
Tobin. .J. F. & Schlril’. R. F. (1990). ./. .l/o/. Ijiol. 211
et al.. 1982: Jinks-Robertson et al.. 1983). Therefore.
75-89.