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,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. IlcKrnnr~-. 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.
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,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. IlcKrnnr~-. 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.
