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Expression of the mcrA gene of escherichia coli is regulated posttranscriptionally, possibly by sequestration of the Shine-Dalgarno region
Gene, 157 (1995) 201-207 © 1995 Elsevier Science B.V. All rights reserved. 0378-1119/95/$09.50
201
GENE 08535
Expression of the mcrA gene of Escherichia coli is regulated posttranscriptionally, possibly by sequestration of the Shine-Dalgarno region* (S1 analysis; primer extension; overexpression; regulation)
R. Shivapriya, Ranjan Prasad, Iyer Lakshmi Narayanan, S. Krishnaswamy and K. Dharmalingam School of Biotechnology, Madurai Kamaraj University, Madurai-625 021, India Received by R.M. Blumenthal: 28 May 1994; Accepted: 20 September 1994; Received at publishers: 20 October 1994
SUMMARY
The polypeptides encoded by the mcrA gene were analysed using a T7 expression system. Cloned fragments of 1.6 and 1.0 kb displayed an McrA +/RglA + phenotype and directed synthesis of a 31-kDa polypeptide. A derivative of these clones altered at an internal HindIII site displayed an McrA +/RglA- phenotype and directed production of a 23-kDa polypeptide. Smaller inserts displayed McrA-/RglA phenotypes, though a 0.7-kb insert did direct production of a 24-kDa polypeptide. A construct carrying the 1.0-kb mcrA insert yielded a single 1.3-kb transcript. The mcrA transcript was found to start from C, G, T and G, namely the fourth, fifth, sixth and seventh nucleotides (nt), respectively, downstream from the last nt of the putative - 10 region. Two mcrA transcriptional/transational fusions were made in the pT7-7 expression vector and the protein encoded by these constructs were analysed. Regulation of mcrA expression was studied by quantitative Northern analysis of RNA from various mcrA clones. Together with a computer analysis of the translation initiation region in these mRNAs, the result~ suggest that the expression of mcrA may be regulated at the translational level.
INTRODUCTION
The Mcr restriction systems of E. coli restrict DNA containing hydroxymethyl cytosine (hmC; Revel, 1967) and methyl cytosine (mC; Blumenthal et al., 1985; Raleigh Correspondence to: Dr. K. Dharmalingam, School of Biotechnology, Madurai Kamaraj University, Madurai-625 021, India. Tel. (91-452) 85-215~ Fax (91-452) 85-205; Telex: 445-337 MKU IN; e-mail: dharm % [email protected] *Presented at the Third New England BioLahs Workshop on Biological DNA Modification, Vilnius, Lithuania, 22 28 May 1994. Abbreviations: A, absorbance (lcm); aa, amino acid(s); Ap, ampicillin; bp, base pair(s); DMSO, dimethylsulfoxide; GCG, Genetics Computer Group (Madison, WI, USA); hmC, hydroxymethylcytosine; IPTG, isopropyl-13-D-thiogalactopyranoside; kb, kilobase(s) or 1000 bp; Kin, kanamycin; nt, nucleotide(s); ORF, open reading frame; RBS, ribosome-binding site(s); SDS-PAGE, sodium dodecylsulfatepolyacrylamide-gel electrophoresis; ss, single strand(ed); Tc, tetracycline; TE, 10 mM Tris.C1/1 mM EDTA pH 7.6; tsp, transcription start point(s); [], denotes plasmid-carrier state. SSDI 0378-1119194)00746-2
and Wilson, 1986; Noyer-Weidner et al., 1986) in specific sequence contexts. The Mcr restriction systems are governed by two loci, mcrA and mcrBC [ Raleigh and Wilson, 1986). The McrA system restricts DNA methylated by M.HpaII (CmCGG; Raleigh and Wilson, 1986) and M.SssI (mCG) (Kelleher and Raleigh, 1991). It also restricts the hmC DNA of all T-even phages (Revel, 1967), though wild-type phages are refractory to restriction as the hmC residues are glucosylated: this restriction of glucose-less phage is known as RglA phenotype. The mcrA gene which has been mapped to 25.24 min on the E. coli chromosome (Ravi et al., 1985; Raleigh et al., 1989) has been sequenced and comprises a 831-bp ORF (Ramalingam, 1990; Hiom and Sedgwick, 1991; Ramalingam et al., 1992). This ORF could encode a polypeptide of 277 aa (31.39 kDa). In this communication the polypeptides from various mcrA constructs were analysed using the T7 expression system. The transcript analysis by primer extension
202 codon introduced by frame shift (Fig. 2, lanes 7 and 8). A slightly larger (24 kDa) polypeptide synthesized by pDRR551.4 could be due to the absence of the stop codon in the insert while the nearest stop codon in the vector is not known, leading to an alternate C terminus.
located the tsp. Comparison of quantitative Northern blots and translation products implied that protein synthesis is possibly regulated during translation. This possibility is supported by theoretical analysis of the secondary structure of mRNA in the translation initiation region.
(b) Low-resolution SI analysis RESULTS AND DISCUSSION
(a) Polypeptides encoded by mcrA Earlier results using mini cells (Ramalingam, 1990) showed that three polypeptides of 29, 18 and 17 kDa were encoded by a 1.6-kb PstI fragment carrying the mcrA gene. In order to confirm this, the T7 promoter expression system was used. Fig. 1 shows the constructs and their McrA/RglA phenotypes. The polypeptides encoded by these constructs are shown in Fig. 2. Plasmids pDRR551.3 and pDRR551.1 produced a single 31-kDa polypeptide (Fig. 2, lanes 2 and 4) as predicted, while pDRR551.2 failed to produce any protein, (Fig. 2, lane 3). These results indicate that the direction of transcription is from left to right as shown in Fig. 1. Plasmid pDRR551.4 yielded a 24-kDa polypeptide (Fig. 2, lane 5), while pDRR551.5 failed to synthesize any polypeptide (Fig. 2, lane 6). Plasmids pDRR551.6 and 551.7 have 4-nt insertional mutation in the H i n d l I l site (cut, filled and ligated) and produce a 23-kDa protein due to the stop
Total RNA isolated from H R l l 2 carrying pBR322 with various mcrA segments were used for analysing the number and size of the transcripts encoded by mcrA. Total RNA including the chromosomal mcrA transcript isolated from W3110 and HR112 were used as controls. In the construct carrying the 1.0-kb mcrA (McrA--, RglA ÷ ) a single transcript of 1.3 kb was detected by the mcrA probe (Fig. 3, lane 1). The size of the hybridising transcript is larger than anticipated, which could be due to the reduced mobility of RNA-DNA hybrid compared to the DNA molecular mass markers. The constructs carrying the 0,7-kb and the 0.9-kb fragments failed to make any detectable rectA-specific transcript (Fig. 3, lanes 3 and 4). It is surprising that the 0.7-kb region did not make any transcript despite having all the promoter sequences intact. Possibly, sequences downstream from the H i n d l I I site have a stabilizing role on the mRNA. It has been suggested by Wong and Chang (1986) and Mott et al. (1985) that specific mRNA structures located downstream from some genes have a stabilizing effect, possibly by acting as barriers to 3' exonuclease activity. The
Phenotype McrA RglA
TTP H I
P
l
P P
m_t ~.~_L
Hp I
P I
pDRR551.1
+
+
P .1 pDRR551,2
÷
+
I
pDRR551.3
+
+
I
pDRR551,4
_
_
I
I pDRR551.5
_
_
I pDRR551.6
+
_
pDRR551,7
+
_
ORF I ORF II Hp I
H
~H I
~tp/Pv
H I ~I
Hp/Pv
I
L
Fig. 1. Restriction map of pDRR551.1 and pDRR551.2, which carry the 1.6-kb PstI fragment in two differentorientations, with respect to the T7 promoter, weremade by ligating the 1.6-kb PstI fragment to pT7-1.3digested with PstI. Plasmid pDRR551.3, carryingthe 1.0-kb PstI-HpaI fragment was constructed by digesting pDRR551.1 with HpaI+ PvulI followed by self ligation. The plasmid pDRR551.4, carrying the 0.7-kb PstI-HindlII region, was constructed by digesting pDRR551.1 with HindlII followed by self ligation. The plasmid pDRR551.5, carrying the 0.9-kb HindlII-Pstl region of rectA, was constructed by ligating the 0.9-kb HindlII fragmentobtained on restrictiondigestion of pDRR451.1 (pBR322 carrying the 1.6-kb mcrA) with HindlII to pT7-1.3 resricted with HindllI and dephosphorylatedusing calf intestinal phosphatase. The plasmid pDRR551.6, carrying the 1.6-kb PstI fragment with a mutation in the HindlII site, was constructed by subjecting pDRR551.1 to partial digestion with HindlII followed by electrophoresisand elution of the full-lengthlinear molecule.The ends were then filledwith dNTPs using Klenow fragment of E. coli DNA polymerase I and ligated. The plasmid pDRR551.7, carrying the 1.0-kb PstI-HpaI region with the HindlII site mutated, was constructed by digesting pDRR551.6 with HpaI+PvulI followedby self ligation. P, Pstl; H, HindlII; H*, Filled-in HindlII site; Hp, HpaI; Pv, PvulI.
203
kDa
1
2
3
4
5
6
7
8
4
kDa
6
kD
66.2 - -
4529-
31 24
23
21.5 - -
-
14.4 - -
Fig. 2. Polypeptides encoded by mcrA constructs. The various mcrA recombinants in the expression vector pT7-1.3 were used to transform E. coli HRll2[pGP1-2], which in turn carries the gene for T7 RNA polymerase under the control of the temperature-sensitive ci857 repressor. The transformants were grown in 20 ml LB containing 50 ~tg Ap/ml and 30 Ixg K m ml at 30°C until A55o was 0.6. Cells from 1.0 ml of culture were harvested, washed thrice in 1 x M9 solution, and resuspended in 1 ml of 1 x M9 solution. The culture was induced by shifting the temperature to 42°C for 30 min. Rifampmm was added to a final concentration of 500 ~tg/ml. and the culture was incubated for additional 15 min before shifting to 30°C. The culture was incubated at 30°C for 30 min. The proteins were labeled with [35S]methionine (20 l~Ci/ml) for 5 min harvested, resuspended in 1 x sample buffer (62.5 mM Tris pH 8/2% SDS/10% glycerol/0.5% 13-mercaptoethanol/0.025% bromophenol blue). The samples were denatured in a boiling water bath for 5 min and analysed by 0.1% SDS-12% PAGE by loading equal counts in each lane. Eventhough there is no chase period in this experiment, the lower molecular mass band could not be due to translational pausing, since we could see only one band in each track. The gel was processed for fluorography using 20%PPO in DMSO. Lanes: 1, pT7-1.3 (control); 2, pDRR551.1 (l.6-kb PstI); 3, pDRR551.2 (l.6-kb PstI); 4, pDRR551.3 (1.0-kb PstI-HpaI); 5, pDRR551.4 (0.7-kb PstI-HindlIl); 6, pDRR551.5 (0.9-kb HindllI-PstI); 7, pDRR551.6 (1.6-kb PstI with the HindlII site mutated); 8, pDRR551.7 (l.0-kb PstI-HpaI with the HindlII site mutated).
absence of transcript m this case is consistent with the lack of McrA + phenotype. The HindIII site mutated construct made two transcripts of sizes 1.3-kb and 0.75-kb (Fig. 3, lanes 2 and 5). The relative abundance of the smaller 0.75-kb transcript could be due to premature termination of transcription. In W3110, the chromosomally located mcrA did not make a detectable level of transcripts, even when a tenfold excess of total RNA was analysed (Fig. 3, lane 6). This could be attributed to either a low transcription rate or to instability of the transcript, or both.
(c) High-resolution transcript mapping Total RNA was isolated from HR112 carrying pRSD1 (1.6-kb mcrA fragment cloned between rrnC and trpA
2.0
-
1.5
-
1.3
-
0.9
-
0.8
-
0.5
iiii
Fig. 3. Total RNA was isolated from HRI12 carrying pBR322 with various mcrA fragments. Low-resolution S1 analysis was carried out as described by Berk and Sharp (1977), with slight modification. Total RNA (25 ~g from recombinants and 100 fag from HRII2 and W3110) was precipitated with 100~tg of ssDNA from 19HL5 (19HL5 is an Ml3mpl9 recombinant carrying the 1.6-kb PstI fragment and the ssDNA from this is complementary to mcrA mRNA). The pellet was air dried and resuspended in 20 lal of hybridisation buffer containing 80% formamide. The sample was incubated at 80°C for 3 min to denature secondary structure and shifted to 47°C for 3 h. The samples were treated with 300 units of nuclease S1 at 37°C for 1 h. The reaction was stopped by the addition of S1 stop solution. The RNA-DNA hybrid was precipitated using cold absolute ethanol, after the addition of carrier tRNA. The pellet was resuspended in 20 p_l of TE and electrophoresed on a 1.2% agarose gel along with DNA digested with HindIIl and EcoRI. The electrophoresis was stopped when the dye reached 3/4 of the length of the gel and transferred to nylon membrane using alkali transfer buffer. The membrane was probed with radiolabeled 1.6-kb PstI fragment of mcrA with high stringency. Lanes: l, pDRR451.3 (1.0-kb PstI-HpaI); 2 and 5, pDRR451.6 (1.6-kb PstI with HindlII site mutated); 3, pDRR451.4 (0.7-kb HindIII-PstI); 4, pDRR451.5 (0.9-kb HindIII-PstI); 6, W3110.
transcription terminators in either direction). A 15-nt primer, homologous to nt 17 to 32 downstream from the ATG start codon, was used to prime synthesis of radiolabeled complementary DNA strands with M-MuLV reverse transcriptase from the RNA template. The strands were separated and loaded onto a sequencing gel along
204 A C GT
TABLE I
PE
Plasmid pPA2 encodes a functional McrA protein Strain a
Efficiency of plating b of LHpalI
Inferred McrA phenotype
BL21 (DE3)[-pT7-7] BL21(DE3)[pPA2]
1 4 x 10 -z
+
a The bacterial cultures were grown in LB medium containing 0.2% maltose and I0 m M MgSO 4 at 37°C, without IPTG. Phage )~HpaII was serially diluted, and 0.1 ml of each dilution was mixed with 0.2 ml of cells and incubated at 37°C for 20 min. Soft agar (4 ml) was added to each tube and the mixture was overlaid on bottom agar plates. Phage )~HpaII is obtained by propagating phage L in a strain carrying M.HpaII methylase clone. b Efficiency of plating was calculated as the ratio of the titer of phage on the test strain to that on the permissive strain HR112. -35
-10
~,~ . . . .
RBS
1TG'rrGcAAITrTATCAATAAAAGTAGTA1"rGTCGTGAAAAA1-rGAITAAAGAIlAATAT slan c,,don Alul TATGCATG i i i i i GATAATAATGGAA'n'GAACTGAAAGCT
ACCTTAACrrGACTr - 5" Fig. 4. High-resolution 5' tsp mapping of mcrA was done as described by Ausubel et al. (1987). Primer extension was done by M-MuLV reverse transcriptase with total RNA preparation from E. coil H R l l 2 carrying pRSD1 as the template source. Plasmid pRSD1 was constructed by cloning the 1.6-kb mcrA BamHI fragment into pFD666 (Denis and Brzezinski, 1992) which possesses rrnC and trpA transcription terminators flanking the multiple cloning site. Primer 5'-TTCAGTTCAATTCCA anneals to a sequence spanning 17 32 nt downstream of the ATG start codon. The newly synthesized strand was labelled by 1-32P]dATP in the reaction. The strands were separated on a 9 M urea-8% polyacrylamide sequencing gel. A sequencing reaction done with the same primer and a mRNA equivalent ss DNA template was loaded alongside. The tsp have been marked with asterisks. Putative - 3 5 , - 1 0 and RBS sequences have been shown. Arrows indicate the two other probable tsp. ATG start codon and primer annealing region are also shown. PE, primer extension.
with a sequence ladder made with the same primer and single stranded DNA template corresponding to mcrA mRNA. Asterisks in Fig. 4 show transcripts originating from C, G, T and G, namely the fourth, fifth, sixth and seventh nt respectively downstream from last nt in a putative - 1 0 sequence. Two other probable tsp have also been shown with arrows. Sequences in the - 10 and - 35 regions are in accordence with the conserved pattern of normal E. coli promoters (Oliphant and Struhl, 1988; Hawley and McClure, 1983).
(d) Overexpression and regulation of mcrA expression Two mcrA translational fusions were made in the expression vector pT7-7, which carries the strong T7 RBS in addition to the T7 promoter. Plasmid pPA2 (Fig. 5a) carries the 1.5-kb AluI-PstI fragment of mcrA gene, cloned between the Sinai and PstI sites of pT7-7. The resulting fusion replaces 13 N-terminal aa from McrA with 6 aa from the pT7.7 vector. The seventh codon (Pro)
is a new one created at the junction. The Plasmid pPN2 (Fig. 5b) carries a 1.3-kb NdeI-PstI fragment of the mcrA gene cloned between the NdeI and PstI sites of pT7-7. This fusion deletes 71 N-terminal aa of McrA and the ORF now starts at the second ATG in the mcrA gene. The polypeptides encoded by these two constructs were labelled with [35S]methionine and analysed on SDSPAGE. Plasmid pPA2 encoded a 29-kDa polypeptide as expected (Fig. 6, lane 2). This 29-kDa McrA polypeptide was found to form inclusion bodies and was visible by Coomassie staining (data not shown). The overexpressed 29-kDa McrA accounted for 4-6% of the total protein based on a densitometer scan of the gel. The McrA phenotype caused by the gene fusion in pPA2 was determined and is shown in Table I. Under uninduced condition due to leaky expression the 29-kDa protein displayed McrA activity but no RglA activity.
(e) Regulation of mcrA expression Quantitative Northern analysis was carried out using total RNA isolated from induced or uninduced cultures of BL21(DE3) carrying pDRR551.1, pPA2 or pPN2. Total RNA from HR112 carrying pBR322 with the 1.6-kb PstI fragment served as control. The results are shown in Fig. 7. Equal amounts of mcrA specific mRNA was made by all the three constructs. This suggested the possibility of regulation at the level of translation, since pPN2 did not make any protein and the 31-kDa McrA from pDRR551.1 is not made in enough quantities to be detected on Coomassie staining (data not shown). Low translational efficiency could be due to either of the following reasons. (i) The native RBS is not strong enough. The proposed RBS sequence for mcrA is 5'-AAGA whereas the consensus sequence found in most of the efficiently expressed proteins is 5'-UAAGGAGG. Gold (1988) reported a threefold higher translational rate in vivo for mRNA containing the sequence
205
Hlnoll
Hincll
/_ ~:~,,,
///
b Z/
~T7
// /
/
/"
/
__
I
C GA'i-r C GAAC'i-rC T C G ATT CG A A C "FI'CT GATAG A C'I'TC(~AAATTA,t, TAC GACT CA C T.8.TAGGG ~ ~
Met AJa Arg
XDal ~ l t I~J
AtIIA AII Pm Qlu ~
R~IS
Ndel
[
1
tie
EcORI
271
Xbal
RB$
NdoI
Fig. 5. The plasmid map of pPA2 (a) and pPN2 (b), with the nt and aa sequence around the fusion region is shown. (a) Plasmid pPA2 was constructed by subjecting the 1.6-kb PstI fragment to partial digestion with AluI and ligating it to pT7-7 (Tabor and Richardson, 1985) cut with Sinai + PstI. Fusion of the first AluI site in the mcrA gene with the SmaI site in pT7-7 maintains the reading frame. This results in deletion of 13 codons from the N terminus of merA and addition of six codons from the vector. In addition, the seventh codon at the junction region codes for a new aa Pro (underlined). The in-frame fusion was verified by sequencing the fusion region. The number denotes aa in fusion products. (b) Plasmid pPN2 was constructed by ligating the 1.3-kb Ndel-PstI fragment of the mcrA gene with pT7-7 cut with NdeI+PstI. This resulted in the fusion of ATG in the ORFII of mcrA gene with that of the vector thus maintaining the reading frame. The numbers denote aa in fusion products.
1
2
INDUCED
3
UNINDUCED
I ! I I I
pDRR 51,
I
I
t ~i~i~ t t
Control t
~
pPA2
l
--29kDa
i
w ,o Control
Fig. 7. Quantitative Northern blot. Total RNA isolated from HR112[pDRR451.1] 11.6 kb, pBR322 construct) and from uninduced and induced culture of BL21(DE3) carrying pDRR551.1, pPA2 or pPN2, was serially diluted (0.325-5.0 lag) in 2 x sample buffer containig 80% formamide. The samples were denatured by keeping at 65°C for 15 min and were then immediately plunged into ice. Two volumes of 20 x SSC were added and the samples were applied to a nitrocellulose membrane, using Hoefer's slot blot apparatus. The membrane was then probed with radiolabelled 1.6-kb mcrA DNA. Arrows show slot blots of 1.6-kb probe DNA control. SSC=0.5 M NaCI/0.015 M Na3.citrate pH 7.6.
!
i !
5'-UAAGGAGG
compared
to t h e m R N A
containing
5 ' - A A G G A as the R B S s e q u e n c e . R i n g q u i s t et al. (1992)
!
h a v e also r e p o r t e d s i m i l a r results for l a c Z e x p r e s s i o n . (ii) T h e R B S is n o t freely accessible for b i n d i n g to t h e r R N A ! Fig. 6. Polypeptides encoded by pPA2 and pPN2 (sse Fig, 5) were analysed using E. coil BL21(DE3), which carries the gene for T7 RNA polymerase under the control of the lacUV5 promoter in an int- prophage. The cultures were grown at 37°C in 1 x M9 medium until A55owas 0.6. A 1-ml culture was induced with IPTG (0.4 raM) for 30 min. Rifampicin was added (500 lag/ml) and incubated for an additional 30 min. after which the proteins were labeled with 20 laCi [ a5S]methionine and analysed by 0.1% SDS-12% PAGE. Lanes: 1, pT7-7; 2, pPA2; 3, pPN2.
b e c a u s e p o s s i b l e s e c o n d a r y s t r u c t u r e s f o r m in the t r a n s l a t i o n a l s t a r t region. It is k n o w n that, a p a r t f r o m the R B S , the nt s e q u e n c e s p r e s e n t u p s t r e a m a n d d o w n s t r e a m f r o m t h e R B S p l a y a r o l e in t r a n s l a t i o n (Steitz, 1975; S c h n e i d e r et al.,
1986;
Bingham
and
Busby,
1987;
Olins
and
R a n g w a l a , 1989). Possible secondary structure around i n i t i a t i o n r e g i o n in m c r A m R N A
the translation
was assessed u s i n g the
206 p r o g r a m F O L D of the G C G package (Devereux et al., 1984). The results are s h o w n in Fig. 8. It is evident that
pressed using the pPA2 construct. The t r u n c a t i o n of the N - t e r m i n a l 13 aa a n d s u b s e q u e n t fusion with 6 aa from
the RBS in p P N 2 m a y be sequestered in the secondary
pT7.7 abolishes RglA activity a n d so does the H i n d I I I
structure, whereas it appears to be freely accessible in pPA2 a n d partially free in pDRR551.1, consistent with
frame-shift m u t a t i o n that occurs at the C - t e r m i n a l end. However, the M c r A p h e n o t y p e is retained in both
the levels of expression obtained.
situations. (5) The expression of m c r A is regulated at the level of
(f) Conclusions (I) The m c r A gene encodes a 31-kDa protein.
translation.
(2) Frame-shift m u t a t i o n at the H i n d l I I
site within
m c r A leads to the p r o d u c t i o n of a t r u n c a t e d polypeptide
ACKNOWLEDGEMENTS
of 23 kDa, a n d the strains carrying these m u t a t i o n s are R g l A - b u t M c r A ÷.
This work was s u p p o r t e d by D e p a r t m e n t of Science
(3) T r a n s c r i p t i o n of the m c r A gene starts from C,G,T a n d G, n a m e l y the fourth, fifth, sixth a n d seventh nt, respectively, d o w n s t r e a m from the last n t of the putative - 10 sequence.
and
Technology,
Government
of
India
grant,
SP/SO/D-50/91. We wish to t h a n k Dr. E,A. Raleigh for the i m p r o v e m e n t of the m a n u s c r i p t a n d
Dr.
M.G.
M a r i n u s for his suggestions.
(4) A t r u n c a t e d (29 kDa) M c r A p r o t e i n can be overex-
REFERENCES
(
o A
( START
<
I
UI G"
START A'(
~"
s__p__D
AAGGA
so
Fig. 8. Secondary structure around the translation initiation region in mcrA mRNA from pDRR551.1, pPA2 and pPN2 was predicted using the program FOLD of the GCG package. The nt sequence from - 32 to + 25 was used for the analysis. The minimum free energy (AG) for the folded structure is calculated using the base stacking and helix loop destabilisingenergies. 1, Wild type, low expression,AG= - 3.2 kcal/mol; 2, pPA2, high expression, AG = -- 3.9 kcal/mol; 3, pPN2, no expression, AG= --4.0 kcal/mol.
Ausubel, F. M., Brent, R., Kingston, R.E., Moore, D.D., Smith, J.A., Seidman, J.G. and Struhl, K.: Current Protocols in Molecular Biology. Wiley, New York, NY, 1987. Berk, A.J. and Sharp, P.A.: Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease digested hybrids. Cell 12 (1977) 721-723. Bingham, A.H.A. and Busby, S.J.W.: Translation of galE and co-ordination of galactose operon expression in Escherichia coil: effects of insertions and deletions in the non-translated leader sequence. Mol. Microbiol. 1 (1987) 117 124. Blumenthal, R.M., Gregory, S.A. and Cooperider, J.S.: Cloning of a restriction-modification system from Proteus vulgaris and its use in analyzinga methylase-sensitive phenotype in Escherichia coli. J. Bacteriol. 164 (1985) 501 509. Denis, F and Brzezinski, R.: A versatile shuttle cosmid vector for use in Escherichia coli and actinomycetes. Gene 111 (1992) 115-118. Devereux, J., Haeberli, P. and Smithies, O.: A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12 (1984) 387-395. Gold, L.: Post-translational regulatory mechanisms in E. coil Annu. Rev. Biochem. 57 (1988) 199-233. Hawley, D.K. and McClure, W.R.: Compilation and analysis of Escherichia coli prometer sequences. Nucleic Acids Res. 11 (1983) 2237 2255. Hiom, K. and Sedgwick, S.G.: Cloning and structural characterization of the mcrA locus of Escherichia coli J. Bacteriol. 173 (1991) 7368-7373. Kelleher, J. and Raleigh, E.A.: A normal activity in Escherichia coli K12 that directs restriction of DNA modified at CG dinucleotides. J. Bacteriol. 173 (1991) 5220-5223. Mott, J.E., Galloway, J.L. and Platt, T.: Maturation of Escherichia coli tryphtophan operon mRNA: evidence for 3' exonuclolytic processing after rho-independent termination. EMBO J. 4 (1985) 1887-1891. Noyer-Weidner, M., Diaz, R. and Reiners, L.: Cytosine specific DNA modification interferes with plasmid establishment in Escherichia coli K-12: involvement of rgIB. Mol. Gen. Genet. 205 (1986) 469 475. Olins, P.O. and Rangwala, S.H.: A novel sequence element derived from
207 bacteriophage T7 mRNA acts as an enhancer of translation of the lacZ gene in Escherichia coll. J. Biol. Chem. 264 (1989) 16973-16976. Oliphant, A.R. and Struhl, K.: Defining the consensus sequences of E. coli promoter elements by random selection. Nucleic Acids Res. 16 (1988) 7673-7683. Raleigh, E.A. and Wilson, G.: Escherichia coli K-12 restricts DNA containing 5-methylcytosine. Proc. Natl. Acad. Sci. USA 83 (1986) 9070-9074. Raleigh, E.A., Trimarchi, R. and Revel, H.: Genetic and physical mapping of the mcrA (rglA) and mcrB (rgIB) loci of Escherichia coil K12. Genetics 122 (1989) 279 296. Ramalingam, R.: Molecular and Recombinant DNA Analysis of the Rgl system in E. coli;II-rglA locus. Ph.D Thesis, Madurai, 1990. Ramalingam, R., Prasad, R., Shivapriya, R. and Dharmalingam, K.: Molecular cloning and sequencing of mcrA locus and identification of McrA protein in Escherichia coll. J. Biosci. 17 (1992) 217 232. Ravi, R.S., Sozhamannan, S. and Dharmalingam, K.: Transposon mutagenesis and genetic mapping of the rglA and rglB loci of Escherichia coll. Mol. Gen. Genet. 198 (1985) 390-392.
Revel, H.R.: Restriction of non-glucosylated T-even bacteriophage: properties of permissive mutants of Escherichia coil B and K-12. Virology 31 (1967) 688 701. Ringquist, S., Shinedling, S., Barrick, D., Louis, G., Binkley, J., Stormo, G.D. and Gold, L.: Translation initiation in Escherichia coli: sequences within the ribosome binding site. Mol. Microbiol. 6 (1992) 1219-1229. Schneider, T.D., Stormo, G.D., Gold, L. and Ehrenfeuchs, A.: Information content of binding sites on nucleotide sequences. J. Mol. Biol. 188 (1986) 415 431. Steitz, J.A.: Ribosome recognition of initiator regions in the RNA bacteriophage genome. In: RNA phages, 1975, pp. 319 352. Tabor, S. and Richardson, C.C.: A bacteriophage T7 RNA polymerase/ promoter system for controlled exclusive expession of specific genes. Proc. Natl. Acad. Sci. USA 82 (1985) 1074 1078. Wong, H.C. and Chang, S.: Identification of a positive retro-regulator that stabilises mRNAs in bacteria. Proc. Natl. Acad. Sci. USA 83 (1986) 3233 3237.
201
GENE 08535
Expression of the mcrA gene of Escherichia coli is regulated posttranscriptionally, possibly by sequestration of the Shine-Dalgarno region* (S1 analysis; primer extension; overexpression; regulation)
R. Shivapriya, Ranjan Prasad, Iyer Lakshmi Narayanan, S. Krishnaswamy and K. Dharmalingam School of Biotechnology, Madurai Kamaraj University, Madurai-625 021, India Received by R.M. Blumenthal: 28 May 1994; Accepted: 20 September 1994; Received at publishers: 20 October 1994
SUMMARY
The polypeptides encoded by the mcrA gene were analysed using a T7 expression system. Cloned fragments of 1.6 and 1.0 kb displayed an McrA +/RglA + phenotype and directed synthesis of a 31-kDa polypeptide. A derivative of these clones altered at an internal HindIII site displayed an McrA +/RglA- phenotype and directed production of a 23-kDa polypeptide. Smaller inserts displayed McrA-/RglA phenotypes, though a 0.7-kb insert did direct production of a 24-kDa polypeptide. A construct carrying the 1.0-kb mcrA insert yielded a single 1.3-kb transcript. The mcrA transcript was found to start from C, G, T and G, namely the fourth, fifth, sixth and seventh nucleotides (nt), respectively, downstream from the last nt of the putative - 10 region. Two mcrA transcriptional/transational fusions were made in the pT7-7 expression vector and the protein encoded by these constructs were analysed. Regulation of mcrA expression was studied by quantitative Northern analysis of RNA from various mcrA clones. Together with a computer analysis of the translation initiation region in these mRNAs, the result~ suggest that the expression of mcrA may be regulated at the translational level.
INTRODUCTION
The Mcr restriction systems of E. coli restrict DNA containing hydroxymethyl cytosine (hmC; Revel, 1967) and methyl cytosine (mC; Blumenthal et al., 1985; Raleigh Correspondence to: Dr. K. Dharmalingam, School of Biotechnology, Madurai Kamaraj University, Madurai-625 021, India. Tel. (91-452) 85-215~ Fax (91-452) 85-205; Telex: 445-337 MKU IN; e-mail: dharm % [email protected] *Presented at the Third New England BioLahs Workshop on Biological DNA Modification, Vilnius, Lithuania, 22 28 May 1994. Abbreviations: A, absorbance (lcm); aa, amino acid(s); Ap, ampicillin; bp, base pair(s); DMSO, dimethylsulfoxide; GCG, Genetics Computer Group (Madison, WI, USA); hmC, hydroxymethylcytosine; IPTG, isopropyl-13-D-thiogalactopyranoside; kb, kilobase(s) or 1000 bp; Kin, kanamycin; nt, nucleotide(s); ORF, open reading frame; RBS, ribosome-binding site(s); SDS-PAGE, sodium dodecylsulfatepolyacrylamide-gel electrophoresis; ss, single strand(ed); Tc, tetracycline; TE, 10 mM Tris.C1/1 mM EDTA pH 7.6; tsp, transcription start point(s); [], denotes plasmid-carrier state. SSDI 0378-1119194)00746-2
and Wilson, 1986; Noyer-Weidner et al., 1986) in specific sequence contexts. The Mcr restriction systems are governed by two loci, mcrA and mcrBC [ Raleigh and Wilson, 1986). The McrA system restricts DNA methylated by M.HpaII (CmCGG; Raleigh and Wilson, 1986) and M.SssI (mCG) (Kelleher and Raleigh, 1991). It also restricts the hmC DNA of all T-even phages (Revel, 1967), though wild-type phages are refractory to restriction as the hmC residues are glucosylated: this restriction of glucose-less phage is known as RglA phenotype. The mcrA gene which has been mapped to 25.24 min on the E. coli chromosome (Ravi et al., 1985; Raleigh et al., 1989) has been sequenced and comprises a 831-bp ORF (Ramalingam, 1990; Hiom and Sedgwick, 1991; Ramalingam et al., 1992). This ORF could encode a polypeptide of 277 aa (31.39 kDa). In this communication the polypeptides from various mcrA constructs were analysed using the T7 expression system. The transcript analysis by primer extension
202 codon introduced by frame shift (Fig. 2, lanes 7 and 8). A slightly larger (24 kDa) polypeptide synthesized by pDRR551.4 could be due to the absence of the stop codon in the insert while the nearest stop codon in the vector is not known, leading to an alternate C terminus.
located the tsp. Comparison of quantitative Northern blots and translation products implied that protein synthesis is possibly regulated during translation. This possibility is supported by theoretical analysis of the secondary structure of mRNA in the translation initiation region.
(b) Low-resolution SI analysis RESULTS AND DISCUSSION
(a) Polypeptides encoded by mcrA Earlier results using mini cells (Ramalingam, 1990) showed that three polypeptides of 29, 18 and 17 kDa were encoded by a 1.6-kb PstI fragment carrying the mcrA gene. In order to confirm this, the T7 promoter expression system was used. Fig. 1 shows the constructs and their McrA/RglA phenotypes. The polypeptides encoded by these constructs are shown in Fig. 2. Plasmids pDRR551.3 and pDRR551.1 produced a single 31-kDa polypeptide (Fig. 2, lanes 2 and 4) as predicted, while pDRR551.2 failed to produce any protein, (Fig. 2, lane 3). These results indicate that the direction of transcription is from left to right as shown in Fig. 1. Plasmid pDRR551.4 yielded a 24-kDa polypeptide (Fig. 2, lane 5), while pDRR551.5 failed to synthesize any polypeptide (Fig. 2, lane 6). Plasmids pDRR551.6 and 551.7 have 4-nt insertional mutation in the H i n d l I l site (cut, filled and ligated) and produce a 23-kDa protein due to the stop
Total RNA isolated from H R l l 2 carrying pBR322 with various mcrA segments were used for analysing the number and size of the transcripts encoded by mcrA. Total RNA including the chromosomal mcrA transcript isolated from W3110 and HR112 were used as controls. In the construct carrying the 1.0-kb mcrA (McrA--, RglA ÷ ) a single transcript of 1.3 kb was detected by the mcrA probe (Fig. 3, lane 1). The size of the hybridising transcript is larger than anticipated, which could be due to the reduced mobility of RNA-DNA hybrid compared to the DNA molecular mass markers. The constructs carrying the 0,7-kb and the 0.9-kb fragments failed to make any detectable rectA-specific transcript (Fig. 3, lanes 3 and 4). It is surprising that the 0.7-kb region did not make any transcript despite having all the promoter sequences intact. Possibly, sequences downstream from the H i n d l I I site have a stabilizing role on the mRNA. It has been suggested by Wong and Chang (1986) and Mott et al. (1985) that specific mRNA structures located downstream from some genes have a stabilizing effect, possibly by acting as barriers to 3' exonuclease activity. The
Phenotype McrA RglA
TTP H I
P
l
P P
m_t ~.~_L
Hp I
P I
pDRR551.1
+
+
P .1 pDRR551,2
÷
+
I
pDRR551.3
+
+
I
pDRR551,4
_
_
I
I pDRR551.5
_
_
I pDRR551.6
+
_
pDRR551,7
+
_
ORF I ORF II Hp I
H
~H I
~tp/Pv
H I ~I
Hp/Pv
I
L
Fig. 1. Restriction map of pDRR551.1 and pDRR551.2, which carry the 1.6-kb PstI fragment in two differentorientations, with respect to the T7 promoter, weremade by ligating the 1.6-kb PstI fragment to pT7-1.3digested with PstI. Plasmid pDRR551.3, carryingthe 1.0-kb PstI-HpaI fragment was constructed by digesting pDRR551.1 with HpaI+ PvulI followed by self ligation. The plasmid pDRR551.4, carrying the 0.7-kb PstI-HindlII region, was constructed by digesting pDRR551.1 with HindlII followed by self ligation. The plasmid pDRR551.5, carrying the 0.9-kb HindlII-Pstl region of rectA, was constructed by ligating the 0.9-kb HindlII fragmentobtained on restrictiondigestion of pDRR451.1 (pBR322 carrying the 1.6-kb mcrA) with HindlII to pT7-1.3 resricted with HindllI and dephosphorylatedusing calf intestinal phosphatase. The plasmid pDRR551.6, carrying the 1.6-kb PstI fragment with a mutation in the HindlII site, was constructed by subjecting pDRR551.1 to partial digestion with HindlII followed by electrophoresisand elution of the full-lengthlinear molecule.The ends were then filledwith dNTPs using Klenow fragment of E. coli DNA polymerase I and ligated. The plasmid pDRR551.7, carrying the 1.0-kb PstI-HpaI region with the HindlII site mutated, was constructed by digesting pDRR551.6 with HpaI+PvulI followedby self ligation. P, Pstl; H, HindlII; H*, Filled-in HindlII site; Hp, HpaI; Pv, PvulI.
203
kDa
1
2
3
4
5
6
7
8
4
kDa
6
kD
66.2 - -
4529-
31 24
23
21.5 - -
-
14.4 - -
Fig. 2. Polypeptides encoded by mcrA constructs. The various mcrA recombinants in the expression vector pT7-1.3 were used to transform E. coli HRll2[pGP1-2], which in turn carries the gene for T7 RNA polymerase under the control of the temperature-sensitive ci857 repressor. The transformants were grown in 20 ml LB containing 50 ~tg Ap/ml and 30 Ixg K m ml at 30°C until A55o was 0.6. Cells from 1.0 ml of culture were harvested, washed thrice in 1 x M9 solution, and resuspended in 1 ml of 1 x M9 solution. The culture was induced by shifting the temperature to 42°C for 30 min. Rifampmm was added to a final concentration of 500 ~tg/ml. and the culture was incubated for additional 15 min before shifting to 30°C. The culture was incubated at 30°C for 30 min. The proteins were labeled with [35S]methionine (20 l~Ci/ml) for 5 min harvested, resuspended in 1 x sample buffer (62.5 mM Tris pH 8/2% SDS/10% glycerol/0.5% 13-mercaptoethanol/0.025% bromophenol blue). The samples were denatured in a boiling water bath for 5 min and analysed by 0.1% SDS-12% PAGE by loading equal counts in each lane. Eventhough there is no chase period in this experiment, the lower molecular mass band could not be due to translational pausing, since we could see only one band in each track. The gel was processed for fluorography using 20%PPO in DMSO. Lanes: 1, pT7-1.3 (control); 2, pDRR551.1 (l.6-kb PstI); 3, pDRR551.2 (l.6-kb PstI); 4, pDRR551.3 (1.0-kb PstI-HpaI); 5, pDRR551.4 (0.7-kb PstI-HindlIl); 6, pDRR551.5 (0.9-kb HindllI-PstI); 7, pDRR551.6 (1.6-kb PstI with the HindlII site mutated); 8, pDRR551.7 (l.0-kb PstI-HpaI with the HindlII site mutated).
absence of transcript m this case is consistent with the lack of McrA + phenotype. The HindIII site mutated construct made two transcripts of sizes 1.3-kb and 0.75-kb (Fig. 3, lanes 2 and 5). The relative abundance of the smaller 0.75-kb transcript could be due to premature termination of transcription. In W3110, the chromosomally located mcrA did not make a detectable level of transcripts, even when a tenfold excess of total RNA was analysed (Fig. 3, lane 6). This could be attributed to either a low transcription rate or to instability of the transcript, or both.
(c) High-resolution transcript mapping Total RNA was isolated from HR112 carrying pRSD1 (1.6-kb mcrA fragment cloned between rrnC and trpA
2.0
-
1.5
-
1.3
-
0.9
-
0.8
-
0.5
iiii
Fig. 3. Total RNA was isolated from HRI12 carrying pBR322 with various mcrA fragments. Low-resolution S1 analysis was carried out as described by Berk and Sharp (1977), with slight modification. Total RNA (25 ~g from recombinants and 100 fag from HRII2 and W3110) was precipitated with 100~tg of ssDNA from 19HL5 (19HL5 is an Ml3mpl9 recombinant carrying the 1.6-kb PstI fragment and the ssDNA from this is complementary to mcrA mRNA). The pellet was air dried and resuspended in 20 lal of hybridisation buffer containing 80% formamide. The sample was incubated at 80°C for 3 min to denature secondary structure and shifted to 47°C for 3 h. The samples were treated with 300 units of nuclease S1 at 37°C for 1 h. The reaction was stopped by the addition of S1 stop solution. The RNA-DNA hybrid was precipitated using cold absolute ethanol, after the addition of carrier tRNA. The pellet was resuspended in 20 p_l of TE and electrophoresed on a 1.2% agarose gel along with DNA digested with HindIIl and EcoRI. The electrophoresis was stopped when the dye reached 3/4 of the length of the gel and transferred to nylon membrane using alkali transfer buffer. The membrane was probed with radiolabeled 1.6-kb PstI fragment of mcrA with high stringency. Lanes: l, pDRR451.3 (1.0-kb PstI-HpaI); 2 and 5, pDRR451.6 (1.6-kb PstI with HindlII site mutated); 3, pDRR451.4 (0.7-kb HindIII-PstI); 4, pDRR451.5 (0.9-kb HindIII-PstI); 6, W3110.
transcription terminators in either direction). A 15-nt primer, homologous to nt 17 to 32 downstream from the ATG start codon, was used to prime synthesis of radiolabeled complementary DNA strands with M-MuLV reverse transcriptase from the RNA template. The strands were separated and loaded onto a sequencing gel along
204 A C GT
TABLE I
PE
Plasmid pPA2 encodes a functional McrA protein Strain a
Efficiency of plating b of LHpalI
Inferred McrA phenotype
BL21 (DE3)[-pT7-7] BL21(DE3)[pPA2]
1 4 x 10 -z
+
a The bacterial cultures were grown in LB medium containing 0.2% maltose and I0 m M MgSO 4 at 37°C, without IPTG. Phage )~HpaII was serially diluted, and 0.1 ml of each dilution was mixed with 0.2 ml of cells and incubated at 37°C for 20 min. Soft agar (4 ml) was added to each tube and the mixture was overlaid on bottom agar plates. Phage )~HpaII is obtained by propagating phage L in a strain carrying M.HpaII methylase clone. b Efficiency of plating was calculated as the ratio of the titer of phage on the test strain to that on the permissive strain HR112. -35
-10
~,~ . . . .
RBS
1TG'rrGcAAITrTATCAATAAAAGTAGTA1"rGTCGTGAAAAA1-rGAITAAAGAIlAATAT slan c,,don Alul TATGCATG i i i i i GATAATAATGGAA'n'GAACTGAAAGCT
ACCTTAACrrGACTr - 5" Fig. 4. High-resolution 5' tsp mapping of mcrA was done as described by Ausubel et al. (1987). Primer extension was done by M-MuLV reverse transcriptase with total RNA preparation from E. coil H R l l 2 carrying pRSD1 as the template source. Plasmid pRSD1 was constructed by cloning the 1.6-kb mcrA BamHI fragment into pFD666 (Denis and Brzezinski, 1992) which possesses rrnC and trpA transcription terminators flanking the multiple cloning site. Primer 5'-TTCAGTTCAATTCCA anneals to a sequence spanning 17 32 nt downstream of the ATG start codon. The newly synthesized strand was labelled by 1-32P]dATP in the reaction. The strands were separated on a 9 M urea-8% polyacrylamide sequencing gel. A sequencing reaction done with the same primer and a mRNA equivalent ss DNA template was loaded alongside. The tsp have been marked with asterisks. Putative - 3 5 , - 1 0 and RBS sequences have been shown. Arrows indicate the two other probable tsp. ATG start codon and primer annealing region are also shown. PE, primer extension.
with a sequence ladder made with the same primer and single stranded DNA template corresponding to mcrA mRNA. Asterisks in Fig. 4 show transcripts originating from C, G, T and G, namely the fourth, fifth, sixth and seventh nt respectively downstream from last nt in a putative - 1 0 sequence. Two other probable tsp have also been shown with arrows. Sequences in the - 10 and - 35 regions are in accordence with the conserved pattern of normal E. coli promoters (Oliphant and Struhl, 1988; Hawley and McClure, 1983).
(d) Overexpression and regulation of mcrA expression Two mcrA translational fusions were made in the expression vector pT7-7, which carries the strong T7 RBS in addition to the T7 promoter. Plasmid pPA2 (Fig. 5a) carries the 1.5-kb AluI-PstI fragment of mcrA gene, cloned between the Sinai and PstI sites of pT7-7. The resulting fusion replaces 13 N-terminal aa from McrA with 6 aa from the pT7.7 vector. The seventh codon (Pro)
is a new one created at the junction. The Plasmid pPN2 (Fig. 5b) carries a 1.3-kb NdeI-PstI fragment of the mcrA gene cloned between the NdeI and PstI sites of pT7-7. This fusion deletes 71 N-terminal aa of McrA and the ORF now starts at the second ATG in the mcrA gene. The polypeptides encoded by these two constructs were labelled with [35S]methionine and analysed on SDSPAGE. Plasmid pPA2 encoded a 29-kDa polypeptide as expected (Fig. 6, lane 2). This 29-kDa McrA polypeptide was found to form inclusion bodies and was visible by Coomassie staining (data not shown). The overexpressed 29-kDa McrA accounted for 4-6% of the total protein based on a densitometer scan of the gel. The McrA phenotype caused by the gene fusion in pPA2 was determined and is shown in Table I. Under uninduced condition due to leaky expression the 29-kDa protein displayed McrA activity but no RglA activity.
(e) Regulation of mcrA expression Quantitative Northern analysis was carried out using total RNA isolated from induced or uninduced cultures of BL21(DE3) carrying pDRR551.1, pPA2 or pPN2. Total RNA from HR112 carrying pBR322 with the 1.6-kb PstI fragment served as control. The results are shown in Fig. 7. Equal amounts of mcrA specific mRNA was made by all the three constructs. This suggested the possibility of regulation at the level of translation, since pPN2 did not make any protein and the 31-kDa McrA from pDRR551.1 is not made in enough quantities to be detected on Coomassie staining (data not shown). Low translational efficiency could be due to either of the following reasons. (i) The native RBS is not strong enough. The proposed RBS sequence for mcrA is 5'-AAGA whereas the consensus sequence found in most of the efficiently expressed proteins is 5'-UAAGGAGG. Gold (1988) reported a threefold higher translational rate in vivo for mRNA containing the sequence
205
Hlnoll
Hincll
/_ ~:~,,,
///
b Z/
~T7
// /
/
/"
/
__
I
C GA'i-r C GAAC'i-rC T C G ATT CG A A C "FI'CT GATAG A C'I'TC(~AAATTA,t, TAC GACT CA C T.8.TAGGG ~ ~
Met AJa Arg
XDal ~ l t I~J
AtIIA AII Pm Qlu ~
R~IS
Ndel
[
1
tie
EcORI
271
Xbal
RB$
NdoI
Fig. 5. The plasmid map of pPA2 (a) and pPN2 (b), with the nt and aa sequence around the fusion region is shown. (a) Plasmid pPA2 was constructed by subjecting the 1.6-kb PstI fragment to partial digestion with AluI and ligating it to pT7-7 (Tabor and Richardson, 1985) cut with Sinai + PstI. Fusion of the first AluI site in the mcrA gene with the SmaI site in pT7-7 maintains the reading frame. This results in deletion of 13 codons from the N terminus of merA and addition of six codons from the vector. In addition, the seventh codon at the junction region codes for a new aa Pro (underlined). The in-frame fusion was verified by sequencing the fusion region. The number denotes aa in fusion products. (b) Plasmid pPN2 was constructed by ligating the 1.3-kb Ndel-PstI fragment of the mcrA gene with pT7-7 cut with NdeI+PstI. This resulted in the fusion of ATG in the ORFII of mcrA gene with that of the vector thus maintaining the reading frame. The numbers denote aa in fusion products.
1
2
INDUCED
3
UNINDUCED
I ! I I I
pDRR 51,
I
I
t ~i~i~ t t
Control t
~
pPA2
l
--29kDa
i
w ,o Control
Fig. 7. Quantitative Northern blot. Total RNA isolated from HR112[pDRR451.1] 11.6 kb, pBR322 construct) and from uninduced and induced culture of BL21(DE3) carrying pDRR551.1, pPA2 or pPN2, was serially diluted (0.325-5.0 lag) in 2 x sample buffer containig 80% formamide. The samples were denatured by keeping at 65°C for 15 min and were then immediately plunged into ice. Two volumes of 20 x SSC were added and the samples were applied to a nitrocellulose membrane, using Hoefer's slot blot apparatus. The membrane was then probed with radiolabelled 1.6-kb mcrA DNA. Arrows show slot blots of 1.6-kb probe DNA control. SSC=0.5 M NaCI/0.015 M Na3.citrate pH 7.6.
!
i !
5'-UAAGGAGG
compared
to t h e m R N A
containing
5 ' - A A G G A as the R B S s e q u e n c e . R i n g q u i s t et al. (1992)
!
h a v e also r e p o r t e d s i m i l a r results for l a c Z e x p r e s s i o n . (ii) T h e R B S is n o t freely accessible for b i n d i n g to t h e r R N A ! Fig. 6. Polypeptides encoded by pPA2 and pPN2 (sse Fig, 5) were analysed using E. coil BL21(DE3), which carries the gene for T7 RNA polymerase under the control of the lacUV5 promoter in an int- prophage. The cultures were grown at 37°C in 1 x M9 medium until A55owas 0.6. A 1-ml culture was induced with IPTG (0.4 raM) for 30 min. Rifampicin was added (500 lag/ml) and incubated for an additional 30 min. after which the proteins were labeled with 20 laCi [ a5S]methionine and analysed by 0.1% SDS-12% PAGE. Lanes: 1, pT7-7; 2, pPA2; 3, pPN2.
b e c a u s e p o s s i b l e s e c o n d a r y s t r u c t u r e s f o r m in the t r a n s l a t i o n a l s t a r t region. It is k n o w n that, a p a r t f r o m the R B S , the nt s e q u e n c e s p r e s e n t u p s t r e a m a n d d o w n s t r e a m f r o m t h e R B S p l a y a r o l e in t r a n s l a t i o n (Steitz, 1975; S c h n e i d e r et al.,
1986;
Bingham
and
Busby,
1987;
Olins
and
R a n g w a l a , 1989). Possible secondary structure around i n i t i a t i o n r e g i o n in m c r A m R N A
the translation
was assessed u s i n g the
206 p r o g r a m F O L D of the G C G package (Devereux et al., 1984). The results are s h o w n in Fig. 8. It is evident that
pressed using the pPA2 construct. The t r u n c a t i o n of the N - t e r m i n a l 13 aa a n d s u b s e q u e n t fusion with 6 aa from
the RBS in p P N 2 m a y be sequestered in the secondary
pT7.7 abolishes RglA activity a n d so does the H i n d I I I
structure, whereas it appears to be freely accessible in pPA2 a n d partially free in pDRR551.1, consistent with
frame-shift m u t a t i o n that occurs at the C - t e r m i n a l end. However, the M c r A p h e n o t y p e is retained in both
the levels of expression obtained.
situations. (5) The expression of m c r A is regulated at the level of
(f) Conclusions (I) The m c r A gene encodes a 31-kDa protein.
translation.
(2) Frame-shift m u t a t i o n at the H i n d l I I
site within
m c r A leads to the p r o d u c t i o n of a t r u n c a t e d polypeptide
ACKNOWLEDGEMENTS
of 23 kDa, a n d the strains carrying these m u t a t i o n s are R g l A - b u t M c r A ÷.
This work was s u p p o r t e d by D e p a r t m e n t of Science
(3) T r a n s c r i p t i o n of the m c r A gene starts from C,G,T a n d G, n a m e l y the fourth, fifth, sixth a n d seventh nt, respectively, d o w n s t r e a m from the last n t of the putative - 10 sequence.
and
Technology,
Government
of
India
grant,
SP/SO/D-50/91. We wish to t h a n k Dr. E,A. Raleigh for the i m p r o v e m e n t of the m a n u s c r i p t a n d
Dr.
M.G.
M a r i n u s for his suggestions.
(4) A t r u n c a t e d (29 kDa) M c r A p r o t e i n can be overex-
REFERENCES
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Fig. 8. Secondary structure around the translation initiation region in mcrA mRNA from pDRR551.1, pPA2 and pPN2 was predicted using the program FOLD of the GCG package. The nt sequence from - 32 to + 25 was used for the analysis. The minimum free energy (AG) for the folded structure is calculated using the base stacking and helix loop destabilisingenergies. 1, Wild type, low expression,AG= - 3.2 kcal/mol; 2, pPA2, high expression, AG = -- 3.9 kcal/mol; 3, pPN2, no expression, AG= --4.0 kcal/mol.
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