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 post-

transcriptionally, 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 and Wilson, 1986; Noyer-Weidner et al., 1986) in specific

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 % bic-mku@dbt.ernet.in
*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 dodecylsulfate-
polyacrylamide-gel electrophoresis; ss, single strand(ed); Tc, tetracy-
cline; TE, 10 mM Tris.C1/1 mM EDTA pH 7.6; tsp, transcription start
point(s); [], denotes plasmid-carrier state.
sequence contexts. The Mcr restriction systems are gov-
erned 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 restric-
tion 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 poly-
peptide 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

SSDI 0378-1119194)00746-2
202

located the tsp. Comparison of quantitative Northern
blots and translation products implied that protein syn-
thesis is possibly regulated during translation. This possi-
bility is supported by theoretical analysis of the
secondary structure of mRNA in the translation initia-
tion region.
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 transcrip-
tion 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
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.
(b) Low-resolution SI analysis
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 car-
rying 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 down-
stream from some genes have a stabilizing effect, possibly
by acting as barriers to 3' exonuclease activity. The

TTP Phenotype
P H Hp P McrA RglA
I I I pDRR551.1 + +

l ORF I

ORF II ~H
P Hp P
I I .1 pDRR551,2 ÷ +
P H ~tp/Pv
I pDRR551.3 + +

I pDRR551,4 _ _

I I pDRR551.5 _ _
H
m_t I I pDRR551.6 + _
~I Hp/Pv
~.~_L I L pDRR551,7 + _

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.

1 2 3 4 5 6 7 8
kDa kDa
66.2 - -
45-
31
29-
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 repres-
sor. 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 temper-
ature 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 con-
struct 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 ter-
mination of transcription. In W3110, the chromosomally
located mcrA did not make a detectable level of tran-
scripts, 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
203
4 6
kD
- 2.0
- 1.5
- 1.3
- 0.9
iiii
- 0.8
- 0.5
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 dena-
ture 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 electro-
phoresed 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 radiola-
beled 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 PE
-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 con-
structed by cloning the 1.6-kb mcrA BamHI fragment into pFD666
(Denis and Brzezinski, 1992) which possesses rrnC and trpA trans-
cription 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 puta-
tive - 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)
TABLE I
Plasmid pPA2 encodes a functional McrA protein
Strain a Efficiency of plating b Inferred McrA
of LHpalI phenotype
BL21 (DE3)[-pT7-7] 1
BL21(DE3)[pPA2] 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.
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 SDS-
PAGE. 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 possi-
bility 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

Hincll Hlnoll

/_ ~:~,,,

/
/
/"
//
/// ~T7

__
b Z/ [
/ I 1
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 tie

XDal R~IS Ndel EcORI

AtIIA AII Pm Qlu ~ ~ l t I~J

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 3 INDUCED UNINDUCED
I ! I I I I pDRR
51,
I
t ~i~i~ t t Control
t ~ pPA2

l 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
--29kDa 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
!

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
! 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 -
Fig. 6. Polypeptides encoded by pPA2 and pPN2 (sse Fig, 5) were ana- 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 ,
lysed using E. coil BL21(DE3), which carries the gene for T7 RNA poly- 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
merase under the control of the lacUV5 promoter in an int- prophage. 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
The cultures were grown at 37°C in 1 x M9 medium until A55owas 0.6. A et al., 1986; Bingham and Busby, 1987; Olins and
1-ml culture was induced with IPTG (0.4 raM) for 30 min. Rifampicin
R a n g w a l a , 1989).
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 Possible secondary structure around the translation
0.1% SDS-12% PAGE. Lanes: 1, pT7-7; 2, pPA2; 3, pPN2. 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 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
the RBS in p P N 2 m a y be sequestered in the secondary
structure, whereas it appears to be freely accessible in
pPA2 a n d partially free in pDRR551.1, consistent with
the levels of expression obtained.
(f) Conclusions
(I) The m c r A gene encodes a 31-kDa protein.
(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
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 ÷.
(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.
(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-
( o
A Biology. Wiley, New York, NY, 1987.
( sequence. Mol. Microbiol. 1 (1987) 117 124.
I G"
U-
START START A'( ~"
<
I 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.
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
pT7.7 abolishes RglA activity a n d so does the H i n d I I I
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
situations.
(5) The expression of m c r A is regulated at the level of
translation.
ACKNOWLEDGEMENTS
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
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.
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