1. PLASMID 6, 112-118 (1981)
Construction and Fine Mapping of Recombinant Plasmids Containing
the rrnB Ribosomal RNA Operon of E. co/i
JCJRGEN BROSIUS,*~~ AXEL ULLRICH,~‘ MARY ALICE RAKER,* ALANE GRAY,?
THOMAS J. DULL,*3 ROBIN R. GUTELL,* AND HARRY F. NOLLER*
*Thimann Laboratories, University of Californiu. Sunta Cruz, CaliJornia 95064, and Wenentech. Inc. I
460 Point San Bruno Boulevard, South San Fruncisco, Culijornia 94080
Received October 28, 1980: revised February 25, 1981
We have constructed recombinant plasmids containing the entire Escherichiu co/i
rrnB ribosomal RNA operon and segments thereof. Cloning of the 7.5kb BamHI frag-
ment, from Artin which contains this operon, in plasmid vectors pBR 313 or pBR 322
is described. The 3.2-kb EcoRIIBamHI fragment containing the 3’ two-thirds of the 23 S
rRNA gene, the SS rRNA gene, and the terminator region has been cloned separately
in pBR 313. As the nucleotide sequences of pBR 322 and the 7.5kb fragment carrying the
rrnB operon have been established, the entire 11.9-kb sequence of pKK 3535 is now known.
This makes possible precise rearrangements and site-specific alterations of the ribosomal
RNA operon; thus, pKK 3535 becomes a powerful tool for studies such as initiation
and termination of transcription, processing of rRNA precursors, and investigations of
the structure, function, and assembly of the ribosome itself. A detailed physical map
of pKK 3535 is presented.
Initially, our rationale in cloning rRNA
genes from Escherichia coli was the de-
termination of the 16 S RNA and 23 S RNA
primary structures (Brosius et al., 1978,
1980). Subsequently, sequences of tran-
scriptional signals and spacer regions flank-
ing rRNA and tRNA genes in rrnB and
their comparison with homologous regions
of other rRNA operons sequenced in other
laboratories have raised questions concern-
ing aspects of initiation and termination of
transcription, as well as steps involved in
the processing of primary transcripts. A po-
tential use of the recombinant plasmids
described here, especially of pKK 3535
for which we now know the entire nucleotide
sequence, lies in specific alteration of func-
tional sequences and subsequent in vitro
and in viva analysis of the impact on: (a)
the control of expression of a ribosomal
RNA operon, (b) processing mechanisms for
1 Present address: The Biological Laboratories, Har-
vard University, 16 Divinity Ave., Cambridge, Mass.
02138.
rRNAs and tRNAs, and (c) the structure,
function, and assembly of the ribosome.
MATERIALS AND METHODS
Isolation of DNA
Strains harboring plasmids pBR 322, pBR
313, and pTUB 2 were kindly provided by
R. Rodriguez, M. Betlach, and Y. Kaziro,
respectively. Plasmid DNA was prepared
in CsCl-ethidium bromide buoyant density
gradients (Clewell, 1972). Bacteriophage
hrifd18 was isolated from an E. cofi K-12
strain by Kirschbaum and Konrad (1973).
Phage DNA was a gift from R. Young.
Restriction Enzyme Digestion
Most of the enzymes were purchased
from New England Bio-Labs or from
Bethesda Research Laboratories. EcoRI
was purified as described by Palmer et al.
(1979). Reactions were carried out under
the conditions recommended by the suppliers.
0147-619X/81/040112-07$02.00/0
Copyright 0 1981 by Academic Press, Inc.
AU rights of reproduction in soy form reserved.
112

2. rRNA OPERON PLASMIDS 113
Ligation, Transformation, and Selection of
Recombinant Plasmid-Containing Cells
Ligation was carried out on appropriately
digested and phenol-extracted DNA under
the conditions of Sgaramella (1972) as de-
scribed by Palmer et al. (1979). For the con-
struction of pKK 123 we ligated 2 pg of
EcoRIIBamHI-digested pTUB 2 DNA, which
carries the 18.6% EcoRI fragment of Xrifd18
in pRSF 2124 (Miyajima et al., 1979) with
1 pg of pBR 313 vector DNA cut with the
same enzymes using 1.5 units of T4 DNA
ligase (Bethesda Research Laboratories)
at 12°C overnight. For construction of pKK
2361, 2.4 pg of BamHI-cut hrifd 18 DNA
was ligated with 1 pg of BamHI-cut pBR
313 DNA under the same conditions. Plas-
mid pKK 3535 was obtained by mixing 1
pg BamHI-cut pKK 2361 DNA with 2 kg
of BamHI-cut and bacterial alkaline phos-
phatase (Sigma)-treated pBR 322 DNA
(Ullrich et al., 1977). After ligation the DNA
was ethanol precipitated and dissolved in 10
mM Tris/HCl, pH 7.5, 5 mM MgC&, 50 mM
CaCl,, and 200 ~1 cells were transformed
with 0.2 pg of DNA according to the pro-
cedure of Bolivar et al. (1977b). E. coli
strains RR1 (F-pro leu thi lacy Str’ r,-
mk- endoII) (Bolivar et al., 1977a) and
HBlOl (F- pro lea thi lacy Str’ r,- mB-
endoI-, recA-) (Boyer and Roulland-Dus-
soix, 1969) were used as recipients for
the recombinant plasmids.
After transformation the cells were selected
on Luria broth plates, containing 20 E.Lg/ml
ampicillin and picked onto Luria broth
plates containing 10 pg/ml tetracycline.
Ampicillin-resistant and tetracycline-sensi-
tive colonies were screened for recombinant
plasmids of increased size (Barnes, 1977)
and small amounts of plasmid DNA were
isolated according to Meagher et al. (1977)
for further characterization by restriction
enzyme mapping. Fragmented DNA was
analyzed on 1% horizontal agarose gels or
6 or 8% polyacrylamide gels as described
elsewhere (Palmer et al., 1979).
Preparation of Plasmid inserts
Inserts from recombinant plasmids were
separated from their vector DNA by su-
crose gradient centrifugation as described
by Valenzuela et al. (1977).
RESULTS AND DISCUSSION
pKK 123
The BamHIIEcoRI-digested DNA from
plasmid pTUB 2 (Miyajima et al., 1979)
containing the 3’ two-thirds of the 23 S RNA
gene on the 18.6% EcoRI fragment of Arifd 18
(Lindahl et al., 1977) was ligated to pBR
313 digested with the same enzymes. We
chose the larger plasmid pBR 313 (9.2 kb)
over pBR 322 (4.3 kb) as vector because
the desired 3.2-bp insert containing part of
the 23 S RNA gene is then more easily re-
solved from the vector DNA by sucrose
gradient centrifugation. E. coli strain RR1
was transformed. Out of 50 ampicillin-
resistant colonies, 14 were tetracycline
sensitive. We isolated the DNA from 8 colo-
nies in a “miniscreen” procedure (Meagher
et al., 1977). Four out of eight samples,
which were double digested with BamHI
and EcoRI, carried the 3.2-kb fragment
and three carried fragments in the size range
of 5-6 kb, which are probably the larger
BamHIIEcoRI fragment from the 18.6%
EcoRI fragment from tiz?lS carried by
pTUB 2 or a BamHIIEcoRI fragment from
the pRSF 2124 vector used for the con-
struction of pTUB 2 (Miyajima et al., 1979).
One plasmid, pKK 123, carries the 3.2-bp
fragment, shown schematically in Fig. 1.
Bernardi and Bernardi (1979) have inde-
pendently constructed a plasmid (pAB 99)
which carries the same 3.2-bp fragment
inserted in pBR 322.
pKK 2361
Because of low yields of plasmid DNA
from strains carrying plasmid PER 24
(Palmer et al., 1979), which contains the
promoter region of the rrnB operon, we at-

3. 114 BROSIUS ET AL.
0 I 2 3 4 5 6 7 6 kbp
I L 1 I ,1 1I r, 4
1
Ram HI
I A t t
Hindm Eco RI Eco RI Barn HI
pER24
I
I I I
pERi pKK 123
I
pKK 2361, pKK 3535
pKK 116
FIG. 1. Schematic of the region of A$‘18 used in these studies. the orientation of the phage DNA
map is reversed from the usual convention, to show the rRNA operon in its conventional orientation.
Wild-type A sequence is shown by hatching , and mature rRNA sequence is shown by black bars. The
scale shows DNA length in kilobase pairs. Sites of restriction enzyme cleavage used in cloning are
shown. Open bars at the bottom show the cloned segments present in the recombinant plasmids. The
pER24 and pER18 segments were cloned in Co1 El (Palmer et al., 1979), pKKll5 in pBR322
(Brosius et nl., 1978), pKK123 and pKK2361 in pBR313, and pKK3535 in pBR322 (cf. Fig. 2).
The orientation of the insert in pKK2361 is analogous to that of pKK3.535. P, and P, are the two
tandem rRNA promoters for rmB. T, and T, are putative transcriptional terminators (Brosius et al.,
1981).
tempted to clone the entire vrnB operon,
which is included in the 7.5kb BumHI frag-
ment of transducing phage hrifd 18 (Boros
and Sain, 1977). We initially chose pBR
313 as vector, because its larger size facili-
tates the isolation of the desired BarnHI/
EcoRI fragment (2189 bp), or the BamHIl
Hind111 fragment (1596 bp), carrying the
rrnB promoter region. After ligation of a
mixture of the Xrifdl8 BumHI fragments
with pBR 313, we transformed E. coli strain
HBlOl and screened colonies for plasmids
of the predicted size range (Barnes, 1977).
Plasmid DNA from eight such colonies was
isolated by the “miniscreen” procedure
(Meagher et al., 1977). The plasmids were
digested with Hind111 and the resulting
fragments were electrophoresed on a 6%
polyacrylamide gel to identify plasmids con-
taining the unique 0.6-kb Hind111 fragment
located at the 5’ end of the 16 S RNA gene
of the rrnB operon (Brosius et al., 1978).
Three transformants contained the fragment
and were further tested by digestion of
plasmid DNA with EcoRI or double diges-
tion with EcoRI/BumHI orHindIIIIBamH1.
The resulting fragments were electrophoresed
on a 1% agarose gel with size markers
including the 2.2-kb insert from PER 18
(Palmer et al., 1979), and the 3.2-kb insert
from pKK 123 (not shown). Plasmid DNA
from the tested colonies gave rise to the
predicted fragments, indicating that all three
contain the 7.5kb BumHI fragment from
kifd18, carrying the entire rrnB operon in
the same orientation with respect to the
vector.
One of the colonies (containing plasmid
pKK 2361) was grown in supplemented
M9-glucose medium (Bolle et al., 1968).
We obtain this plasmid in a yield of about
2 mg/liter. There is no indication of segrega-
tion of the plasmid as in the case of pER
24 (Palmer et al., 1979).

4. SacII
Sac II 1000
:k
Sac II
Bal I
Xma III
115rRNA OPERON PLASMIDS
Pst I
Tth 111 I
FIG. 2. Schematic map of hybrid plasmid pKK 3535. Positions of vector DNA or inserts from other
plasmids carrying parts of the rrnB operon are indicated on the inner circle. The A portion of the
7.5kb fragment is hatched. The genes for the rRNAs and tRNA2’” are represented by filled bars. Two
open reading frames (ORF I and ORF II) flanking the rrnB operon are indicated. The tandem rRNA
promoters PI and P2 and their sites of initiation of transcription are indicated by arrows. A putative
promoter proximal to ORF II is indicated as PORFii. Putative terminators for the rrnB operon are
marked as Tl and T2. The ampicillin and tetracycline genes (the latter is interrupted by the 7.5-kb
BamHI insert) of plasmid vector pBR 322 are dotted. The direction of transcription is indicated
by arrows under the genes. The location of these landmarks and the location of restriction
enzyme (those which recognize a sequence of six nucleotides) sites are based on the known sequence
of pKK 3535 via the primary structures of pBR 322 (Sutcliffe, 1978a) and the 7.5-kb insert (Brosius
et al., 1981). Locations of all but the sites AvrII, &/I, BclI, ClaI, SphI, Trh 1111, and XmaIII have
been confirmed by digestion of pKK 3535 with various enzymes (see Fig. 2; Table 1).
The identity of the plasmid insert was parently identical to pKK 2361 (also with
further confirmed by restriction mapping respect to the orientation of the insert).
with restriction enzymes recognizing se-
quences of four or five nucleotides and by
DNA sequencing (Brosius et al., 1981).
pKK 3535
Kiss et al. (1978) have independently As the nucleotide sequence of both the
constructed a plasmid (2/12) which is ap- 7.5kb insert of pKK 2361 and the 4.3-kb

5. 116 BROSIUS ET AL.
plasmid pBR 322 have recently become
available (Brosius et al., 1981; Sutcliffe,
1978a) it was desirable to subclone the 7.5
kb BarnHI fragment from pKK 2361 into
theBamH1 site of pBR 322. After ligation E.
coli strain HBlOl was transformed and
ampicillin-resistant, tetracycline-sensitive
colonies were screened for plasmids with
the predicted size (Barnes, 1977). Three
out of twelve transformants were further
tested by digestion of their plasmid DNA
with EcoRI. In all cases three fragments
(6.2, 3.5, and 2.2 kb) were resolved on a
1% agarose gel, indicating that the 7.5kb
fragment carrying the rrnB operon was
cloned into vector pBR 322 in the same
orientation as in pKK 2361 (see Fig. 2 for
the detailed physical map of pKK 3535).
Cells from one of the three positive colonies
were grown and plasmid pKK 3535 was iso-
abcdefghi jk lmnopq
FIG. 3. Restriction patterns resolved on a 1% agarose
gel of pKK 3535 digested with various enzymes:
Lanes (a) BarnHI; (b) uncut pKK 3535 (c) Egll; (d)
BglII: (e) BstEII; (f) EcoRI: (g) HindIII; (h) HpI:
(i) PstI; (j) PvuI; (k) PruII: (1) SalI: (m) SmuI: (n)
WI: (0) SacII; (p) XbaI; (q) hDNA digested with
HindIII. The patterns in lanes (a) and(k) reveal in addi-
tion to the expected fragments linear pKK 3535 DNA
due to incomplete digestion. The smear in lane (m) is
caused by exonuclease activity present in the SmaI
preparation. The larger of the expected bands dis-
appeared completely, while the smaller 0.77-kb frag-
ment is still visible. Lane (c) shows the expected
restriction pattern of A DNA cut with HindIII; the
additional band above the 23.7-kb band and the low
yield of the 4.3-kb band is due to the cohesive ends of
ADNA. Fragments smaller than 0.5 kb are not visible.
TABLE 1
SIZES OF FRAGMENTS GENERATED BY DIGESTION
OF pKK3535 DNA WITH VARIOUS
RESTRICTION ENZYMES”
Fragment size
W)
BamHI 7.5, 4.4
BglI 5.0, 2.9, 2.3, 1.4 (0.2)
BglII 11.9
Bst EII 11.9
EcoRI 6.2, 3.5, 2.2
Hind111 5.7, 5.6, 0.6
HpaI 11.9
PSI 8.3, 3.5
PVUI 11.9
PVUII 7.0, 4.9
SulI 6.8, 2.6, 2.5 (0.1)
SmClI 11.0, 0.8
SstI 11.9
Sac11 6.8, 3.5, 1.4 (0.2)
XbaI 11.9
Hind111 (ADNA) 23.7,9.5,6.7,4.3,2.3,2.0(0.6)(0.1)
fl Numbers in parentheses represent predicted frag-
ments not visible on the gel (Fig. 3) due to small size.
lated. The yield of pKK 3535 is about 0.2
mg/liter, much lower than that of pKK 2361.
However we do not observe segregation
of the plasmid. The basis for the differ-
ence in yield between pKK 2361 and pKK
3535 is unknown.
DNA from plasmid pKK 3535 was further
analyzed by digestion with BarnHI, Bgll,
BglII, BstEII, EcoRI, HindIII, HpaI, PstI,
PvuI, PvuII, SacII, SalI, SstI, and XbaI
on a 1% agarose gel (Fig. 3). The resulting
fragments are summarized in Table 1 and
are in complete agreement with the nucleo-
tide sequence (11,864 bp) of pKK 3535.
The 7.5-kb insert of pKK 3535 and various
subfragments thereof (isolated in part from
other described plasmids) have been ex-
tensively mapped with about 35 restriction
enzymes, mainly as a prerequisite for gen-
erating fragments for DNA sequence de-
termination. The results are in accord with
the nucleotide sequence of the 7.5-kb insert
(Brosius et al., 198l), except where the

6. rRNA OPERON PLASMIDS 117
digest pattern indicates that predicted sites
remain uncut. These sites include in the
7508bp fragment: (a) HphI at positions
3049, 4180, and 7098; (b) TaqI at position
5385; and (c) Sau96I and AvaII at position
7419. In cases (a) and (b) an MboI site
(G-,A-T-C) overlaps the HphI sites (G-
G-T-G-A or T-C-A-C-C) or the TuqI
site (T-C-G-A) thus including the methyl-
ated A residue in the HphI or TuqI recog-
nition site. This is a likely reason for the
failure of these enzymes to cut if the DNA,
as in our case, is isolated from a dum+,dcm+
strain. Digestion at a much slower rate was
also observed at the TuqI site at position
1125 of pBR 322 (G. Sutcliffe, personal com-
munication), which is similarly overlapped
by an MboI site. In case (c) an EcoRII site
(C-,C-$-G-G) overlaps the Suu961 or
AvuII site (G-G-N-C-C/G-G-$-C-C)
including the methylated C residue in their
recognition site. It has been described pre-
viously (Sutcliffe and Church, 1978) that the
AvuII site at position 1438 of pBR 322 is
digested at a rate about 10 times slower
than the remaining AvuII sites. The Suu961
site at position 3247 of the 7.5-kb fragment,
which is also overlapped by an EcoRII site,
has not yet been tested by enzymatic
digestion.
For an extensive restriction map of pBR
322 see Sutcliffe (1978b). All restriction sites
occurring in pKK 3535, including the 7.5-
kb fragment carrying the rmB operon, have
been compiled and are available from the
authors on request.
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