Volume 11 Number 21 1983
Nucleic Acids Research
Transcription regulatory elements In the late region of bacteriophage T5 DNA
Franchise Brunei, Vinh Ha Thi, Marie-Francpise Pilaete and John Davison
Unit of Molecular Biology, International Institute of Cellular and Molecular Pathology, 75 Avenue
Hippocrate, B-1200 Brussels, Belgium
Received 27 June 1983; Accepted 23 September 1983
ABSTRACT
Transcription promoters and terminators have been cloned from the late
region of bacteriophage T5 DNA and their strengths determined in vivo in
plasmid derivatives.DNA sequence analysis shows these transcription signals
to be remarkable in that,in all four cases studied in detail,the promoters
and terminators overlapped or were very close together.
INTRODUCTION
The ' 'in vivo" transcription of bacteriophage T5 DNA is a well regulated
phenomenon which occurs in three steps (1). Immediately after infection,
the pre-early mRNAs are synthesized from the left-hand 8X of the genome,
which is the first to penetrate the bacterial cell (first step transfer) (2,
3 ) . As soon as the rest of the molecule has been injected (four minutes
later) the transcription of early mRNAs also takes place. Late mRNA transcripts,on the contrary, only begin to appear some 10 to 12 minutes after
infection. Experiments using rifampicin (4) and RNA polymerase purified from
T5 infected cells (5,6) suggest that the E.Coli RNA polymerase serves to
transcribe the T5 DNA throughout infection though,at late tiroes,it does so
in association with T5 coded polyoeptides.Analysis of T5 mutants shows that
at least three T5 gene products (C2, D5, D15) are needed for late transcription.The C2 gene codes for a 90K polypeptide that binds to RNA polymerase,
as do two unidentified polypeptides of 1 IK and 15K (5,6). The D15 product
is a 5'-3' exonuclease (7,8) able to introduce endonucleolytic scissions in
T5 DNA (9). The T5 gene product is a DNA binding protein,involved in DNA
synthesis and turn-off of early gene expression as well as turn-on of late
gene expression (10,11).
The molecular mechanisms by which C2, D5 and D15 positively control
transcription are unknown. However,efficient expression of late genes may
be obtained in the absence of these nositive control factors when the genes
are cloned on phage
or plasmid vectors. In several cases,it is clear that
© IRL Press Limited, Oxford, England.
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the expression is from a promoter located on the cloned T5 fragment rather
than on the vector (12, 13, 14).
If late promoters normally interact with the modified RNA polymerase in
vivo, it is possible that their DNA sequences may be somewhat different to
those of classical RNA polymerase recognition sites or that their neighbouring sequences carry special information.
In order to test this, we isolated
late regulatory sequences and characterized their structures and biological
properties.
To do so, advantage was taken of the observation (12, 14) that
they are functional
proteins.
in a plasmid in the absence of any T5 regulatory
Thus, the late T5 DNA fragments Hindlll-L and Pstl-G were sub-
cloned in the expression probe vectors pKO-1 and pKG1800 (15). Promoters
(and terminators) were then characterized by their ability to turn-on (or off)
the galactokinase gene carried by these plasmids and by DNA sequence analysis.
MATERIALS AND METHODS
Plasmids and bacteria
pBR322::T5HindIII-L and pBR322::T5PstI-G have been described previously
(12, 13, 14). Plasmids pKO-1, pKG1800 and E.coli C600 galK were obtained
from McKenney et al., (15). C600 galK derivatives carrying various plasmids
are defined in Table 1.
Cloning and DNA sequencing
General procedures for the cloning of DNA fragments, rapid extraction of
recombinant DNA, purification of the plasmid DNA and characterization of the
clones by restriction enzyme analysis have been described previously (12, 13,
16).
The subcloning into pKO-1 and pKG1800 blunt-end Smal sites of fragments
with sticky ends was achieved in three steps : firstly, the sticky ends were
filled in using E.coli DNA polymerase Klenow
fragment and deoxynucleoside
triphosphates using the method of Wartell and Reznikoff (17); secondly, they
were separated on agarose gels and extracted by the method of Dretzen et al.,
(18); finally, they were ligated to the Smal site by blunt-end ligation.
DNA sequencing followed the method of Maxam and Gilbert (19) and was analysed
on a Textronix 4051 micro-computer using a programme designed by Mr. Richard
Robert-Shaw.
Galactokinase assays
The galactokinase assays were performed following the method of Wilson
14
C galactose was
and Hogness (20) except that the specific activity of the
1.77 x 10
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dpm/ymole and the final concentration of galactose in the assay
Nucleic Acids Research
_2
mixture was 2 x 10 M.
A galactokinase unit represents the amount of enzyme
necessary to convert one nanomole of galactose to galactose-1 P in 1 hour at
32° C.
Enrymes and chemicals
All restriction enzymes and DNA ligase were purchased from New England
Biolabs, Boehringer Mannheim or Amershaa and were used according to the
manufacturerft instructions.
Biochemicals.
32
Polynucleotide kinase was obtained from P. L.
B-dATP
P-dATP (3000
(3000 Ci/t
Ci/mmole) and D- C galactose (40-60 mCi/mmole)
were purchased from Amersham Ltd.
RESULTS
Regulatory signals in T5
The present study makes use of two vectors designed to facilitate the
screening of promoters and terminators : pKO-1 and pKG1800 (15). In pK0-l,
the gene coding for galactokinase is not expressed so that E.coli FDBIOO
(C600 galK carrying pKO-1) gives white colonies on McConkey galactose plates.
The cloning of a promoter upstream of galK activates galactokinase expression
and results in the formation of red colonies.
In contrast, pKG1800 already
carries a promoter (pgal) transcribing the galK gene and the cloning of a
terminator between galK and its promoter changes the colony colour from red
to white.
The T5 Hindlll-L fragment (3.9 kb) has been cloned and characterized
previously (12, 14, 21). It is known to contain at least one promoter since
the corresponding recombinant clones express the gene D21 as well as another
unidentified gene (14). The work presented here concentrates on the 1.9 kb
PvuII fragment contained within Hindlll-L (Fig. 1). This PvuII fragment was
cut into four sub-fragments of 504, 490, 135 and 770 bp respectively (Fig. lc)
and these were subcloned into the Smal site of the two expression probevectors.
They were recovered in each plasmid in both possible orientations
except for the Taql-PvuII fragment (770 bp) which was cloned in both directions in pKG1800 but only in one direction in pKO-1.
When in pKO-1, all of
the fragments (except the 135 bp Xbal-TaqI) showed a red phenotype irrespective of orientation, suggesting promoter activity for each of them in both
directions.
2-9).
The galactokinase assays confirm these results (Table 1A, lines
The promoters are of moderate strength causing a 10-12 fold increase
over the negative control and producing about 20 X of the level of galactokinase synthesized from the pgal promoter (Table IB, line 1).
All the pKG1800 sub-clones retained the parental FDB200 red (galK+)
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Nucleic Acids Research
F
B
E
I
A
I
E
A
D
J I O H L K C P H I
I
O
F
H
C
N
K
B
P O 1 J L
>-%•
Fig. 1 Physical and regulatory maps of the T5 PvuII fragments subcloned in
pKO-1 and pKG1800. A- PstI and Hindlll restriction maps of the bacteriophage
T5 DNA Showing the location of the cloned restriction fragments (thick lines)
from which the PvuII fragments were obtained. The arrows above the diagram
represent the pre-early (PE), early (E), and late (L) parts of the genome.
B- The regulatory map of the subcloned PvuII (564 bp) fragment derived from
Pstl-G showing the promoters ("iw—-) and terminators (|—) and the restriction
sites used for the sequencing. The dotted line shows the pKO-1 vector and
the position of galK in orientation £ (Table 1). C- Restriction map of the
PvuII-PvuII (1.9 kb) fragment derived from Hindlll-L (21 and unpublished data).
D- The regulatory map of the subcloned PvuII-Xbal (504 bp) fragment showing
promoters (vWV—) and terminators ( I — ) and the restriction sites used for
sequencing. The dotted line shows the vector and the position of galK in
orientation a. (Table 1). Abbreviations : Hd, Hindlll; Hf, Hinfl; Be, Bell;
Ec, EcoRI; Pv, PvuII; Xb, Xbal, Sm, Smal.
phenotype.
Such a result can be obtained in three different ways : either
there, are no terminators on these fragments; of terminators are present but not
fully efficient so that transcriptional read-through takes place from the
pgal promoter.
Finally, it is also possible that the terminators are followed
by active T5 promoters responsible for galK expression.
Measuring the
quantity of galactokinase synthesized by each strain enabled us to differentiate between the first hypothesis and the other two, as can be seen in Table
IB (lines 2-9). All FDB200 derivatives make the enzyme in sufficient amounts
to account for their red phenotype.
nant to recombinant.
In FDB205 and FDB206 they are identical to that of the
parental strain (FDB200).
Therefore, the Xbal-TaqI 135 bp fragment does not
contain any terminator sequences.
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However, theae amounts vary from recombi-
In FDB2O7 and FDB2O8, the quantity of galac-
Table 1. Galactokinase levels in the pKO-1 and pKG1800 aubclones
B. DNA inserted in pKG1800
A. DNA inserted in pKO-1
Strain
fragment
number
size
source
o r i 5 kinase
bP
.raits
1 FDB100
2 FDB101 PvuII-Xbal
it
ii
3 FDB102
4 FDB103 Xbal-Xbal
II
II
5 FDB104
6 FDB105 Xbal-TaqI
*i
n
7 FDB106
8
9
10
11
504
tt
490
II
135
H
FDB107 Taql-PvuII
II
n
FDB108
770
FDB109
564
FDB110
PvuII-PvuII
II
II
It
II
Hindlll-L
II
Hindlll-L
ti
Hindlll-L
11
Hindlll-L
II
Pstl-G
II
a)
o)
a)
number
295
317
279
FDB201
FDB202
PvuII-Xbal
FDB203
FDB204
FDB205
Xbal-Xbal
a)
b)
a)
238
nt
110
b)
20
FDB206
source
o r i 6 kinase
units
1630
II
II
II
II
Xbal-TaqI
it
II
FDB207 Taql-PvuII
II
II
FDB208
FDB209 PvuII-PvuII
FDB210
size
bp
FDB200
b)
a)
fragment
25
311
33
15
P)
Strain
II
11
504
II
490
11
135
II
770
II
564
II
Hindlll-L
II
Hindlll-L
tl
Hindlll-L
It
Hindlll-L
II
Pfltl-G
II
a)
b)
a)
b)
a)
b)
1520
469
.1350
580
1449
1367
403
110
a)
b)
a)
1450
b)
50
CD
n'
Galactokinase units are given per OD
of bacteria and represent the mean of five independent measurements.
Orientations a and b of the fragments are defined in Fig. 1.
Q)
O
Nucleic Acids Research
tokinase made is reduced, independently of the orientation of the 770 bp TaqlPvuII fragment, suggesting terminator
activity in both orientations. Follo-
wing the same reasoning, the PvuII-Xbal 504 bp and Xbal-Xbal 490 bp fragments
should contain terminator sequences in only one orientation (FDB202, FDB204).
The DNA sequence of the 504 bp PvuII-Xbal fragment that contains both a
promoter and a terminator was examined for the presence of sequences analogous
to the consensus promoter and terminator structures as defined by Rosenberg
and Court (22). Regions of DNA were defined as potentially capable of promoter activity when their sequences followed three criteria. Firstly, they contain a Pribnow box (-10 region) having the general sequence TAtaaT (where the
final T ia always present, the other upper case letters usually present and
the lower case letters often present).
Secondly, they have a -35 region with
the general structure TTGaca. Thirdly, the distance between the end of the
-35 region and the beginning of the Pribnow box is not larger than 18 bp or
smaller than 16 base pairs as these have been shown to correspond to the maximal and minimal spacings compatible with promoter activity (23, 24). Studying
the 504 bp PvuII-Xbal fragment along these lines shows two promoter-like
sequences, one in each orientation : p . situated between bp 385 and 412 and
p. - situated between bp 222 and 193
(Fig. 2), p j and p,_ are therefore most
probably responsible for galK expression in FDB101 (Table IA, line 2) and
FDB102 (Table IA, line 3) respectively.
The presence of terminators on the fragment was investigated by looking
for the characteristic stem and loop structure followed by a series of Ts
(22).
Two such sequences can be found; one between coordinates 225-212 (t.)
and the other between coordinates 397-381
(t T1 ) (Fig. 2 ) .
1J
These are posi-
1
tioned such that either one or the other would cause termination of transcription from pgal in FDB202.
The residual transcription in FDB202 would be from
p.„ which is on the galK proximal side of these terminators.
Indirect confir-
mation of such a transcriptional pattern may be found in the fact that the
amounts of galactokinase produced in FDB102 and FDB202 are very nearly identical.
No terminator-like sequences were found in the reverse orientation,
in agreement with the observation that FDB201 shoved no terminator activity.
Regulatory sequences in T5 Patl-G
The 7000 bp T5 Pstl-G DNA is cleaved by PyuII to give a subset of four
fragments of 6000, 564, 250 and 150 bp (data not shown).
Cloning of the 564
bp fragment into pKO-1 gives galactokinase expression in one orientation
(FDB109) but not in the other (FDB110) (Table IA, lines 10 and 11). Conversely cloning this fragment into pKG1800 shows transcription terminator
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Nucleic Acids Research
TGCTACTGCTTAATAATCTAncCAAATCTG€lTrilirc:AGGTAATAAAAGACGATTATTT^
250
300
CAArcCTCAAGAAAAGACACAACTCAACACTCAAACAAAATCAGCATTCGCAACGTTCTTATAATGGC
CTTAGGACTT€TU ICTCTCTlEACTTCTGAll 1101 lUACTCCTAAGCGTTCC^GAATATTACCCAAAATTTTCCACCTAATGACTTTTCCATTTAG
*"
"*~
350
400
CGGCTt^CCTATTATAAC^CATGCACCAGTTAGTCATCGCACTAACCGTAAGCCTTTTACI^CTCKACAACC^
450
500
AACTGCTAATATtXrrTATAGAIAGTGCGGCGGACTGTTCTTATACTCTCCCAGATAAATATAATATTGTTAC^
TTGACGATTATACCAATATCTATCACGCCGCCTCACAAGAATATGACACCCTCTATTTATATTATAACAATGCATCCCATTACCGCAOl
H I G H 1CTAA
CTAG 3"
CATC 5'
Figure 2. The DNA sequence of the 504 hp PvuII-Xbal fragment. The
promoter-like sequences are enclosed in boxes and the terminator-like
sequences are underlined by arrows, indicating the hairpin loop. The
sequencing strategy is given in Figure 1.
activity in one orientation (FDB210) but not in the other (FDB209) (Table IB,
lines 11 and 10). These results suggest a promoter on one strand and a
transcription terminator in the other.
expectation.
The sequencing data confirm this
Two terminator-like sequences (t^. and t^o) can be found on the
Gl
GZ
fragment (coordinates
484-501 and 194-213, Fig. 3)
any obvious promoter-like sequence.
cause termination in FDB210.
and are not followed by
Both are orientated such that they would
On the other hand, two promoter-like sequences
(pG1 and p G 2 ) are present on the other strand (coordinates 203-232 and
437
466, Fig. 3). These are appropriately orientated so as to promote galactokinase expression in FDB109 (Table 1).
DISCUSSION
This publication describes the identification of transcription promoter
and terminator signals from the late region of the T5 genome.
These were
defined by two criteria : their capacity to initiate (or prevent) in vivo
transcription and their similarities to the generally accepted consensus DNA
sequences for such regulatory elements.
Several sequences were found in sub-
clones of Hindlll-L and Pstl-G that fulfill these requirements.
The two promoters from the 504 bp PvuII-Xbal fragment (Fig. 2) of
Hindlll-L cause a 10-12 fold increase in galactokinase production (FDB101 and
FDB102, Table 1) compared to the negative control (FDB100) but are only of
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Nucleic Acids Research
M
3
.
100
' CACCTTCATACATTCCCATTATC<XTTCTC(rrCGAATTaTACCATTACTCTAMTATrrTGAACTCCTC(^AATCTCGTCGAAl
111 ICCTCCCTCm
150
200
GTCAMTGCAGCAACAarrAaiATCTrCTirrTGCCCCTiCTTATACTCCTACTACTGGGTTAATCGCACAAt^TAAAMTTTTCCTCATATCCA^
250
AGTTCTCTCAGCCATCTrTACATACTACCTCTATC^TTCCTTCCGGTATGCAGACTGTAGCTTCTATGATTCAAT^^
TCAACAGAGTCCCTAGAAATCTATGATCCAGATACTAACCAAGCCCATAIXTCTGACATCCAAGATACTAACTTATCTCATGATCAGTCCTCCMTCAM
450
500
AATTCATCAGCCTATTCCACCAGAACACAAGCGTCAlTGGCAAATCTGAGGCATCTAAACCTAAG^
TTAACTAGTCCGATAACCTCCTCTTCTCTTCGCACTACCGTTTAGACTCCGTAGATTTCCATTCCAll111 UAATCTTCCACn TTCGACTTCTAACTC
550
CAAGATGCAGCIAAGAAACAGATCATCATTCAGACACCACTAGCACTAATGCMGCAGCAACAG 3 '
CTTCTACGTCCATTCTTTCTCTAGTACTAACrnrrGTCCTCATCGTCArrACCTTCGTCCTTCTC 5 '
Figure 3. The DNA sequence of the 564 bp PvuII-PvuII fragment.
in Figure 2.
moderate strength compared to that of pgal (FDB200).
Legend as
However, comparison of
their DNA sequences with the consensus sequence for promoters indicates that
the overall general structure is well preserved as 17/22 (p..) and 15/22
(pT_ ) bases are found in the expected positions.
-35 region and the Pribnow box is in one
The spacing
between the
case shorter (pLj:16 bp) and in the
other, longer (p^.MS bp) than the optimal spacing (pgal : 17 bp) (23, 24, 25).
And this may be the reason for the promoter's relative lack of strength.
more observations may be made about p
and P L2 -
Two
Firstly, they differ in that
p.. shows more similarities to the consensus sequence than does P^2> P art i- Cu ~
larly in the -35 and Pribnow box regions, yet, their capacities to initiate
transcription are about the same.
terminator sequences (t_
Secondly, they both overlap with potential
or t,~) . In the case of p., t^e terminator-like
structure ( L . ) is on the same strand and in the case of p ^ the terminatorlike structure L.
is on the opposite strand.
Association of promoters and
terminators has been previously described (26,27), and taken as an indication
that certain features are common to sites where RNA polymerase begins or ends
transcription (22). The functional reality of the t . and Lj, a s terminators
is suggested by the fact that the PvuII-Xbal fragment causes termination in
the b_ orientation (FDB202), the true magnitude of this effect being disguised
by the p . promoter that initiates in the same direction after the termination
s equences (FDB102).
The 564 bp PvuII-PvuII fragmept (Fig. 3) derived from the Pstl-C fragment
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Nucleic Acids Research
likewise has two promoter-like structures (p,,. and p _ ~ ) , both orientated in the
same direction, and one or both of these is probably responsible for the increased
amount of galactokinase made in FDB109.
In these promoters the most
conserved bases are present but others are badly represented, and the distance
between the end of the -35 region and the beginning of the Pribnow box is 18
bp, possibly accounting for the weak promoter activity observed in FDB109.
The 564 bp PvuII-PvuII fragment also shows terminator activity (FDB210), this
being reflected in the DNA sequence by two terminator-like sequences t . and
t ^ , characterized by an inverted repeat followed by a run of Ts. As in the
case with L , and t^2' t G ] overlaps with the promoter-like sequence p
t j is located within ~ 20 bp of P r 2 ) •
(while
Both t . and t.,. are orientated in the
same way, resulting in efficient transcription termination (FDB210, Table IB).
One of the surprising results of this study is the high frequency with
which regulatory signals have been identified.
Four potential promoters and
four terminators have been found in the two small fragments (504 and 564 bp)
which were completely studied.
The biological data obtained for the other
fragments indicate the presence of promoters in all but the smallest (Table
1).
Another unexpected observation is the overlap of the terminators and
promoters in three of the four cases studied, while the fourth case showed
them in very close proximity.
The in vivo significance of the promoters and terminators described
here is difficult to assess since T5 late transcription normally takes place
in the presence of T5 coded positive control factors, that are missing in the
clones studied.
It is not known why late T5 genes are not transcribed in the
absence of these factors, nor the way in which the factors overcome this
transcriptional block.
The overlapping arrangement of the promoters and
terminators could conceivably be part of this mechanism.
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