volume e Number 51979
Nucleic A c i d s Research
Nucleotide sequence of the three major early promoters of bacteriophage T7
Ulrich Siebenlist
Harvard Biological Laboratories, Cambridge, MA 02138, USA
Received 4 December 1978
ABSTRACT
I have determined the nucleotide sequences of the three
major early promoters of bacteriophage T7 (Al, A2, A 3 ) . The
sequences confirm the two main homologies found between
other known promoters for E. coli RNA polymerase (nucleoside
triphosphate:RNA nucleotidyl transferase, E.C. 2.7.7.6). In
particular, all three T7 promoters show a very good match
with the -35 region homology; the A2 and A3 promoters share
a 17 basepair sequence in this region. On the other hand,
the match with the Pribnow Box homology is much less pronounced and different for each T7 promoter.
INTRODUCTION
RNA polymerase initiates transcription at specific
sites (promoters) on the DNA template. The enzyme first
forms a stable binary complex at such sites. This association involves a limited unwinding of the DNA double helix (1).
In the presence of ribonucleoside triphosphates, transcription ensues rapidly.
Nucleotide sequences from several promoters for F. coli
RMA polymerase reveal two regions of at least partial homoloqy.
The first such region is commonly referred to as the Pribnow
Box, and consists of seven base pairs centered approximately
one helical turn upstream from the RNA start site (2,3,4).
The second region with a partially conserved sequence is
located about 35 base pairs in front of the first transcribed
base; it is therefore called the -35 region. Several promoter mutations have been mapped within these two regions
(5,6,7,8,9,10).
It has been suggested that the -35 region is involved
in the initial recognition by RNA polymerase and that the
© Information Retrieval Limited 1 Falconberg Court London W1V5FG England
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Pribnow Box provides a binding site possibly involved with
the melting of DNA (4,5). Direct evidence is missing,
however.
One therefore would like to identify the roles the two
main regions of homology play and to pinpoint the precise
bases RNA polymerase recognizes. To this end I have first
located the three major early promoters of bacteriophage T7
(Al, A2 and A3) on specific restriction fragments and then
sequenced them.
These three promoters are considered strong ones, where
almost all early RNA from T7 is initiated (11). They form a
unique system in that they are very closely spaced.
MATERIALS AND METHODS
T7 DNA
DNA of bacteriophage T7 was prepared as described by
Minkley and Pribnow (11)
ENZYMES
Alu I restriction enzyme from Athrobacter luteus was
prepared according to Roberts et. a_l (12) . Several enzymes
were received as gifts: E. coli RNA polymerase (R. Simpson);
T4 polynucleotide kinase (A. Maxam); restriction enzymes Taq
I and Hpa II (D. McConnell); restriction enzyme Hindll/III
(J. Sims); restriction enzyme Hinf I (G. Sutcliffe and D.
Hourcade); restriction enzyme Hae III (F. Ausubel). The
following enzymes were purchased: restriction enzyme Hha I
(New England BioLabs); alkaline phosphatase (Worthington);
ribonucleases Tl and U2 (Sankyo, Calbiochem). I am grateful
to R. Roberts for enabling me to screen several of his
restriction enzymes.
ENZYMATIC CONDITIONS
Restriction digests were generally performed in .01 M
MgCl 2 , .01 M Tris-HCl pH 7.5, .1 mM EDTA, 1 mM dithiothreitol and .01 - .05 M KC1.
Conditions for synthesis of and
32
labelling at 5' ends with [gamma- P] ATP were as described
in Maxara and Gilbert (13). Briefly, to label the 5' ends,
DNA was treated with alkaline phosphatase to remove 51
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terminal phosphates and then labelled with
P by transfer
from gamma-labelled ATP with T4 polynucleotide kinase. As a
slight modification, the boiling step immediately preceding
the transfer reaction was omitted during labelling of whole
T7 molecules. Also, to complete the reannealing process of
a previously boiled restriction digest of T7 after the
transfer reaction, the mixture was brought to 50°C and
allowed to cool slowly.
GEL ELECTROPHORESIS AND ASSOCIATED TECHNIQUES
Constituents of sequencing gels, their electrophoresis,
autoradiography and the elution of DNA from gels have recently been described by Maxam and Gilbert (13).
NITROCELLULOSE FILTER BINDING ASSAY
Filter binding procedures are essentially those of
Hinkle and Chamberlin (14). RNA polymerase was incubated
with DNA for 2-5 minutes at 37°C in 50 -100 microliters of
binding buffer (10 mM Tris-HCl pH 8, 10 mM MgCl 2 , .lmM EDTA,
05 M KC1, 1 mM dithiothreitol). The volume was then brought
to 1 ml with binding buffer and rapidly passed through
nitrocellulose filters (Schleicher and Schuell), which were
presoaked in binding buffer. Filters were extracted with a
1% SDS, 10 mM Tris-HCl pH 7.5 solution.
NUCLEOTIDE SEQUENCING
DNA sequencing was according to the dimethylsulfate/
hydrazine technique of Maxam and Gilbert (13). RNA sequencing was according to Donis-Keller e_t. a_l (15) . Briefly,
end-labeled RNA was partially digested with ribonucleases Tl
(G specific) and U2 (A specific); also NaOH hydrolysis
provided a complete set of partials representing all nucleotides. Electrophoresis of the Tl, U2 and NaOH partials on a
sequencing gel allowed the reading of a G, A, pyrimidine
sequence.
Al mRNA SYNTHESIS
.2 micrograms of the 520 basepair Hpa II fragment were
incubated with 2-3 raicrograms of RNA polymerase in synthesis
buffer for 30 minutes at 37°C (synthesis buffer: .15 M KC1,
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.1 mM EDTA, 10 mM MgCl , 1 mM dithiothreitol, 10 mM Tris-HCl
pH 8 ) . The concentration of the ribonucleoside triphosphates
was 50 micromolar; the only labeled triphosphate was [gammaPi ATP at a specific activity of about 300 Ci/mM. This
labels the Al mRNA specifically at 5'. The products were
then electrophoresed in a 12% TBE polyacrylamide, 7 M urea
gel (TBE = .1 M Trisborate pH 8.3, 1 mM EDTA); the approximately 40 nucleotide long Al mRNA was the only visible band
on an autoradiograph.
RESULTS
RESTRICTION MAPPING
In order to identify restriction fragments carrying
promoters, I bound these fragments to a nitrocellulose
filter by forming specific binary complexes between RNA
polymerases and promoter fragments. Only DNA bound to RNA
polymerase will stick to nitrocellulose when filtered (14).
Figure la shows the restriction fragments that were retained
Figure 1. A 5% TBE polyacrylamide slab gel, stained with ethidium bromide, showing the top
portion of an Alu I digest of
2 1/2 micrograms of T7 in lane
(b), and the fragments of 10
micrograms of the same Alu I
digest that were filterbound
with 3 micrograms of RNA polymerase in lane (a). See Materials and Methods for filter
binding procedure.
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on and then extracted from such a filter. Thus I identified
an 815 basepair long Alu I fragment as a promoter fragment.
Similarly, several other promoter fragments were identified: an approximately 1300 basepair long Hae III fragment,
three Hpa II fragments of 520, 105 and approximately 1100
basepairs and two Hind II fragments 105 and 280 basepairs
long.
(A 795 basepair long Hind II fragment from the E.
coli lac region and its Hae III digest served" as size markers
(16) .)
Increasing the molar ratio of RNA polymerase to DNA
causes the retention of further specific fragments on filters
(data not shown). I presume that such fragments contain
weaker promoters. I focused attention on the strongest
promoters; those that bound RNA polymerase first.
Since only one promoter fragment was obtained with both
Alu I and Hae III, these fragments may contain all three
major promoters. Hpa II, on the other hand, cut between
these promoters, creating three filterbound fragments. The
major promoters are located within approximately 400 - 750
basepairs from the left end of T7 by electron microscopic
mapping (most recent measurements: 17,18). Therefore, I
reasoned that at least some of the filterbound fragments
might contain the left terminus of T7, which would provide
an ideal reference point for further mapping.
To test this possibility, whole T7 DNA was labelled at
the 5' termini with
P (see Materials and Methods), and
then digested with Alu I, for example. By electrophoresing
this digest in parallel with an Alu I digest endlabelled
after cutting, I identified the two terminal fragments of
T7.
Several of the filterbound promoter fragments are left
terminal fragments of T7: the 815 basepair long Alu I
fragment, the 1300 basepair Hae III and the 520 basepair
Hpa II fragments (see Fig. 3 for final map).
I now easily obtained a restriction map of the early
(left terminal) region. For example. Fig. 2 shows a partial
Hind II restriction digest of the 815 basepair long Alu I
band labelled at the left terminus only, which establishes
the location of the recognition sites of this enzyme.
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/.Q|O"O
6 3 0
Figure 2. Autoradiograph of a
5% TBE polyacryl amide gel,
showing Hind II partiais of the
left terminal 815 basepair long
Alu I fragment of T7, labelled at
the left terminus only (whole T7
was endlabelled and then digested
with Alu I) . Sizes in numbers of
basepairs are indicated on the
right. Partiais were obtained by
severely limiting digestion times.
245
80
70
Further restriction experiments led to the map shown in Fig. 3.
With this map I deduced the approximate positions of
the promoters and I proceeded to sequence the relevant DNA
fragments. Since Pribnow had previously determined the
sequences of portions of A2 and A3 these promoters were easy
to identify on my sequences (2,4). For Al, however, I had
to sequence the corresponding mRNA as well.
SEQUENCING
The previously published portion of the A3 sequence
contained a Hinf I site about 15 nucleotides into its trans-
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Basapairs from thm laft end of T7
O
10O
200
9OO
3OO
Alu I
Hint I
Hpa II
Rha I
Toq I
: in
AD
"T"" :
I
Al
—fr
Figure 3. Restriction map of the leftmost region of T7 , indicating initiation sites for the three early promoters of Al, A2
and A3 and the approximately position of the minor promoter AO
(17,18,19). Sequencing work for AO is in progress. Hsieh and
Wang have established some of the restriction sites as well (19).
cribed portion (2). The only such site in the early region,
at about 740 basepairs from the left end of T7, this was a
convenient restriction cut from which to sequence A3. Also,
the map in Fig. 3 shows two convenient Hpa II cuts flankinq
the A2 promoter.
Microgram quantities of the 815 basepair Alu I fragment
were digested with Hpa II and Hinf I to obtain thus two
promoter fragments, the 104 basepair long Hpa II - Hpa II piece
and the 112 basepair long Hpa II - Hinf I piece, containing
A2 and A3 respectively. After labelling and electrophoresis
I eluted both bands from the gel, and separated the strands
by denaturing them in .3 NaOH and electrophoresing them on a
5% TBE polyacrylamide gel (the strand separation procedure
is described by Haxam and Gilbert (13)). I isolated" the
separated strands, and sequenced them according to the
dimethylsulfate/ hydrazine technique of Maxam and Gilbert
(13). The strands representing the sense strands of T7 (r
strand) ran as the faster of the two for both fraamfints.
Fig. 4 shows the sequences of A2 and A3 (the sequence
obtained with one strand is confirmed by its counterpart on
the other strand). The underlined portions are the sequences
which Pribnow had elucidated previously (2,4) and which I
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-35
-10
-1+1
ACAAMCGGTTGACAACATGA AGTAAACACGGTACGATGTACCACATGAAACGACAGTGAGTC
T7A3
AAACAGGTATTGACAACATGAAGTAACATGCAGTAAGATACAAATCGCTAGGTAACACTAGCAG
T7A2
AAAA6A6TATTGACTTAAAGT CTAACCTATAGGATACTTACAGCCATCGAGAGGGACACGGCG
pppAUCGACACGGA
T7A1
T7AlmRHA
Figure 4. Promoter sequences of Al, A2 and A3. The underlined portions of A2 and A3 are protected by HNA polymerase
from pancreatic DNase digestion and have been sequenced pre=
viously (2,4). The Al mRNA is indicated below the DNA sequence.
Sequences are shown to -43, about the farthest base of A3 protected by RNA polymerase against exonuclease III (to be published) . Cutting with Hind II around the -35 region of A3
abolishes both binding to and transcription of that promoter
(unpublished observation); a similar phenomonon has been
observed in several other promoters (5).
confirmed here. Hsieh and Wang (19) established independently
the sequence contained in the Hind II - Hinf I portion of A3
as well.
From the filter binding and restriction data presented
above I located the Al promoter somewhere to the left of the
Hind 11/ Hpa II restriction sites 525 basepairs from the
left T7 terminus (see Fig. 3 ) . The Taq I site at position
480 was the only other restriction enzyme site in this
region.
The 480 basepair long Taq I fragment was obtained by
digesting the terminal Alu I piece. After labelling its
ends with 3 2 P , I cut with Hind II to get a 240 basepair
fragment, labelled at its Taq I end only; this I sequenced
directly.
To sequence across and to the right of the Taq I site
at position 480, I labelled the ends of the 280 basepair
Hind II fragment (see Fig. 3) and separated its strands as
described above.
After sequencing both strands, I located the Taq I site
and the beginning of the previous sequence on one of them.
I now had a continuous sequence surrounding the Taq I site
region.
To show the Al promoter was indeed located in the region
sequenced, I partially sequenced the RNA initiated at Al
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and matched this sequence with my DNA sequence to find the
RNA initiation point. Fig. 5 shows the partial RNA sequence,
which was determined according to Donis-Keller e_t. a^. (15)
(see Materials and Methods). To achieve the necessary
labelling at the 5' end of the message, I transcribed it off
the 520 basepair Hpa II fragment (see Fig. 3) in the presence
of [gamma-32P] ATP (see Materials and Methods). This RNA
was 39 nucleotides in length and its start site agrees with
the initation studies of Minkley and Pribnow (11). Fig. 4
Figure 5. Showing autoradiogram
of the Al mRNA sequence (see Materials and Methods) , with NaOH
hydrolysis, Tl and U2 partials
in l a n e s
*b' ' ' c ' a n d ' d ' respectively; lane (a) represents a
control without any added RNases
and shows background degradation
of RNA. Bromophenol blue (BPB)
runs with about 8 basepairs and
xylene cyanol (XC) runs with
about 26 basepairs.
G
G
-^
.BPB
G
A
G
A
G
Py
Py
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shows the Al promoter sequence and its transcript.
DISCUSSION
In order to detect nucleotides which might be relevant
to promoter function, I have lined up all available promoter
sequences in Fig. 6. (Similar analyses have recently been
undertaken by others as well (37).) The sequences were
aligned for maximum homology in the Pribnow Box and -35
region, treating each region separately, however. Due to
this novel alignment, the distance between the two DNA
regions varies among promoters, but only by + one basepair;
whether this is significant cannot yet be assessed.
Fig.
6 shows that the most probable sequences to be
-35
c
A
G
C
T
•30
AAAA C G G TTG ACA A CA T G A
A
T
C
T
T
C
C
A
A
A
T
A
G
T
T
C
T
A
C
A
G
G
G
C
A
G
A
T
T
C
A
C
T
A
C
T
A
G
6
1
6
7
7
5
4
4
A
G
T
C
T
T
T
T
G
C
T
C
A
C
A
C
T
T
T
4
2
5
9
GA G
C GG
GC G
C GA
TT T
T AA
CA G
T GT
A G C
G AA
CA T
A G G
C GG
T CT
AT A
C AG
AT T
CT C
C
A A
6
3
7
4
T
T
T
A
T
C
G
T
T
T
G
T
T
T
G
G
C
C
A
A
G
G
G
G
G
A
G
G
G
T
A
G
G
C
C
C
G
C
T
T
T
T
A
T
T
T
T
G
T
T
T
T
T
T
A
T
T
T
T
T
T
T
T
T
T
T
C
T
T
T
T
T
T
T
T
T
G A C
G A C
G A C
GAG
G C A
T A C
G A C
G T T
G A C
G CA
G A C
G A C
A G A
G A C
G C A
T A C
G T C
G T A
T A C
S 4 2 3 2 0 1 1 3 5
6 7 5 1 2 1 0 16 1
2 4 3 4 0 1 0 3 1 3
4
5 O 1 1 7 19 3 3 1
T
A
T
T
A
A
A
A
A
A
A
A
T
A
A
A
A
C
A
T
T
A
A
T
A
C
A
A
A
G
A
A
T
A
C
C
T
G
AA
A A
T T
TT
TC
TT
CC
CT
TT
AC
CT
CA
TT
TT
AT
TT
A C
TT
CG
A GT
ATA
T TA
T TT
G CT
T A A
T CC
T GT
A AT
C TT
TAT
T GA A
T A T C
T AA A
A C G T
T AT G
T T T T
G TT
G C G
1 5 10 5 4 4 6 6
1 0 2 0 1 3 4 2
1 3 6 4 1 4 1
4 S 9 1 1 1 2 6 1I
G
T
C
C
C
T
T
A
T
A
C
A
GT
CC
AG
GG
CT
CG
T
C
3
4
6
7
-20
T AA
A AC
A CT
T C T
T G T
GC T
A TT
A TT
A TT
T CG
GCG
T CG
A A C
T TG
A GC
C CT
T CC
C A T
C GC
G TC
7
3
3
7
4
3
8
5
1
4
6
9
-10
A
C
G
G
A
T
T
G
G
A
G
A
A
C
G
T
G
C
G
A
6
9
3
3
C
T
G
G
T
C
G
T
C
A
T
T
T
G
T
A
G
T
C
T
2
5
4
9
A
A
C
C
T
T
C
A
A
C
A
A
G
G
G
T
C
T
T
T
C
T
G
G
T
G
T
T
G
T
T
A
C
T
G
G
T
T
T
T
G
A
G
G
G
A
T
G
C
A
G
G
A
G
A
G
C
G
G
G
G T
GG
TG
TG
TC
CT
AT
T T
T T
G T
GC
C T
GT
AT
TT
TT
GT
T T
G T
AT
A CG
ATA
ATA
ATA
ATA
ATA
A C A
T TC
ATA
T AA
A TG
T TA
A AG
A G A
ACT
A C A
ATA
A TG
ATA
A TG
0+1
A
C
C
A
A
A
A
A
A
C
A
A
A
T
A
G
A
C
A
A
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
6 1 5 3 0 17 2 13 U
3 6
12 7 3 O 1 5 1
5 2 1 2 2 0 4 1 4 0
6 11 2 8 1 5 3 1 3 1 1
G
T
G
G
G
A
C
G
G
A
A
G
A
T
C
A
G
A
C
G
0
O
3
2
T
A
A
G
A
G
A
C
G
G
G
C
C
A
T
T
T
T
G
C
6
9
4
0
A C C A C ® T T7A1
C A G C C ® T T7A1
G C A C ® T C XPL
T T G C @ T G \PR
C T C C T © T XPO
A C A © © © T fdX
T C C T © T T fdll
C T C C ® A A *IA
T T A C ® A A SV40
T A C G C ® A trp
C G C C C Q G Ucl
G G T A C T T tit
A A A T C © C T7A2
A C G T ® T G XPrm
G A G T C C©
G C C C®T C
G T G © ® A T loo UVS
G G T T ® T T gal(-cap)
C T©©©©T
fdVIII
G C C C©C T TyrtRUA
5 4
6 7
5 7
2 5 4
4
3
6
6
4
6
9
2
2
4
6
5
8 6 3
5 5 4
2 3
1 7 1 0
Figure 6. The available promoter sequences for E. coli RNA
polymerase are lined up for maximum homology in the --35 and
Pribnow Box regions respectively; consequently the distance
between these two regions varies by + one basepair. The
X Prm and lac I sequences also allow a different alignment
around -35"! Additional, recently published promoters support
the homologies detected here. The sequences are shown out to
-43, about the farthest base protected by RNA polymerase
from exonuclease III in the case of A3 (to be published).
UPL(20), APR (21,22), XPO(23), fdX(24,25), fd II (26,27),
<f>XA(28) , SV40(29), trp (30), lac 1(31) tet(32), XPrm(33,34),
4>XD(28), (t>XB(28), lac UV5(5), gal (35) fdVIII(27), Tyr
tRNA(36)).
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Nucleic Acids Research
recognized by RNA polymerase are TTGACA and TATAAT for the -35
ion and the Pribnow Box, respectively. But not one of the
promoters listed shows a perfect match with both 'ideal'
sequences.
Other regions exhibit some degree of homology as well
(see Fig. 6 ) . I found relatively conserved stretches of
basepairs immediately preceeding the Pribnow Box and following
the -35 homology; the latter region is high in AT content
and possibly plays an indirect role during the binding
process. Although principal points of contact with RNA
polymerase probably lie in the more highly preserved sequences,
weaker homologies and even nonhomologus regions may define
the promoters as well. Only experiments which directly
probe the interaction between RNA polymerase and DMA on the
nucleotide level will yield more information on this point.
Since the existence of three closely spaced major early
promoters of T7 may be to assure transcription under a
variety of conditions (38), it is not surprising to observe
extensive variance in their Pribnow Box sequences (except
the second and the universally conserved sixth position),
but all contain 'good' -35 sequences. Indeed, A2 and A3
share a seventeen basepair sequence in this region and
detailed studies have revealed a particularly striking
difference in the temperature and salt sensitivities between
these two promoters (3 8). Two explanations for the lower
transition temperature of A3 seem possible. The distance
between the -35 region and the Pribnow Box is larger for A2
than for A3, and A3 has a slightly better match with the
'ideal' Pribnow Box sequence and the weak homology preceding
it. Indeed, in the case of lac UV5 versus lac P S , a single
base difference at the fifth position in the Pribnow Box (TA
goes to AT) causes a larqe transition temperature change
(37).
Combining kinetic and structural approaches to the
interaction of E. coli RNA polymerase with its various promoters, one should ultimately be able to 'read' any promoter
code. The complete primary structure of the three strong
promoters Al, A2 and A3 provide the basis for such experiments
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on T7.
ACKNOWLEDGEMENTS
I thank Walter Gilbert for advice and for help with the
manuscript. This work was supported by a National Institutes
of Health grant to Walter Gilbert (GM09541-17).
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~~
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