Volume 4 Number 9 September 1977
Nucleic Acids Research
Studies on gene control regions. VI. The 5- methyl of thymine, a lac repressor recognition site
D.V. Goeddel, D.G.Yansura and M.H.Caruthers
Department of Chemistry, University of Colorado, Boulder, CO 80309, USA Received 1 June 1977 ABSTRACT
Three site specific deoxyuridine analogs of lac operator were tested for binding with wild type (SO) and tgIiTt binding (QX86) lac repressors. Insertion of uracil for thymine at site 13T(our nomenclature) significantly reduced the dissociation half-life of QX86 repressor for lac operator DNA (21 vs 1.2 min). Two other sites (6 and 7) are affected to a much lesser extent. INTRODUCTION Our objective is to contribute toward an understanding of how proteins interact with DNA to control gene expression. One system presently being examined intensively in this laboratory (1) and by others (2) using chemically synthesized lac operator segments is the lac operator-lac repressor complex. Initial investigations in our laboratory are directed toward defining the recognition elements on lac operator DNA that are associated with this highly specific interaction. The thymine methyl group protrudes into the major groove of DNA and would appear to be a likely candidate as a protein contact site. Some preliminary results indicated that this may be the case (3). In order to systematically investigate this possibility, several operators with uracil in place of thymine have been constructed. This paper reports (1) the synthesis of three lac operators containing deoxyuridine in place of thymidine at specific sites and (2) the dissociation half-life measurements of these operators with SQ and QX86 repressors. MATERIALS AND METHODS
Wild type (SQ) repressor was purified by a published proceC Information Retrieval Limited I Falconberg Court London W1 V 5FG England
3039
Nucleic Acids Research dure (1). The iSQ strain was obtained from Dr. W. Gilbert. Tight binding (QX86) repressor was provided by Dr. J. Sadler. Xh8O dlac DNA was prepared as previously described (4) and used as a non-radioactive competitor for lac repressor in dissociation half-life measurements. The strain RV/80 used for the preparation of Xh8O dlac was obtained from Dr. J. Sadler. T4-Ligase (5), T4-kinase (5) and E. coli DNA polymerase I (6) were prepared by published procedures. The enzymes snake venom phosphodiesterase, pancreatic deoxyribonuclease, spleen phosphodiesterase, micrococcal deoxyribonuclease and bacterial alkaline phosphatase were commercial preparations. fy-3 PI ATP at a specific activity of 1500 Ci/mmole was prepared by a published procedure (7). Deoxytriphosphates, including dUTP, were purified using a published procedure (8). Repair syntheses with DNA polymerase I were as described elsewhere (9). Gel electrophoresis was carried out using published procedures (10). The gel buffer was 89 mM Tris-borate (pH.8.3) and 2.2 mM EDTA (TBE buffer). The membrane filter assays were performed basically as described by Riggs et. al. (11,12). The composition of binding buffer (BB) and washing buffer (WB) as used in this laboratory has been described elsewhere (13). The ionic strength (I) of the binding buffer was adjusted by addition of KCL. Low (I=0.05 M), medium (I=0.12 M) and high (I=0.20 M) ionic strength binding buffers contained 10 mM, 80 mM, and 160 mM KCL respectively. Washing buffers were of the same ionic strength as binding buffers. The procedure used for measuring dissociation kinetics in this laboratory has been described previously (2). Nitrocellulose filters (BA-85, 27 mM) were obtained from Schleicher and Schuell. Chemical and enzymatic synthesis of segments [1-2-3], [2-3], [5a-6-7], and 15a-6J has been described previously (1,14,15). Chemical synthesis of segment 6c [d(T-A-T-C-C-G-C)] and segment 5a [d(A-A-T-T-G-T)], has been described previously (14). These segments were purified from the completely protected deoxyoligonucleotides by published procedures (16). The sequences were confirmed by the Sanger (17) two dimension sequencing procedure (data not shown). The synthesis of 26 base pair (duplex I) and 21 base pair (duplex II) lac operator DNA has been described previously (1). 3040
Nucleic Acids Research EXPERIMENTAL
Synthesis of segment 15a-6c]: The sequence for 15a-6c] is shown in Figure 1. Segment 5a (847 pmole), t5 '-32p segment 6c (776 pmole) and 15'-3'P] segment 12-3] (774 pmole) in 20 mM TrisHCl (pH 7.6), 10 mM MgCl2 and 27 pM ATP were warmed at 700C and cooled to O°C (1 hour). The reaction volume was 100 p1. Dithiothreitol (10 mM) and 50 units T4 ligase were added. After 120 hours the reaction solution was warmed briefly (two minutes) in a boiling water bath and the product fractionated from unreacted starting materials on a Sephadex G-75-40 column (0.8 x 45 cm). The isolated yield of segment 15a-6c] was 56% (437 pmole).
(3)-DEOXY
[2-33
T-G-T-G-G-A-A-T-T-G-T-G-A-G-C-G-G-A-T-A-A-C-A-A-T-T
t3)-DEOXY
[1-2-33
A-C-A-C-C-T-T-A-A-C-A-C-T-C-G-C-C-T-A-T-T-G-T-T-A-A a L6 e t
(5)-DEOXY
[5a-6-7]
C-A-C-T-C-G-C-C-T-A-T-T-G-T-T-A-A
(5')-DEOXY
C5o-63
C-U-0-G-C-C-T-A-T-T-G-T-T-A-A
M-DEOXY
CMo-6c-U-Cl
C-G-C-C-T-A-T-T-G-T-T-A-A
C5)-DEOXY
M5G-603
A-A-T-T-G-T-G-A-G-C-G-G-A-T-A-A-C-A-A-T-T 1
2 3 4 5 6 7
8 9 10 11
12 13 14 15 16 17 1
19 20 21 22 23 24 25 26
7
Figure 1. A summation of deoxyoligonucleotides that have been enzymatically joined to form lac operator DNA segments used in the present investigation. Segments chemically synthesized are partitioned and numbered. Above the total plan is a partial operator sequence corresponding to the top DNA strand and written 5' to 3'. Below the total plan are three partial operator sequences corresponding to the bottom strand and written 3' to 5'. The row of hyphenated numbers in brackets (right part of figure) correspond to these deoxyoligonucleotides prepared by enzymatic joining of the numbered, chemically synthesized fragments. These bracketed numbers written left to right refer to the deoxyoligonucleotide with polarity 5' to 3'. Numbers followed by lower case letters refer to deoxyoligonucleotides that are only part of the original, chemically synthesized segments referred to by the numbers. The symbol U refers to deoxyuridine. Capitalized letters within brackets refer to deoxyoligonucleotides that have been added to the 3'end of a primer by repair synthesis with E. coli DNA polymerase I.
3041
Nucleic Acids Research Characterization by degradation to 3'- and 5'- mononucleotides is shown in Table 1. The 5'-mononucleotide analysis is consistent with results expected for segment 15a-6c2 where only the internal Table I:
Characterization of Oligonucleotides by Analysis of
Labeling Patternsa,b
segment
[5a-6c]
Nucleoside Analysis (cpm)
5'-Nucleotide Analysis (cpm)
Sample c
pdA 24
pdG 139
pdT
11490
pdC 785
3'-Nucleotide Analysis (cpm) segment L5a-6cJ
dAp
dGp
duplex II(6,7A/U)d
dTp
211
5429
3920(1.1)
3529(1.0)
119
dCp 122
[3H]dUp
[3H]dU
116(0.85)
136(1.0)
a
3'-
b
The numbers in parenthesis after the counts per minute (cpm) are the experimental molar ratios.
and
5'-nucleotide
analysis was by a previously reported procedure
132P] phosphate originally was located on the synthesized segment 6c.
c The d
The
132p]
5'-end
phosphates originally were located on the
synthesized segments 6 and 3.
(18).
of chemically
5'-ends
of chemically
ligation site is radioactive. Since no 1 32P] pdA was observed, unreacted 15'-32P] segment 12-3] had been separated completely during the fractionation. Analysis by degradation to 3'monOnucleotides transferred all radioactivity to dTp as expected for segment 15a-6c]. Finally gel electrophoresis of segment 15a-6c] indicated a single band with mobility expected of a tridecanucleotide. The gel pattern for 15'_32p] segment 15a-6cJ is shown in Figure 2 (gel A, channel I). No slow moving band is present. This is further proof that 15,_32p] segment 12-3] had been removed during the column chromatography. Synthesis of Duplex I (13 A/U): The synthesis required two steps as shown in Figure 2. The first step was as follows. The reaction mixture (100 pl) contained 44 pmole segment 15a-6c], 75 pmole segment 12-3], 100 pM dUTp, 100 iM dCTP, 8 mM MgCl2, 0.12 M potassium phosphate (pH 6.9) and 5 mM dithiothreitol. After warming to 70°C, the reaction mixture was cooled slowly (1 hour) 3042
4
Nucleic Acids Research C2-33
T-AA-C-A-A-T-T
)-DEOXY
CG-C-C-T-A-T-T-G-T-T-A-A
(5)-DEOXY
Ca-6,0
(3)-DEOXY
C2-3 3
AA-T-T-G-T- G-FA-C-
dUTP,dCTP
STEP i
M')-DEOXY
C-U
T--T--G-A-A-T-T-T-T-G-A-G- C-G-G-A-T-A-A-C-A-A-T-T C-"U
M5a-6c-U-CO
(3)-DEOXY
CI-2-33
C')DEOXY
C5a-6c-U-C3
STEP 2 j dTTP,dATP,dCTP
(3*)-DEOXY X(13 A/U)
cW)-DEOXY
U
STEP I
STEP 2
I(13 A/U)---E ft -6c-U-C3
~
:. ..:.
*:
. ....
t 5a-6c3 -
Figure 2. Synthesis of duplex I (13 A/U). The top part of the figure outlines the steps for repair synthesis of duplex I (13 A/U) with E. coli DNA polymerase I. Extent of reaction was monitored by ger electrophoresis. The denaturing gel was 20% acrylamide, 1% N,N-methylenebisacrylamide in TBE containing 7 M urea. The first gel shows the analysis of step 1. Channel I contained segment 15a-6c] and channel II contained the reaction mixture after 4 hours. The second gel shows the analysis of step 2. Channel III contained segment [5a-6c-U-CJ and channel IV the reaction mixture after 6 hours. The [32p] phosphate was in the phosphodiester bond joining segment 5a to 6c. See Fig. 5 for the duplex nomenclature. 3043
Nucleic Acids Research to OOC. E. coli DNA polymerase I (28 units) was added and the reaction allowed to proceed four hours at OOC. Analysis of this reaction is shown in Figure 2. The reaction mixture was next adjusted to 40 mM EDTA and 50% formamide. After heating in a boiling water bath, the reaction mixture was fractionated on a Sephadex G-75-40 column (0.8 x 45 cm) using 10 mM, triethylammonium bicarbonate (TEAB) as eluting buffer. The isolated yield of repaired segment 15a-6c] (segment 15a-6c-U-CJ) was 37 pmole. The completely repaired duplex was prepared next (step 2 synthesis). The reaction mixture (100 p1) contained 37 pmole segment [5a-6c-U-C], 43 pmole segment 11-2-3], 100 pM dCTP, 100 pM dATP, 100 pM dTTP, 8 mM MgCl2, 0.12 M potassium phosphate (pl 6.9) and 5 mM dithiothreitol. After warming to 70°C, the reaction mixture was cooled slowly (1 hour) to OOC. E. coli DNA polymerase I (28 units) was added and the reaction allowed to proceed six hours at OOC. The analysis of this reaction is shown in Figure 2. The reaction mixture was adjusted to 50 mM EDTA and 50% formamide. After heating in a boiling water bath, the reaction mixture was fractionated on a Sephadex G-100-40 column (0.8 x 50 cm) using 10 mM TEAB as eluting buffer. The lyophilized single strands corresponding to duplex I (13 A/U) were reannealed in 10 mM MgCl2 and 10 mM Tris-HCl (pH 7.5). The procedure was to first warm the solution in a boiling water bath and then slowly cool (1 hour) to OOC. Fractionation of the duplex was on a Bio Gel A-0.5 M column (1 x 100 cm) using 10 mM TEAB as eluting buffer. The yield was 9.9 pmoles. Synthesis of duplex I (6,7,13 A/U): The reaction mixture (100 p4) contained 22 pmole segment [5a-6c], 22.5 pmole segment 11-2-3], 100 pM dUTP, 100 pM dCTP, 100 pM dATP, 8 mM MgCl2, 0.12 M potassium phosphate (pH 6.9) and 5 mM dithiothreitol. After warming to 370C, the reaction mixture was cooled slowly (1 hour) to OOC. E. coli DNA polymerase I (35 units) was added and the reaction allowed to proceed.4.5 hours at O°C. Analysis of the reaction by gel electrophoresis (Figure 3) indicated that repair was complete. The reaction mixture was then fractionated on a Bio Gel A-0.5 m column (1 x 100 cm) using 10 mM TEAB as eluting buffer. The isolated yield was 10 pmoles. Synthesis of duplex II (6, 7 A/U): The reaction mixture (40 3044
Nucleic Acids Research
A B C D
+ Figure 3. Synthesis of duplex I(6,7,13 A/U). The synthesis was described in the experimental section. The denaturing gel was 20% acrylamide, 1% N,N-methylenebisacrylamide in the TBE containing 7 M urea. Channel A, segment 15a-6c]; channel B, duplex II as part of an unpurified repair reaction; channel C, duplex I as part of an unpurified repair reaction; channel D, duplex I (6,7, 13 A/U) as part of the reaction mixture described in the experimental section. Synthesis of duplexes I, II as shown on this gel used segment [5a-6cJ as primer. For all the results shown on this gel, the [32pj phosphate was in the phosphodiester bond joining segment 5a to 6c.
p4)
contained 54 pmole segment [5a-6J, 54 pmole segment [2-3J, 100 PM [3HJdUTP, 100 pM dATP, 8 mM MgCl2, 0.12 M potassium phosphate (pH 6.9) and 5 mM dithiothreitol. After warming to 70°C, the reaction mixture was cooled slowly (1 hour) to O°C. E. coli DNA polymerase I (3.5 units) was added and the reaction allowed to proceed for 3 hours at O°C. The reaction mixture was then fractionated on a Bio Gel A-0.5 m column (1 x 100 cm) using 10 mM TEAB as eluting buffer. The isolated yield was 48 pmole. 1 32P 3045
Nucleic Acids Research phosphate of equivalent specific activity was in the phosphodiester bond joining segment 2 to segment 3 and segment 5a to 6. Analysis by degradation to 3'- and 5'-mononucleotides were consistent with the calculated results. Analysis for [ 3H] labeling was also consistent since essentially equivalent radioactivity was found in dUp and dU. The characterization data is shown in Table 1. RESULTS Duplex I (13 A/U), duplex I (6,7,13 A/U) and duplex II (6,7 A/U) were synthesized by repair replication using DNA polymerase I The procedure outlined by Kleppe et. al. (9) was used for all repair reactions. The synthesis of duplex I (13 A/U) required two repair replication steps. These are outlined in Figure 2. The first step utilized segment 12-31 as template, segment [5a-6c] as primer, dUTP and dCTP. Insertion of dCTP and dUTP appeared to go to completion (Figure 2, gel A). No intermediate partially repaired product was present. Step two used segment 11-2-3] as template, segment [5a-6c-U-C] as primer, dTTP, dCTP and dATP. The repair reaction appeared approximately 80% complete. One partially repaired product was also present (Figure 2, gel B). Duplex I (13 A/U) was first fractionated as denatured single strands. This step removed the partially repaired material. The final step involved chromatography of the reannealed duplex I (13 A/U). Duplex I (6,7,13 A/U) was synthesized using one repair replication step. Segment 11-2-3] was the template and segment [5a-6c] was the primer. Repair was carried out with dUTP, dCTP and dATP. Gel electrophoresis of the repair reaction after 4.5 hours is shown in Figure 3. Several markers were also included on this gel. The repair to duplex operator DNA was essentially complete. No bands corresponding to intermediate size, partially repaired operators were observed. The only contaminants were unrepaired primer and partially degraded primer. These impurities were easily removed by column chromatography on Bio Gel A-0.5 m. The synthesis of duplex II (6,7 A/U) required segment 12-3] as template and segment 15a-6] as primer. As a check on complete
repair, 1 H]dUTP
was
used.
If
repair synthesis
was
complete,
tritium radioactivity should be evenly divided between dUp and dU when the duplex is degraded to 3'-mononucleotides. Although 3046
Nucleic Acids Research the counts are low, the results indicated that repair was essentially complete (Table 1). As a final, rigorous test of purity, these duplexes were phosphorylated with [y- 32P]ATP and examined on denaturing gels. For all duplexes, two single, sharp bands corresponding to the separated strands were observed. No partially repaired lac operator segments could be detected. These gel patterns are shown in Figure 4. The kinetics of dissociation for preformed repressoroperator duplexes were examined. The operator duplexes used for this study are shown in Figure 5. Duplexes I and II were
B
A I
=
I.
n
+ Figure 4. Analysis of 15'-32P] phosphate labeled duplexes by polyacrylamide gel electrophoresis. The denaturing gels were 20% acrylamide, 1% N,N-methylenebisacrylamide in TBE containing 7 M urea. Gel A shows the analysis of duplex II (6,7 A/U). Channel I, duplex II (6,7 A/U); Channel II 15'-32p] segment [2-3]. Gel B shows the analysis of duplex I (13 A/U) in channel I, and duplex I (6,7,13 A/U) in channel II. The two bands correspond to the separated strands. The slow migrating strand is segment
11-2-3]
.
3047
Nucleic Acids Research 2
a
4 6 6 7 8 9 loll 12 1314 15 1678 1920212223242526
T-G-T-G-6-A-A-T-T-G-T-G-A-G-C-G-G-A-T-A-A-C-A-A-T-TT)-DEOXY A-C-A-C-C-T-T-WAA-C-A-C-T-C-G-C-C-T-A-T-G-T-T-A-A
C-DEOXY 3)-DEOXY
U
(5')-DEOXY
1(13 A/U)
(3')-DEOXY
________U
C5)-DEOXY
A-A-T-T-G-T-G-A-G-C-G-G-A-T-A-A- C-A-A-T-T
(3)-DEOXY
T-T-A-A-C-A-C-T-C-G-C-C-T-A-T-T- G-T-T- A-A
(5)-DEOXY
1(6,7j3
A/U)
(C)-DEOXY U-U
C5)-DEOXY
It (6,7
A/U)
Figure 5. A summation of lac operator DNA duplexes that have been used in the present investTgjation. The Roman Numerals I and II designate the 26 mer and 21 mer duplexes respectively. Parentheses following these numerals indicate sequence modifications within these duplexes. The position number of a modification site is followed by the base pair at that site. The synbol U refers to deoxyuridine. As an example, duplex I (13 A/U) refers to duplex I where position 13 has been-altered to contain an adenine-uracil base pair. The lines are used as a substitute for nucleotide symbols in the modified operators. In this way, the modified site(s) can be clearly designated with the appropriate symbol(s).
used as standards for the three uracil modified operators. Two of these modified duplexes, I (13 A/U) and I (6,7,13 A/U), contained 26 base pairs. Therefore half-lives should be compared to duplex I. The third modified operator, II (6,7 A/U), contained 21 base pairs. Thus duplex II was the control for studies with this operator. The results are shown in Figure 6 and Table 2. Each dissociation experiment was repeated twice. Furthermore, as outlined previously (2), triplicate samples were analyzed for each dissociation experiment. Therefor a total of six data points were available for each sampling time. In all these cases, the half-lives measured were within 3048
Nucleic Acids Research
0.8
0.6 0
2 0.4 0
0C
0. 0.2
5
10 MINUTES
15
Figure 6. Dissociation kinetics of various operator DNAs from QX86 repressor. The conditions for dissociation kinetic measurements are referenced in the Materials and Methods section. The ionic strength was 0.05 M. Each point is the average of two dissociation experiments (six individual assays). o, duplex I; *, duplex II; *, duplex II (6,7 A/U);- duplex I (6,7,13 A/U); U, duplex I (13 A/U). ,
Table 2
Comparison of Dissociation Rates of Repressor-Operator Complex Using SQ and QX86 Repressors Duplex Analyzed
I I (13 A/U) I (6,7,13 A/U) II II
a.
(6,7 A/U)
t½ (sec) SQ Repressor (I=0.05 M)
47