Volume 6 Number 7 1979
Nucleic Acids Research
Studies on gene control regions X. The effect of specific adenine-thymine transversions on the lac repressor-lac operator interaction
H.S.Sista, R.T.Loder and M.H.Caruthers Department of Chemistry, University of Colorado, Boulder, CO 80309, USA Received 26 March 1979 ABSTRACT
Chemical and enzymatic methods were used to synthesize a transition (AT to GC) and a transversion (AT to TA) at a lac operator site known to interact with lac repressor through the thymine 5 methyl group. These operators also contained a poly(dA).poly(dT) tail 8 to 12 base pairs in length at one end. Results suggest that the steric constraints of lac repressor relative to the position of the 5 methyl group are quite critical. For example a seven fold reduction in stability was observed for the transversion. Results also suggest that the operator spans at least 21 base pairs. INTRODUCTION
Previous results from this laboratory indicate that the 5 methyl group on thymine at position 13 (Figure 1) is a major lac repressor recognition site (1). Additional data also suggests that the methyl groups at positions 6 and 7 are minor but specific recognition sites (5,6). The transversion from adenine-thymine (A-T) to thymine-adenine (T*A) at any of these sites is therefore of interest. Such a transversion shifts the methyl group approximately 13 A and to the opposite side of the major groove. The consequence of this shift is unknown since this transversion was not found among the sequenced operator constitutive (Oc) mutants (3). The objective of the research outlined in this paper was therefore two-fold. (1) Prepare lac operator containing the base pair change A-T to T-A at sites 6, 7 and 13. (2) Measure the stability of these lac repressor-lac operator (RO) complexes. As a consequence of these experiments, additional information on the size of the lac operator and the steric constraints on the interaction at sites 6,7 and 13 has been obtained. C) Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
2583
Nucleic Acids Research 2 3 4 5
6 7 8 9 10 If 12 13 14 15 16 17 18 19 2021 2223 242526
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
(3')-DEO XY
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
(5')-DEOXY
- --
+ ++ +
Figure 1.
+-
-
-
A
T GT T A
C
T
T
ACAAT
G
A
+
+
++
-
METHYLATION
OC MUTATIONS ++++
CROSS-LINKED THYMINE
Chemically and enzymatically synthesized lac operator DNA. Below the sequence is shown the increase (+) or decrease (-) in methylation of purines by dimethylsulfate in the presence of lac repressor (2). Also shown directly below the appropriate positions are the seauence changes for variolus operator constitutive mutants (3) and the thymine sites crosslinked with repressor in the presence of uv light when 5-bromouracil replaces thymine (4). The heavy lines above and below the sequence delineate 2-fold symmetric regions. The dyad axis is indicated by the arrow.
MATERIALS AND METHODS Commercial sources for various chemicals, reagents, and enzymes have been listed elsewhere (5,7). Deoxynucleotidyl terminal transferase was purchased from P. L. Biochemicals. One unit of enzyme catalyzes the incorporation of one nanomole of deoxyadenylate into oligonucleotide per hour at 370 C, pH 6.8 with d(pT)6 as primer (8). The P. L. Biochemicals minimal nuclease preparation was used. T4-ligase, T4-kinase, E. coli DNA Polymerase I and lac repressor were isolated as described previously (9). Thymidylic acid polymers of defined sizes were a gift from Dr. M. Downing. These polymers were prepared using a published procedure (10). The chemical and enzymatic syntheses of lac operator deoxyoligonucleotides were carried out according to published procedures (7,11). The membrane filter assays were performed basically as described previously (12,13,14). Adaptations of this method to our system have been published (9). The duplexes used 2584
Nucleic Acids Research En
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Nucleic Acids Research
Studies on gene control regions X. The effect of specific adenine-thymine transversions on the lac repressor-lac operator interaction
H.S.Sista, R.T.Loder and M.H.Caruthers Department of Chemistry, University of Colorado, Boulder, CO 80309, USA Received 26 March 1979 ABSTRACT
Chemical and enzymatic methods were used to synthesize a transition (AT to GC) and a transversion (AT to TA) at a lac operator site known to interact with lac repressor through the thymine 5 methyl group. These operators also contained a poly(dA).poly(dT) tail 8 to 12 base pairs in length at one end. Results suggest that the steric constraints of lac repressor relative to the position of the 5 methyl group are quite critical. For example a seven fold reduction in stability was observed for the transversion. Results also suggest that the operator spans at least 21 base pairs. INTRODUCTION
Previous results from this laboratory indicate that the 5 methyl group on thymine at position 13 (Figure 1) is a major lac repressor recognition site (1). Additional data also suggests that the methyl groups at positions 6 and 7 are minor but specific recognition sites (5,6). The transversion from adenine-thymine (A-T) to thymine-adenine (T*A) at any of these sites is therefore of interest. Such a transversion shifts the methyl group approximately 13 A and to the opposite side of the major groove. The consequence of this shift is unknown since this transversion was not found among the sequenced operator constitutive (Oc) mutants (3). The objective of the research outlined in this paper was therefore two-fold. (1) Prepare lac operator containing the base pair change A-T to T-A at sites 6, 7 and 13. (2) Measure the stability of these lac repressor-lac operator (RO) complexes. As a consequence of these experiments, additional information on the size of the lac operator and the steric constraints on the interaction at sites 6,7 and 13 has been obtained. C) Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
2583
Nucleic Acids Research 2 3 4 5
6 7 8 9 10 If 12 13 14 15 16 17 18 19 2021 2223 242526
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
(3')-DEO XY
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
(5')-DEOXY
- --
+ ++ +
Figure 1.
+-
-
-
A
T GT T A
C
T
T
ACAAT
G
A
+
+
++
-
METHYLATION
OC MUTATIONS ++++
CROSS-LINKED THYMINE
Chemically and enzymatically synthesized lac operator DNA. Below the sequence is shown the increase (+) or decrease (-) in methylation of purines by dimethylsulfate in the presence of lac repressor (2). Also shown directly below the appropriate positions are the seauence changes for variolus operator constitutive mutants (3) and the thymine sites crosslinked with repressor in the presence of uv light when 5-bromouracil replaces thymine (4). The heavy lines above and below the sequence delineate 2-fold symmetric regions. The dyad axis is indicated by the arrow.
MATERIALS AND METHODS Commercial sources for various chemicals, reagents, and enzymes have been listed elsewhere (5,7). Deoxynucleotidyl terminal transferase was purchased from P. L. Biochemicals. One unit of enzyme catalyzes the incorporation of one nanomole of deoxyadenylate into oligonucleotide per hour at 370 C, pH 6.8 with d(pT)6 as primer (8). The P. L. Biochemicals minimal nuclease preparation was used. T4-ligase, T4-kinase, E. coli DNA Polymerase I and lac repressor were isolated as described previously (9). Thymidylic acid polymers of defined sizes were a gift from Dr. M. Downing. These polymers were prepared using a published procedure (10). The chemical and enzymatic syntheses of lac operator deoxyoligonucleotides were carried out according to published procedures (7,11). The membrane filter assays were performed basically as described previously (12,13,14). Adaptations of this method to our system have been published (9). The duplexes used 2584
Nucleic Acids Research En
ro OL
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W)
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I ( 0 ( T a)) -H h4 bh
4-
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