Lac repressor binding to operator analogues: Comparison of poly[d(A-T)], poly[d(A-BrU)], and poly[d(A-U)]

doi:10.1016/0006-291X(71)90195-1

Biochemical and Biophysical Research Communications

Volume 45, Issue 6, 17 December 1971, Pages 1542-1547

https://doi.org/10.1016/0006-291X(71)90195-1 Get rights and content

Abstract

Previous work has established that Escherichia coli lac repressor binds strongly to poly[d(A-T)]. Here we use competition experiments to measure repressor binding to synthetic DNA's related to this polymer. Poly[d(A-BrU)] competes about forty times more effectively for repressor than does poly[d(A-T)], whereas poly[d(A-U)] competes twenty fold less effectively. This result suggests that the atom or group occupying the 5-position of the pyrimidine ring is important for recognition by repressor.

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Production of the glycoprotein hormone common α-subunit and placental alkaline phosphatase activity can be modulated in HeLa cells by a variety of deoxynucleosides. Dose response curves for thymidine (Thd), fluorodeoxyuridine (FdUrd), bromodeoxyuridine (BrdUrd) and iododeoxyuridine (IdUrd) demonstrate that, in general, alkaline phosphatase was increased by lower concentrations of inducer than was α-subunit. The deoxynucleosides were not as effective as sodium butyrate as inducers of either protein. Whereas Thd and the halogenated dUrd derivatives enhanced protein expression, deoxycytidine (dCyd) had negative effects. Induction by deoxynucleosides of both alkaline phosphatase and α-subunit was inhibited by dCyd, but induction of alkaline phosphatase by butyrate was more sensitive to dCyd inhibition than was the buryrate-mediated induction of α-subunit. These results suggest that the two proteins are not regulated in a coordinate manner. Reversal of alkaline phosphatase induction by dCyd was not observed in cells preincubated with sodium butyrate for 6–24 h before the addition of dCyd, indicating that the deoxynucleoside interferes with an early event in the butyrate-mediated response. Combinations of butyrate with Thd, BrdUrd or IdUrd were synergistic with respect to the induction of HeLa-α. It is concluded that incorporation of the deoxynucleosides into DNA may not be required for the synergistic response since 2′,5′-dideoxythymidine was an effective as Thd. Cytoplasmic dot hybridizations demonstrate that a primary effect of the various effectors is to increase the steady-state levels of α-subunit mRNA. There was a good correlation between α-subunit accumulation and corresponding levels of α-mRNA, suggesting that regulation occurs at a pretranslational site. Although the mechanism(s) is not understood, these data provide evidence that nucleosides or their derivatives can significantly affect gene expression.
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5-Bromo-2′-deoxyuridine induces placental alkaline phosphatase biosynthesis in cultured choriocarcinoma cells
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5-Bromo-2′-deoxyuridine (BrdUrd) stimulated the biosynthesis and hence increased the activity of placental alkaline phosphatase in choriocarcinoma cells. While BrdUrd had no effect on the rate of degradation or processing of placental alkaline phosphatase, it increased the rate of phosphatase synthesis. The stimulation of enzyme activity could be completely accounted for by the increase in alkaline phosphatase protein. Both control and BrdUrd-induced cells contained polypeptides of 61,500 and 64,500 Da, identified as the precursor and fully processed forms of placental alkaline phosphatase monomer. The half-life of this enzyme monomer in both control and BrdUrd-treated cells was estimated to be 36 h. BrdUrd induced a specific increase in the placental alkaline phosphatase mRNA leading to the observed enhancement of biosynthesis. The continued rise in alkaline phosphatase biosynthesis in BrdUrd-induced cells following BrdUrd removal indicated that this analog acted by incorporation into DNA.
Nonspecific binding of lac repressor to DNA. I. An absorption and circular dichroism study
1983, Biophysical Chemistry
Nonspecific binding of lac represser on DNA has been studied by absorption and circular dichroism (CD) spectroscopies. In a first step. the complex formation is accompanied by an absorption difference spectrum and a change of the CD signal of the DNA- The absorption difference spectrum is mainly due to a spectral change of the DNA- The variation of the CD signal has been analyzed according to a model calculation, which takes into account the fact that the excluded site is shorter than the perturbed site. We found that in this first step one represser can bind every 14 ±2 base-pairs, whereas one represser perturbs 22 ± 2 base-pairs. In a second step, more represser can bind on DNA. but without further change in the absorption and CD spectrum, indicating that another binding process occurs. The model calculation developed here is general for all binding processes inducing a perturbation over a length of DNA longer than that of the excluded site,
Studies on synthetic chromatins containing poly(dA-dT)·poly(dA-dT) and poly(dG-dC)·poly(dG-dC)
1983, BBA - Gene Structure and Expression
Core histones, (H2A,H2B,H3,H4)₂, were reconstituted with the synthethic polynucleotides poly(dA-dT)·poly(dA-dT) and poly(dG-dC)·poly(dG-dC) to yield synthetic chromatins containing 200 basepairs per octamer. These synthetic chromatins displayed a 36% decrease in the circular dichroism (CD) peak ellipticity from the value of the polynucleotide free in solution; the poly(dA-dT)·poly(dA-dT)/chromatin showed an increase in the complexity of the thermal denaturation profile compared to that of the polynucleotide. Both the temperature of maximum $d h d T$ for each transition (T_m) and the relative amount of poly(dA-dT)·poly(dA-dT) in the synthetic chromatin melting in each of the four thermal transitions is a function of the ionic strength over the 0–5 mM sodium phosphate range (0.25 mM EDTA, pH 7.0); a shift of material toward higher melting transitions was observed with increasing ionic strength. The CD peak ellipticity value for both synthetic chromatins was ionic strength-independent over the 0–5 mM sodium phosphate range. These results are in contrast to those observed with $H 1 H 5$ stripped chicken erythrocyte chromatin (Fulmer, A. and Fasman, G.D. (1979) Biopolymers 18, 2875–2891), where an ionic strength dependence was found. Differences in the CD spectra between poly(dA-dT)·poly(dA-dT)/chromatin, poly(dG-dC)·poly(dG-dC)/chromatin and $H 1 H 5$ stripped chicken erythrocyte chromatin suggest subtle differences in assembly. Finally, the temperature dependence of the CD spectra of poly(dA-dT)·poly(dA-dT)-containing synthetic chromatin, which is similar to that for the polynucleotide, suggests the core histone bound polynucleotide has a large degree of conformational flexibility allowing it to undergo the premelt transition.
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The androgen receptor from mouse kidney cytosol has been studied for its nucleic acid binding properties by DNA-cellulose centrifugation assay. The receptor appears to bind to RNA (mRNA, tRNA, rRNA) as well as to DNA. Salt and heat activation of the androgen receptor enhances both DNA and RNA binding. The receptor binds slightly better to denatured DNA than to native DNA. The androgen receptor binds about 2-fold tighter to poly(dG-dC) than to poly(dA-dT). The interaction of the receptor with DNA is not greatly affected by the BrdUrd substitution. The observation that androgen receptor shows a significant affinity to RNA may imply that androgen receptor-RNA interaction could play a role in gene regulation.

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^☆: Supported by grants from the National Institutes of Health (1-R01-HD04420) and the National Science Foundation (GB-26517).

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Lac repressor binding to operator analogues: Comparison of poly[d(A-T)], poly[d(A-BrU)], and poly[d(A-U)]☆

Abstract

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