Studies of the structure of bacteriophage λ <i>cro</i> protein in solution

Bolotina, I. A.; Kurochkin, A. V.; Кирпичников, М. П.

doi:10.1016/0014-5793(82)80623-6

Cited by 15 publications

(9 citation statements)

References 17 publications

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“…The percentages of amide I intensity contributed by the curve-fitted a-helix markers, interpreted as the percentages of a-helix secondary structure in the proteins, are summarized in Table 3. For X repressor and X Cro, the results of Table 3 are consistent with the deconvolution results and are in reasonably good agreement with the available X-ray crystal structures (Pabo & Lewis, 1982;Jordan & Pabo, 1988;Anderson et al, 1981) andNMR solution structure (Bolotina et al, 1983). The threedimensional structures indicate =60% helix in X repressor and =40% helix in X Cro.…”

Section: Results and Interpretationsupporting

confidence: 86%

Secondary structure and interaction of phage D108 Ner repressor with a 61-base-pair operator: Evidence for altered protein and DNA structures in the complex

Benevides¹,

Kukolj²,

Autexier³

et al. 1994

Biochemistry

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Ner repressors of the transposable phages Mu and D108 play a central role in regulating the expression of the early (transposase) operon and in ensuring that phage growth proceeds along a lytic pathway. The latter function is analogous to that performed by the Cro protein of phage lambda. Unlike lambda Cro, however, the structural basis of operator recognition is not known for the Ner repressors. In order to elucidate the structural features underlying operator recognition by Ner repressors, we have employed Raman spectroscopy as a probe of the solution secondary structures of both D108 Ner and Mu Ner. Additionally, we have obtained Raman spectra of the D108 Ner repressor when bound to a 61-base-pair oligodeoxynucleotide containing the 55-base-pair D108 ner binding site. Conformation-sensitive Raman bands show that both D108 and Mu Ner contain similar, highly alpha-helical (approximately 45%) secondary structures. The Raman markers also show that the substantial nonhelical secondary structure of both D108 Ner and Mu Ner is largely beta-stranded. The protein-free 61-bp D108 ner operator exhibits Raman marker bands diagnostic of an uninterrupted B DNA duplex. In the D108 Ner:DNA complex, we find the following: (i) B DNA stereochemistry is fully conserved, although with significant perturbations to the B form backbone geometry, particularly in AT-rich regions of the bound operator. (ii) The specific interactions that occur between Ner repressor and operator involve B DNA major groove sites. (iii) A small (8 +/- 3%) increase in alpha-helix content of the Ner repressor is detected upon operator binding. (iv) Finally, the local environments of many aromatic amino acids are substantially altered in the D108 Ner:DNA complex. We propose a molecular model for binding of D108 Ner to its operator that is consistent with both the present spectroscopic findings and the results of recent biochemical studies. Essential features of this model are bending of the DNA double helix and contact of operator sites with repressor domains bearing sequence homologies with the helix-turn-helix (HTH) motifs of other DNA-binding proteins. The Raman fingerprint of the Ner:DNA complex is shown to be clearly distinguishable from that of the lambda cI:DNA complex, even though both gene regulatory complexes are presumed to employ HTH recognition motifs. The unique Raman signatures observed for these repressor complexes suggest that the Raman methodology may be useful in discriminating different modes of operator recognition by the HTH motifs of regulatory proteins.

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Section: Results and Interpretationsupporting

confidence: 86%

Secondary structure and interaction of phage D108 Ner repressor with a 61-base-pair operator: Evidence for altered protein and DNA structures in the complex

Benevides¹,

Kukolj²,

Autexier³

et al. 1994

Biochemistry

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“…However, other interpretations are possible. Analysis of the CD spectra of the cro indicated a secondary structure distribution of35% a helix, 20% ~-strand and 45% undefined conformation (37). This is in agreement with a published secondary structure distribution derived from X-ray analysis of cro crystals (3).…”

Section: Spectra Of Complexes Between Cro Protein and Dna In The Fsupporting

confidence: 68%

Interaction of Lambda cro Repressor with Synthetic Operator 0_R3 Studied by Competition Binding with Minor Groove Binders

Gursky

Surovaya

Kurochkin

et al. 1992

Journal of Biomolecular Structure and Dynamics

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In the present work, we employ a combination of CD spectroscopy and gel retardation technique to characterize thermodynamically the binding of lambda phage cro repressor to a 17 base pair operator OR3. We have found that three minor groove-binding antibiotics, distamycin A, netropsin and sibiromycin, compete effectively with the cro for binding to the operator OR3. Among these antibiotics, sibiromycin binds covalently to DNA in the minor groove at the NH2 of guanine, whereas distamycin A and netropsin interact preferentially with runs of AT base pairs and avoid DNA regions containing guanine bases in the two polynucleotide strands. Only subtle DNA conformation changes are known to take place upon binding of these antibiotics. Both the CD spectral profiles and the results of the gel retardation experiments indicate that distamycin A and netropsin can displace cro repressor from the operator OR3. The binding of cro repressor to the OR3 is accompanied by considerable changes in CD in the far-UV region which appear to be attributed to a DNA-dependent structural transition in the protein. Spectral changes are also induced in the wavelength region of 270-290 nm. The CD spectral profile of the cro-OR3 mixture in the presence of distamycin A can be represented as a sum of the CD spectrum of the repressor-operator complex and spectrum of distamycin-DNA complex at the appropriate molar ratio of the bound antibiotic to the operator DNA (r). When r tends to the saturation level of binding the CD spectrum in the region of 270-360 nm approaches a CD pattern typical of complexes of the antibiotic with the free DNA oligomer. This suggests that simultaneous binding of cro repressor and distamycin A to the same DNA oligomer is not possible and that distamycin A and netropsin can be used to determine the equilibrium affinity constant of cro repressor to the synthetic operator from competition-type experiments. The binding constant of cro repressor to the OR3 is found to be (6 +/- 1).10(6)M-1 at 20 degrees C in 10 mM sodium cacodylate buffer (pH 7.0) in the presence of 0.1 M NH4F.

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“…Cro was isolated and purified according to procedures described previously (15). Cro concentration was determined using the molar extinction coefficient at 276 nm of 4330 (22).…”

Section: Methodsmentioning

confidence: 99%

¹H NMR sudy of the interaction of bacteriophageλ Cro protein with the O_R3 operator. Evidence for a change of the conformation of the O_R3 operator on binding

Кирпичников¹,

Hahn²,

Buck³

et al. 1984

Nucl Acids Res

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The specific complex between the lambda phage OR3 operator and the Cro protein has been studied by proton NMR spectroscopy at 500 MHz. The DNA imino proton resonances of this complex have been assigned to specific base pairs using the known assignments of these resonances for the free operator. Increase of the protein/DNA ratio to complete saturation of the OR3 operator with the Cro protein made it possible to follow the shift changes of the resonances. Ambiguities were resolved by nuclear Overhauser effect measurements on the complex. The shifts of the imino proton resonance positions provide information on the changes induced in the conformation of the operator upon complex formation with a dimer of the Cro protein. The most striking shift occurs for the central (GC 9) base pair, which is known to have no direct contacts with the Cro protein. This shift may be induced by a bend in the OR3 operator DNA at the GC 9 base pair to accommodate the operator for the binding of the Cro protein dimer. The imino proton resonances of two additional base pairs can be observed in the complex, demonstrating an overall stabilization of the DNA structure by the binding of the Cro protein.

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Studies of the structure of bacteriophage λ cro protein in solution

Cited by 15 publications

References 17 publications

Secondary structure and interaction of phage D108 Ner repressor with a 61-base-pair operator: Evidence for altered protein and DNA structures in the complex

Secondary structure and interaction of phage D108 Ner repressor with a 61-base-pair operator: Evidence for altered protein and DNA structures in the complex

Interaction of Lambda cro Repressor with Synthetic Operator 0_R3 Studied by Competition Binding with Minor Groove Binders

¹H NMR sudy of the interaction of bacteriophageλ Cro protein with the O_R3 operator. Evidence for a change of the conformation of the O_R3 operator on binding

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Studies of the structure of bacteriophage λ cro protein in solution

Cited by 15 publications

References 17 publications

Secondary structure and interaction of phage D108 Ner repressor with a 61-base-pair operator: Evidence for altered protein and DNA structures in the complex

Secondary structure and interaction of phage D108 Ner repressor with a 61-base-pair operator: Evidence for altered protein and DNA structures in the complex

Interaction of Lambda cro Repressor with Synthetic Operator 0R3 Studied by Competition Binding with Minor Groove Binders

1H NMR sudy of the interaction of bacteriophageλ Cro protein with the OR3 operator. Evidence for a change of the conformation of the OR3 operator on binding

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Interaction of Lambda cro Repressor with Synthetic Operator 0_R3 Studied by Competition Binding with Minor Groove Binders

¹H NMR sudy of the interaction of bacteriophageλ Cro protein with the O_R3 operator. Evidence for a change of the conformation of the O_R3 operator on binding