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

Abstract

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.

Structural Perturbations Induced in Linear and Circular DNA by the Architectural Protein HU from Bacillus stearothermophilus

“…Hence, the bending of DNA C222 by 1.7°/bp is considered to be the principal source of the Raman difference bands of Figure 3C. These results contrast strikingly with the much larger effects of protein-induced DNA curvature (26,29,52,56). The latter perturbations are characterized by both shifts in wavenumber values and changes in amplitudes affecting numerous Raman bands, including several that are diagnostic of specific base interactions.…”

Raman, polarized Raman and ultraviolet resonance Raman spectroscopy of nucleic acids and their complexes

“…A case in point is the specific complex between phage D108 Ner repressor and a 61 base-pair operator site. 144 The empirical database can also be used to characterize complexes of related molecules such as mutant transcription factors or operators. 25,143 Other protein-DNA complexes In addition to the gene regulatory proteins discussed above, several other DNA binding proteins have been probed by Raman spectroscopy.…”

Raman spectroscopy of DNA modified by intrastrand cross-links of antitumor cisplatin

“…The 782 cm À1 peak in the Raman spectra of DNA is assigned to cytosine and the 791 cm À1 peak is assigned to a stretching vibration of backbone phosphodiester groups and diagnostic of B-form DNA backbone geometry (Benevides et al, 1994;Benevides et al, 2000;Movileanu et al, 1999). The 782 cm À1 cytosine and 791 cm À1 backbone bands overlap in the spectrum of DNA to form a large 784 cm À1 peak (Dostal et al, 2004).…”