Radboud Repository of the Radboud University Nijmegen

The 3'-5' circular trinucleotide cr(GpGpGp) was studied by means of 1 D and 2D high resolution NMR techniques and molecular mechanics calculations. Analysis of the J-couplings, obtained from the 1H and 13C-NMR spectra, allowed the determination of the conformation of the sugar rings and of the 'circular' phosphate backbone. In the course of the investigations it was found that the Karplus-equation most recently parametrized for the CCOP J-coupling constants could not account for the measured J(C4'P) of 11.1 Hz and a new parametrization for both HCOP and CCOP coupling constants is therefore presented. Subsequent analysis of the coupling constants yielded 'fixed' values for the torsion angles ,B and 6 (with : = 1780 and 6 = 1390). The value of the latter angle corresponds to an S-type sugar conformation. The torsion angles ey and e are involved in a rapid equilibrium in which they are converted between the gauche(+) and trans and between the trans and gauche( -) domain respectively. We show that the occurrence of E in the gauche( -) domain necessitates S-type sugar conformations. Given the aforementioned values for (, ay, 6 and e the ring closure constraints for the ring, formed by the phosphate backbone can only be fulfilled if a and r adopt some special values. After energy minimization with the CHARMm force field only two combinations of a and r result in energetically favourable structures, i.e. the combination a(t)/I(g -) in case ey is in a gauche(+) and e is in a trans conformation, and the combination a(t)/I(g +) for the combination 7(t)/E(g -). The results are discussed in relation to earlier findings obtained for cd(ApAp) and cr(GpGp), the latter molecule being a regulator of the synthesis of cellulose in Acetobacter xylinum. INTRODUCTION Low molecular weight nucleosides and nucleotides such as cyclic molecules in which the nucleotide units are connected by 3'-5' linkages, play important roles in many cellular functions. For example, the circular ribo-dinucleotides cr(UpUp) and cr(ApUp) are effective inhibitors of the DNA dependent RNA-polymerase of the bacterium Escherichia coli during the initiation phase of transcription (1,2). A completely different activity is exhibited by the circular dinucleotide cr(GpGp), which is an activator of the enzyme cellulose synthase in the bacterium Acetobacter xylinum (3). Some structural analogs, like cr(IpIp), cd(GpGp) and two diastereomers of the monophosphothioates cr(Gp(S)Gp) were found to be relatively potent activators of the cellulose synthase as well. On the other hand, closely related molecules were found to be only marginally stimulatory, like cd((Gp)r(Gp)) and cr(GpGpGp) or entirely devoid of stimulatory activity, like cd(ApAp), cr(CpCp), cr(UpUp) and cr(GpCp) (4). The structural reason for this is not clear. Therefore we investigated the solution structure of the circular trinucleotide cr(GpGpGp). This molecule is schematized in Fig. 1. A second reason for studying cr(GpGpGp) is that such circular molecules contain structural elements which may enhance the understanding of folding in larger DNA and RNA molecules. To obtain the solution structure of cr(GpGpGp) we have employed high resolution NMR and molecular mechanics calculations. The most salient finding is that the ribose rings of the molecule adopt a pure S-conformation at the temperatures studied, contrary to what is usually found for these types of sugars. MATERIALS AND METHODS