Conformaticm of viroids

Viroids are uncoated infectious RNA molecules (MW 107 OOO127 000) known as pathogens of certain higher plants. Thermodynamic and kinetic studies were carried out on highly purified viroid preparations by applying UV-absorption melting analysis and temperature jump methods. The thermal denaturation of viroids is characterized by high thermal stability, high cooperativlty and a high degree of base pairing. Two relaxation processes could be resolved; a process in the sec range could be evaluated as an independent all-or-none-transition with the following properties: reaction enthalpy = 550 kcal/mol, activation enthalpy of the dissociation = 470 kcal/mol; G : C content = 72 %. These data indicate the existence of an uninterrupted double helix of 52 base pairs. A process in the msec range involves 1 5 2 5 base pairs which are most probably distributed over several short double helical stretches. A tentative model for the secondary structure of viroids is proposed and the possible functional implications of their physicochemical properties are discussed. INTRODUCTION The term viroid has been used in recent years to describe minimal-sized infectious RNA molecules which behave biologically very similarly to conventional viruses. These RNA molecules are the agents of several economically important diseases of certain higher plants (see Review by T.O. Diener [1] ). In contrast to the RNA of usual viruses, viroid RNA is not protected by a coat protein. Although this does not exclude a possible viroid protein complex inside the host cell, infectivity and pathogenicity are associated with viroids isolated as protein-free RNA molecules. The second striking feature of viroids is their low molecular weight of about 120 OOO daltons, i.e. the RNA is about ten times smaller than the RNA of the smallest known bacterial viruses such as phage MS 2 or R 17. Since the potential genetic Information is not sufficient to © Information Retrieval Limited 1 Falconberg Court London W1V5FG England Nucleic Acids Research code for a protein with a molecular weight larger than 10 000 daltons, viroids cannot directly code for a viroid specific RNA polymerase. Accordingly little is yet understood about their mechanisms of replication and pathogenicity. During the last two years three groups have succeeded in purifying viroid RNA in amounts sufficient for physico-chemical investigations [1 , 2, 3] . Several lines of indirect evidence led Diener 1̂] to the conclusion that viroids may be either single-stranded RNA molecules of hairpin structure and extensive base pairing or doublestranded RNAs with incomplete base pairing. From optical and NMR investigations Semancik et al. [2] inferred that viroid RNA is highly structured, and that 70 80 % of all base pairs are G : C pairs. In a recent study using hydrodynamic, thennodynamic, electron microscopic, and biochemical methods, we described several new structural features of the viroid molecule [3]. First, by equilibrium sedimentation the molecular weights of six different viroids were determined to range between 107 OOO and 127 OOO daltons. Secondly, it was shown that viroids exist as a single nucleotide strand, which is partially refolded and assumes a rod-like shape in its native conformation. Finally, and most surprising, electron microscopic studies on denatured viroids, and viroid RNA endgroup analysis revealed that viroids are covalently closed circular molecules. In the present work we report physico-chemical investigations on two viroids, CEV [4] from tomato and CPFV [5] from tomato. From quantitative thermodynamic and kinetic data on the thermal denaturation of viroids relevant conclusions about the secondary structure can be drawn. We provide evidence for the existence of distinct structural regions in viroid molecules which may be responsible for specific biological functions. MATERIALS The purification of the viroids CEV [4] and CPFV [5] , both from tomato, up to gel electrophoretical homogeneity has been described in detail elsewhere [6] . According to the analysis under native and denaturing conditions in gels of different acrylamide concentration their homogeneity was estimated to be