Abstract
BackgroundViroids are the smallest pathogens of plants. To date the structural and conformational details of the cleavage of Avocado sunblotch viroid (ASBVd) and the catalytic role of Mg2+ ions in efficient self-cleavage are of crucial interest.ResultsWe report the first Raman characterization of the structure and activity of ASBVd, for plus and minus viroid strands. Both strands exhibit a typical A-type RNA conformation with an ordered double-helical content and a C3′-endo/anti sugar pucker configuration, although small but specific differences are found in the sugar puckering and base-stacking regions. The ASBVd(-) is shown to self-cleave 3.5 times more actively than ASBVd(+). Deuteration and temperature increase perturb differently the double-helical content and the phosphodiester conformation, as revealed by corresponding characteristic Raman spectral changes. Our data suggest that the structure rigidity and stability are higher and the D2O accessibility to H-bonding network is lower for ASBVd(+) than for ASBVd(-). Remarkably, the Mg2+-activated self-cleavage of the viroid does not induce any significant alterations of the secondary viroid structure, as evidenced from the absence of intensity changes of Raman marker bands that, however exhibit small but noticeable frequency downshifts suggesting several minor changes in phosphodioxy, internal loops and hairpins of the cleaved viroids.ConclusionsOur results demonstrate the sensitivity of Raman spectroscopy in monitoring structural and conformational changes of the viroid and constitute the basis for further studies of its interactions with therapeutic agents and cell membranes.
Highlights
Viroids are the smallest pathogens of plants
Viroids are divided into two families, the Avsunviroidae such as Avocado sunblotch viroid (ASBVd) that all possess a catalytic RNA with a hammerhead ribozyme (HHR) motif responsible for a crucial cleavage step during viroid replication and
Since the magnitude I813 is directly proportional to the number of phosphodiester linkages in ordered configurations and that of I1100 is independent of such structural factors, we have defined the ratio r2 = I813/I1100 to characterize the amount of the secondary structure
Summary
To date the structural and conformational details of the cleavage of Avocado sunblotch viroid (ASBVd) and the catalytic role of Mg2+ ions in efficient self-cleavage are of crucial interest. The analysis of the ASBVd contents in avocado extracts [9] revealed the presence of RNA of both polarities in multimeric forms, from monomers to octamers for ASBVd(+) and monomers to dimers for ASBVd(−) This difference in oligomeric sizes reveals a less efficient in vivo cleavage activity of ASBVd(+) than of ASBVd(−) than was observed by in vitro cleavage. The predicted structures of ASBV(−) and (+) strands have been studied experimentally by Navarro and Flores [11,12] These structural components might represent the driving force necessary for the viroid to penetrate the cell as well as to interact with cell components. From base-sequence alignments it is suggested that proto-tRNAGly was possibly a viroid-like self-cleavable ribozyme with a hammerhead-like motif [13]
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have