Analysis of RNA structures by temperature-gradient gel electrophoresis: viroid replication and processing

Abstract

The structure and structural transitions of single-stranded RNA were investigated by energy calculations and temperature-gradient gel electrophoresis. Most experiments have been carried out on RNA of mature viroids and their replication intermediates, which are RNA (−) strand oligomers and RNA (+) strand oligomers. The technique of temperature-gradient gel electrophoresis proved to be particularly useful for analysing co-existing structures. The secondary structure of lowest free energy for unit length and oligomeric replication intermediates is an extended rod-like structure similar to that of the mature circular viroid. When this structure is used as a model for calculations, there is a large degree of agreement between theoretical and experimental curves. Under particular solution conditions, however, (+) strand oligomers undergo a rearrangement from the extended structure to a branched structure, in which every two units form a region of three helices, together 28 bp long. This structure is called the tri-helical structure. The process of structure formation during the synthesis of oligomers could be followed: at first, a transient multi-branched structure is formed which is then transformed into the extended and the tri-helical structures. The region of the three stable helices serves to divide up the oligomeric (+) strand into structural units which may be recognized by cleavage and ligation enzymes, and be processed into circular mature viroids. Co-transcription of complementary (+) and (−) strands shows that energetically favored double-strand formation may at least partially be prohibited by stable secondary structures of the single strands. Natural replication intermediates have been analysed in respect to their subcellular location and their size distribution. They are associated with the nucleoli as was found earlier for mature viroids. Natural (−) strand oligomers are larger than (+) strand oligomers; both types show a periodicity in the size distribution of two units. The models of the structures, which are involved in viroid processing, are in accordance with recent infectivity data and with the results on natural replication intermediates.