Molecular assembly of tobacco mosaic virus in vitro

Abstract

TMV assembly starts with a specific interaction between the assembly origin on the RNA and a disk aggregate of coat protein. The assembly origin is located in the 30K protein cistron for common and tomato strains of TMV and in the coat protein cistron for cowpea strain of TMV and for CGMMV. All the assembly origins have three essential structures: a long base-paired hairpin loop structure; a target sequence, GAPuGUUG, at the top of the hairpin loop structure; and a tract where every third base is a purine. The protein aggregate responsible for the initiation of TMV assembly is a 20S disk, a two-layered aggregate of 34 protein subunits. The two layers of a disk open apart onto the central hole and this structure may be critical for the disk to interact with the assembly origin on the RNA. The target sequence may bind specifically to this structure. Although only a low concentration of 20S disks exists in the usual assembly condition, one disk is enough to initiate TMV assembly. TMV elongation proceeds in two directions. Elongation to the 5'-end proceeds rapidly by preferential incorporation of protein subunits (or A protein) and in 5-7 min gives rise to 260 nm intermediate particles whose 5'-end is coated. A model of elongation toward the 5'-end is shown in Fig. 15. Protected RNAs from nuclease digestion during the assembly reaction produce a banding pattern on gels by electrophoresis. The banding pattern reflects features of the RNA rather than protein that are used in the assembly reaction, since the pattern was the same for assembly between TMV-RNA and CGMMV protein subunits as for assembly between TMV-RNA and TMV protein containing 20S aggregates. The 20S aggregate in the assembly solution has a helical structure with 39 protein subunits rather than the disk structure. Rapid addition of 20S helical aggregates to the top of the growing rod seems to be impossible because of its topological complexity. Elongation toward the 3'-end does not start for at least the first 4 min after initiation. It probably cannot begin until the 5'-end RNA tail disappears into the intermediate rod. Elongation toward the 3'-end favors 20S aggregates as the protein source and gives rise to the full-length rods in about 30 min after the initiation. There are no topological difficulties in adding 20S helical aggregates to the protruding RNA tail.(ABSTRACT TRUNCATED AT 400 WORDS)