Studies of tobacco mosaic virus reassembly with an RNA tail blocked by a hybridised and cross-linked probe.

Segments of cloned cDNA to tobacco mosaic virus RNA, 150--300-bases long, have been hybridised and cross-linked to the RNA, which has then been used for reassembly experiments. This enables the elongation reaction, which does not encapsidate the double-stranded region generated, to be stopped at specific regions along the RNA and the resulting particles to be characterised, by measuring the lengths of the rods in the electron microscope. With hybridisation to the 3'-tail the entire RNA contiguous to the nucleation region is encapsidated, from the 5'-terminus up to the modified region. When the double-stranded region is on the 5'-side of the nucleation region, the mean length of the particles corresponds to a situation in which the double-stranded region is unable to enter the central hole of the growing rod, but the 3'-tail of the RNA is completely encapsidated. The longest particles hybridised on the 5'-tail (i.e. in a class longer than the mean length) show an effect complementary to those with a 3'-block, and have lengths which correspond to encapsidation from the modified region to the 3'-terminus, despite the continued presence of the 5'-tail up the rod. In all cases where there is a remaining 5'-tail the lengths observed can only be explained if elongation has occurred substantially, or probably completely, along the 3'-tail. Hence elongation must have occurred simultaneously along both the 5' and 3'-tails of the tobacco mosaic virus RNA after initiation on the internal nucleation region.