FIGURE 5.

Analysis of eggplant tRNA ligase role in ELVd RNA circularization in E. coli. Northern blot hybridization analysis of total RNA from E. coli clones in which longer-than-unit ELVd transcripts were co-expressed with eggplant tRNA ligase. RNAs were separated by denaturing PAGE and transferred to a membrane. ELVd + strands were detected with a complementary ³²P-labeled RNA probe. Lanes 1, 2, and 3, E. coli clones in which the wild-type ELVd was co-expressed with the K219A catalytic mutant of eggplant tRNA ligase (lane 1), no tRNA ligase (lane 2) or the wild-type tRNA ligase (lane 3). Lane 4, control clone in which the wild-type tRNA ligase was expressed alone. The positions and sizes (in nt) of RNA markers are indicated on the right of the panel, and the positions of the monomeric circular (mc) and linear (ml) forms of ELVd are indicated on the left. Normalized histograms of band intensities corresponding to monomeric circular and linear ELVd forms are presented on the right.