A.O. Pasternak et al. discontinuous sg RNA synthesis. Remarkably, the effects of single (leader or body) TRS mutations were mostly base speci®c, i.e. different nucleotide substitutions at the same position affected sg RNA7 synthesis to different extents. For example, at position 1, the BU 1 A mutant retained 44% of the wild-type RNA7 synthesis level, whereas both the BU 1 C and BU 1 G mutants lost RNA7 synthesis almost completely. Conversely, when U 1 of the leader TRS was changed to A or G, RNA7 synthesis was completely abolished, whereas 13% of the wild-type level was still maintained by LU 1 C. For position 2, only the BC 2 U mutant retained 30% of the wild-type RNA7 synthesis level, while all the other position 2 single mutants have lost 90% or more of wild-type RNA7 synthesis. Another example is position 6: BU 6 C left only 5% of wild-type RNA7 synthesis, whereas BU 6 A produced much higher RNA7 levels. This implied that for some positions (1, 2 and 6), certain mismatches in the duplex between plus leader TRS and minus body TRS, such as U±U (BU 1 A and BU 6 A) or C±A (LU 1 C and BC 2 U), are allowed to a limited extent. In contrast, no mismatches were allowed for position 5, where all single nucleotide substitutions abolished RNA7 synthesis almost completely. Surprisingly, both body TRS U to C substitutions at positions 1 and 6 (BU 1 C and BU 6 C) resulted in low levels of RNA7, despite the fact that these mutations allow the formation of a G±U base pair between the plus leader TRS, providing the U nucleotide, and the minus body TRS, providing the G. On the other hand, for positions 3 and 4, G±U base pairing was shown to be functional, because mutants LA 3 G and LA 4 G, which can form G±U base pairs between the G in the plus leader TRS and U in the minus body TRS, were the only position 3 and 4 single mutants that produced reasonable levels of RNA7. Taken together, these ®ndings suggest that other factors, besides leader± body base pairing, also play a role in sg RNA synthesis and that the primary sequence (or secondary structure) of TRSs may dictate strong base preferences at certain positions. Our analysis of the degree of complementation by the double mutants provided strong support for this assumption.