Fig. 4.

Activity of tTFIIB mutants with alanine substitutions in in vitro SL RNA transcription assays. (A) Transcription activity in tTFIIB-depleted extracts (lanes 1 and 10) was restored upon addback of wild-type (WT) tTFIIB (lanes 2 and 11). In the loop linking H2 and H3, the R138A mutant had near WT activity (lane 4), but both R135A and the triple mutant nearly abolished activity. Mutations in H5 and H6 (R179A, R194A, R195A; lanes 12–14) reduced activity ≈50%. In the linker between H3′ and H4′, K168A also reduced activity ≈50% (lane 17), while K270 had near WT activity (lane 18). In helix H5′, T292A had increased activity (lane 6), while K293A, N295A, and K296A toward the center of the helix had reduced activity (lane 7–9). (B) Histogram represents averages of 3 replicates for each mutant. Error bars indicate 1 standard deviation.