Figure 5: The impacts of sequence and structure on termination of transcription from type 2 Pol III promoters.

A) A schematic depicting the construction of our reporters with type 2 promoters versus the previously utilized type 3 promoters. Our type 2 promoter encompassed the transcription start site (TSS) as well as the complete tRNA (GLN or GLY) sequence. This diagram illustrates the positioning of predicted RNA secondary structure upstream of the poly-U tract for type 2 Pol III promoters. Constructs were made by fusing each of two type 2 tRNA promoters with a portion of the U6 leader sequence (minus the first 19 nt) to increase the length of the predicted linear region before the poly-U tract. An additional 23 nt portion of the 5S ribosomal RNA (rRNA), predicted to fold into a 9 bp hairpin structure, was either omitted (No Hairpin) or positioned 10 nt upstream of the poly-U tract. In these constructs, the Linear-3 sequence was utilized. B) Constructs using type 2 Pol III promoters show different patterns of termination from those using a type 3 promoter. Significant termination is seen whenever a poly-U tract of 4 uracils is present, independent of the presence of a predicted hairpin structure. Significance was measured using a one-tailed heteroscedastic Welch’s t-test (Supplementary Table 4). ** = p < 0.01, *** = p < 0.001. Colored bars represent the average of 3 biological replicates with individual points plotted as circles. Error bars represent the S.E.M. The dashed line represents the average of three replicates for the vector-only control (v) and the grey horizontal bar represents the S.E.M. of the vector-only control.