Figure 2. RNA pol III type 2_(3A) A box promoter element requirements in the transcription of the γHV68 TMER-1 and TMER-5 genes.

(A) The A box promoter element consensus sequence, the overlapping A box configuration, and the 3A box consensus sequence and its comparison to the γHV68 TMER gene A box consensus sequence is shown. Bases within the A box promoter elements that are highlighted red are invariant in highly expressed human tRNAs (Canella et al., 2010). The underlined R and N bases in the TMER 3A box consensus sequence represent differences between this consensus sequence and the constructed 3A box consensus sequence above it at positions where the bases are predicted to be invariant. (B) Schematic detailing the wildtype and mutant sequences of the RNA pol III type 2_(3A) promoter sequences found within the TMER-1 and the TMER-5 genes. Red underlined bases represent replacement mutations created within each TMER gene. Below each sequence is a schematic of the resulting potential RNA pol III promoter. Light grey boxes represent wildtype A box promoter elements while red boxes represent mutated A box promoter elements. Dark grey boxes represent wildtype B box promoter elements. (C, D) Northern blot analysis of γHV68 TMER-1 and TMER-5. (C) Blot probed using a 5’-biotinylated probe designed to hybridize antisense to the miR-M1-1 sequence. (D) Blot probed using a 5’-biotinylated probe designed to hybridize antisense to the miR-M1-7-3p sequence. Two non-specific bands in the miR-M1-7-3p blot are starred to the right of the blot. Below each blot is the ethidium bromide stained 5S rRNA band from each gel photographed to gauge both RNA quality and loading prior to transfer. Labeled below each gel is the infection or transfection condition from which the total RNA was isolated.