Fig. 4. T7 RNAP can use a template molecule processively to instruct multiple rounds of RNA synthesis.

(A) Two possible mechanisms for RNA dimer generation, with both halves of a dimer synthesized using the same monomer template molecule in uni-templated synthesis but using two different monomer molecules in bi-templated synthesis. (B) Uni-templated synthesis is the dominant mechanism for RNA dimer generation. Two diverse monomer template pools, X₁ and Y2₁, with base randomization at six positions in X RNA and Y2 RNA respectively, were used as templates for dimer synthesis followed by high-throughput sequencing of RNA dimers. Plots show the sequence agreement between the six randomized positions in the first dimer half (S_i) and in the second dimer half (S_k), for the ten most abundant RNA templates present in the sequenced pools. No mismatches were allowed in sequence agreement calculations. Prob = Probability. (C) Model for uni-templated synthesis is an interrupted rolling circle mechanism. In this model, the RNA template is positioned such that T7 RNAP can jump from the template 5’ to 3’ end, and synthesize RNA products that are longer than monomers.