Figure 4.

The −1 position contributes to efficiency of pRNA synthesis for BsEσ^A and EcEσ⁷⁰, but the +2 position has minimal impact. (A) Schematic of Ec6S(iGTP) RNA (indicated as Ec6S* here). The −1 and +2 positions where changes were made for various mutants are indicated in red and blue, respectively. The +1C position, which directs initiation of pRNA synthesis with GTP, is indicated in green. (B) pRNAs generated in vitro by BsEσ^A (right) and EcEσ⁷⁰ (left) from various RNAs (indicated at top) when labeled with ³²P γ-GTP were visualized on a denaturing gel. Lane M contains a 5′ end labeled oligonucleotide 19 nt in length for size comparison. There are slight changes in apparent size of pRNA resulting from changes in sequence at the +2 position consistent with the change in molecular weight of each NTP (i.e. GTP > ATP > UTP > CTP). (C) Growth of B. subtilis cells as monitored by optical density at 595 nm (OD₅₉₅) in an absorbance plate reader after stationary phase cells were diluted ∼1:500 into 2× YT medium. Growth was of B. subtilis ΔbsrAΔbsrB cells (KW590) containing plasmids pSP*-Ec6S(-1A) (blue), pSP*-Ec6S(-1G) (dotted pink), pSP*Ec6S(-1U) (red) or pSP*-Ec6S(-1C) (dotted green). Data shown are from one representative experiment with three biological replicates. Similar results were observed in at least three experiments. Error bars correspond to ± standard deviations from the averages.