Figure 1.

The non-template strand between −4 and +2 is required for DNA melting downstream of the transcription start site and for promoter escape. (A). Structures of M13ori derivatives used in our study. At the top is the initial NTS⁻⁷ promoter, which was progressively shortened from the 5′-end to NTS⁺⁴ (bottom). (B) Synthesis of abortive and the full-length RNA18 on the NTS⁻⁷ promoter and its shorter derivatives by Eσ⁷⁰ in the presence of all NTPs (no short primers were used). Note that the synthesis of RNA20, which originates upstream of the RNA18 start site, is not affected by non-template strand truncations. A number of minor bands running slower than the abortive dinucleotide correspond to tri- and tetra nucleotides as well as small amount of transcripts resulting from non-specific initiation. These products were not taken into consideration during quantification. (C) Quantification of the results in (B). Here and after, promoter escape was quantified as a ratio of RNA18 to the abortive products (pppApG) and normalized to promoter escape on the NTS⁻⁷ promoter. Here, and after, data presented as mean ± SEM from at least three independent experiments. (D) Transcription by Eσ⁷⁰ on pre-melted promoter NTS^−2/+2C,+3C. (E and F) Transcription and promoter escape by holo (Eσ⁷⁰) (black) and core (E) RNAPs (grey) on templates indicated above the gel and below the histogram were analysed in 1 mM NTPs.