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. 1998 Aug 18;95(17):9761–9766. doi: 10.1073/pnas.95.17.9761

Figure 1.

Figure 1

In vitro selection. (A) Synthesis of rrnB P1 promoter fragments with a randomized upstream region. Oligonucleotides were annealed and extended with T7 DNA polymerase to form a library of double-stranded DNA fragments with different UP element regions (−59 to −38). The top strand oligonucleotide (80 nt) contained (from 5′ to 3′) an EcoRI site (RI), rrnB P1 sequence from −66 to −60, random sequence from −59 to −38, and rrnB P1 sequence from −37 to +1. The bottom strand oligonucleotide (81 nt) contained (from 5′ to 3′) a HindIII site (H3) and rrnB P1 sequence from +50 to −17. Each contained a short additional sequence 5′ to the restriction site to ensure enzyme digestion. The randomized region is indicated by a hatched box, and −10 and −35 hexamers of rrnB P1 by open boxes. (B) Theoretical time course of RNAP binding to promoters containing (UP+) or lacking (UP−) an UP element. Broken line represents a time at which RNAP-promoter binding reactions were stopped to enrich for UP element-containing fragments.