![]() Upstream sequence names are the strain numbers of λ lysogens carrying the promoter– lacZ fusions. Wild-type rrnB P1 contained its natural UP element sequence, and the core rrnB P1 promoter contained an upstream sequence with no UP element function. Sequences of the nontemplate strand in the upstream region (−59 to −38) from 31 selected promoters, wild-type rrnB P1, and the rrnB P1 core promoter are shown. ( A) Promoters contained wild-type rrnB P1 sequences (solid line open boxes indicate the −10 and −35 hexamers) and different upstream regions (dotted line). Upstream sequences and relative transcription activities of 31 in vitro-selected promoters used in defining an UP element consensus sequence. Broken line represents a time at which RNAP-promoter binding reactions were stopped to enrich for UP element-containing fragments. ( B) Theoretical time course of RNAP binding to promoters containing (UP +) or lacking (UP −) an UP element. The randomized region is indicated by a hatched box, and −10 and −35 hexamers of rrnB P1 by open boxes. Each contained a short additional sequence 5′ to the restriction site to ensure enzyme digestion. The bottom strand oligonucleotide (81 nt) contained (from 5′ to 3′) a HindIII site (H3) and rrnB P1 sequence from +50 to −17. 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. 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). ( A) Synthesis of rrnB P1 promoter fragments with a randomized upstream region. Based on the evolutionary conservation of the residues in alpha responsible for interaction with UP elements, we suggest that the UP element consensus sequence should be applicable throughout eubacteria, should generally facilitate promoter prediction, and may be of use for biotechnological applications. The identification of the UP element consensus should facilitate a detailed understanding of the alpha-DNA interaction. The most active selected sequence contained the derived consensus, displayed all of the properties of an UP element, and the interaction of this sequence with the alpha C-terminal domain was similar to that of previously characterized UP elements. A set of 31 of these upstream sequences increased transcription from 136- to 326-fold in vivo, considerably more than the natural rrnB P1 UP element, and was used to derive a consensus sequence: -59 nnAAA(A/T)(A/T)T(A/T)TTTTnnAAAAnnn -38. By using a modification of the SELEX procedure for identification of protein-binding sites, we selected in vitro and subsequently screened in vivo for sequences that greatly increased promoter activity when situated upstream of the Escherichia coli rrnB P1 core promoter. The UP element, a component of bacterial promoters located upstream of the -35 hexamer, increases transcription by interacting with the RNA polymerase alpha-subunit.
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