Relevance of UP elements for three strong Bacillus subtilis phage phi29 promoters

Various Escherichia coli promoters contain, in addition to the classical -35 and -10 hexamers, a third recognition element, named the UP element. Located upstream of the -35 box, UP elements stimulate promoter activity by forming a docking site for the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD). Accumulating genetic, biochemical and structural information has provided a detailed picture on the molecular mechanism underlying UP element-dependent promoter stimulation in E.coli. However, far less is known about functional UP elements of Bacillus subtilis promoters. Here we analyse the strong early sigma(A)-RNA polymerase-dependent promoters C2, A2c and A2b of the lytic B.subtilis phage phi29. We demonstrate that the phage promoters contain functional UP elements although their contribution to promoter strength is very different. Moreover, we show that the UP element of the A2b promoter, being critical for its activity, is located further upstream of the -35 box than most E.coli UP elements. The importance of the UP elements for the phage promoters and how they relate to other UP elements are discussed.     

Secretory overproduction of the aminopeptidase from Bacillus licheniformis by a novel hybrid promoter in Bacillus subtilis

Bacillus licheniformis aminopeptidase (BAP) was overexpressed in B. subtilis using a novel expression vector carrying a hybrid promoter, BJ27UP, which was constructed from a strong promoter BJ27Δ88 and a fragment of the tac promoter. When added upstream of the BJ27Δ88 promoter, the tac fragment (including the -10 box) improved the promoter activity of the BJ27Δ88 promoter by approximately threefold. The hybrid promoter, BJ27UP, allowed overexpression of BAP in B. subtilis, and over 95% of the produced BAP was secreted into the culture medium, whereas in E. coli, BAP was poorly expressed, despite the use of the T7 expression system. The volumetric production of BAP mediated by the hybrid promoter BJ27UP was reproducibly over 9.0 U/ml in Luria–Bertani medium after cultivation for 12 h, representing a 20-fold increase over that of the endogenous promoter of the bap gene. Due to its high-yield secretion, the recombinant BAP was purified using a simple inexpensive purification method consisting of ammonium sulfate fractionation and Q-Sepharose column chromatography.     

Repression of the aroP gene of Escherichia coli involves activation of a divergent promoter.

The repression of aroP expression which is mediated by the TyrR protein with phenylalanine, tyrosine, or tryptophan has been shown to be primarily a direct result of TyrR-mediated activation of a divergent promoter, P3, which directs the RNA polymerase away from promoter P1. Evidence which has been presented to support this conclusion is as follows. Repression of P1 does not occur either in vitro or in vivo if wild-type TyrR protein is substituted by the activation-negative mutant RQ10 (with an R-to-Q change at position 10). Repression of P1 is greatly diminished if the P3 promoter is inactivated or if a 5-bp insertion is made between the P3 promoter and the binding sites for TyrR. Repression is also abolished if the promoter strength of P1 is increased or a putative UP element associated with P3 is altered. Repression of the second promoter, P2, still occurs if the wild-type TyrR protein is substituted with RQ10 or EQ274. The tryptophan-mediated repression of aroP does not involve the TrpR protein.     

Interaction of the alpha-subunit of Escherichia coli RNA polymerase with DNA: rigid body nature of the protein-DNA contact

The alpha-subunit of Escherichia coli RNA polymerase plays an important role in the activity of many promoters by providing a direct protein-DNA contact with a specific sequence (UP element) located upstream of the core promoter sequence. To obtain insight into the nature of thermodynamic forces involved in the formation of this protein-DNA contact, the binding of the alpha-subunit of E. coli RNA polymerase to a fluorochrome-labeled DNA fragment containing the rrnB P1 promoter UP element sequence was quantitatively studied using fluorescence polarization. The alpha dimer and DNA formed a 1:1 complex in solution. Complex formation at 25 degrees C was enthalpy-driven, the binding was accompanied by a net release of 1-2 ions, and no significant specific ion effects were observed. The van't Hoff plot of temperature dependence of binding was linear suggesting that the heat capacity change (Deltac(p)) was close to zero. Protein footprinting with hydroxyradicals showed that the protein did not change its conformation upon protein-DNA contact formation. No conformational changes in the DNA molecule were detected by CD spectroscopy upon protein-DNA complex formation. The thermodynamic characteristics of the binding together with the lack of significant conformational changes in the protein and in the DNA suggested that the alpha-subunit formed a rigid body-like contact with the DNA in which a tight complementary recognition interface between alpha-subunit and DNA was not formed.