Spacing mutations between theEscherichia coliPBAD RNA polymerase binding site and thearaC(I) induction site

Abstract

Mutations in the Escherichia coli promoter PBAD have been constructed which alter the spacing of the adjacent RNA polymerase and araC inducer protein binding sites. While deletion of a single base-pair or small insertions do not detectably affect araC protein binding to DNA and they do not alter the conserved sequence of the RNA polymerase binding site, stimulation of PBAD in vivo is greatly reduced. The experiments suggest that the distance or angle between the two proteins on the DNA is critical for promoter function. INTRODUCTIONTranscription from the Escherichia coli L-arabinose operon promoter,

TECS: a toxin expression control strategy as a tool for optimization of inducible promoters

“…2 a) [ 33 ]. The presence of the Bam HI site outside MCS is inconvenient for cloning with restriction enzymes but it was shown that the deletions or mutations within this site affects the activity of the p BAD promoter [ 13 ]. With our protocol we tested effects of single nucleotide substitutions in the first position of G GATCC hexamer.…”

“…However, the development of inducible promoters through randomization is complicated. It is likely due to the fact that in many cases interference with regulatory elements of an inducible promoter results in disruption of its functions [ 13 , 24 , 25 ]. Thus, attempts to optimize inducible promoters are laborious and time-consuming and in practice, the majority of obtained clones are characterized by increased leakage level in comparison to the initial promoter.…”

“…Since p BAD promoter offers remarkably robust gene expression regulation, efforts were made to eliminate all-or-none induction profile and increase the strength of the promoter. However, it was shown that interference with promoter sequence or flanking sequences results in alleviation of AraC-mediated regulation of expression or decreased activity of the p BAD promoter [ 13 ]. Therefore, researchers focused on the elimination of other features like all-or-none induction phenomenon, which was bypassed by constitutive expression of araE encoding arabinose transporter [ 14 ] or engineering the repressor-activator protein AraC [ 15 , 16 ].…”

Flexibility in Repression and Cooperativity by KorB of Broad Host Range IncP-1 Plasmid RK2

“…47 The apparent flexibility of KorB in its interactions is in contrast to other well-studied regulators; for example, in the lac and ara operons of E. coli. [30][31][32] At proximal positions, any helical periodicity in repression should be highly pronounced due to the stiffness of a relatively short looping DNA, in contrast to our observation that KorB can repress equally well from opposing faces of the DNA. 32,48,49 An example is the CRP/FNR family of transcriptional activators found in Gram-negative bacteria, which also interact with RNAP, but exhibit a strict requirement for the spatial relationship between binding site and promoter in class I activation, where only the insertion of integral helical turns is tolerated.…”

“…moving the DNA binding-site halfway around the helix reduces regulation. [30][31][32] Comparison of trbBp sequences of RK2 and the related IncP-1 plasmid R751 reveals that while O B and promoter sequences are highly conserved, the intervening distance differs by 4 bp, i.e. approximately half a helical turn.…”