Part:BBa_K1031911

XylS-Terminator Overview

XylS is an archetype transcriptional activator of AraC/XylS family, mined from the TOL plasmid pWW0 of the bacterium Pseudomonas putida. It is composed of a C-terminal domain (CTD) involved in DNA binding containing two helix-turn-helix motifs and an N-terminal domain required for effector binding and protein dimerization. XylS detects benzoate and its’ derivatives, mainly methyl and chlorine substitutes at 2-, 3- carbon.

Fig. 1 Regulatory circuits controlling the expression from the TOL plasmid pWW0[7]. Squares, XylS; circles, XylR; open symbol,s.transcriptional regulator without aromatics effector binding; closed symbols, effector-bound transcription factors that is in active form. See the main text for the detailed explanation for regulatory loops.

Promoter Structure

The cognate promoter regulated by XylS, Pm, is σ70-dependent in E.coli, while in Pseudomonas putida, it is σ32/38-dependent[6]. It acts as the master regulator to control the ON/OFF expression of meta-operon on TOL plasmid pWW0[2]. In this meta-operon, XylXYZLTEGFJQKIH genes encode enzymes for the degradation of benzoate and its derivatives, generating intermediate products in TCA cycle. (Fig 1)

Mechanism

Fig 2. Mechanism of XylS binding to Pm promoter

Mechanism transcription activation by XylS at Pm promoter. Step 1: Free DNA. The -10/-35 consensus sequence motifs of σ70-dependent promoter and the two XylS binding sites (D: distal; and P: proximal) are depicted. The bending angle is supposed to be 35°, centered at the XylS proximal binding site. Step 2: A first XylS monomer binds to Pm at the proximal site, shifts the bent center to the DNA sequence between the two XylS binding sites, and increases the bending angle to 50°. Step 3: This change favors the binding of a second monomer to the distal site, further increasing the DNA curvature to an overall value of 98° (here schematized as a right angle). Contacts with RNAP, also probably with the σ-subunit, are established through the α-CTD, which dramatically facilitates the open complex formation and transcription initiation as shown in Step 4.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 32
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 754
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 217