Mutations in TrpI binding site II that differentially affect activation of the trpBA promoter of Pseudomonas aeruginosa.

Abstract

In vitro, Pseudomonas aeruginosa TrpI protein activates transcription initiation at the trpBA promoter (trpPB) and represses initiation at its own promoter (trpPI), which diverges from, and overlaps, trpPB. Indoleglycerol phosphate (InGP) reduces the TrpI concentration required for binding to its strong binding site (site I), as measured by repression of trpPI; it also facilitates activation of trpPB, presumably because it enables TrpI to bind to a weaker binding site (site II) and thereby interact with RNA polymerase. The role of site II and InGP in regulation of the two promoters was investigated by constructing site II mutants. A 2 bp substitution affected the ability of TrpI to activate trpPB, but did not significantly affect TrpI binding to site II. A more extensive (8 bp) substitution inhibited TrpI‐mediated activation of trpPB and TrpI‐mediated protection of site II in a DNase I footprinting assay. However, the mutation did not alter the pattern of TrpI binding observed in gel retardation experiments. In particular, a more slowly‐migrating complex (Complex 2) whose appearance was correlated with TrpI binding to site II was formed equally well on a wild‐type or substituted DNA fragment. Based on the mutant phenotypes, we propose that a particular sequence of protein‐‐protein and protein‐‐DNA interactions is required for activation of trpPB by TrpI and InGP.

Glycine binds the transcriptional accessory protein GcvR to disrupt a GcvA/GcvR interaction and allow GcvA-mediated activation of the Escherichia coli gcvTHP operon

“…These low molecular mass co-effectors are believed to directly bind their respective LTTR proteins, resulting in altered binding site affinity or relaxed DNA bending by the LTTR proteins at the regulated promoters (Schell, 1993). These changes are necessary for appropriate transcriptional regulation (Gao & Gussin, 1991 ;Wek & Hatfield, 1988 ;Ogawa et al, 1999 ;Rhee et al, 1998 ;van Keulen et al, 1998). The addition of glycine to GM medium results in GcvAmediated activation of the gcv operon (Plamann et al, 1983).…”

“…The presence of the co-inducer molecule usually results in either an altered DNA binding affinity of the LTTR, observed as an increase in the number of protein monomers bound to a specific site, or altered DNA bending of the promoter region by the LTTR (Gao & Gussin, 1991 ;Wek & Hatfield, 1988 ;Ogawa et al, 1999 ;Rhee et al, 1998 ;van Keulen et al, 1998). These effects result in appropriate transcriptional regulation of the respective operons.…”

“…Co-inducer binding by LTTRs is manifested in several ways including altered DNA binding and DNA bending, which facilitate LTTR activation or repression of transcription (Gao & Gussin, 1991 ;Wek & Hatfield, 1988 ;van Keulen et al, 1998 ;Ogawa et al, 1999 ;Hryniewicz & Kredich, 1994 ;Wang et al, 1992). How glycine and purines affect GcvA and GcvR to allow an appropriate response at the gcv promoter is unknown.…”

Identification and characterization of the ilvR gene encoding a LysR-type regulator of Caulobacter crescentus

“…Alternatively, they may represent a single site that is partially or fully saturated by the IlvR protein, respectively. Although the biochemical mechanism that underlies gene expression by LysR-type proteins is not fully understood, activation of gene expression is mediated through binding of the regulatory proteins to the promoter regions of the genes they regulate (9,10,21,32). The DNA nucleotide sequence between the translation initiation codons of the ilvD and ilvR genes contains a T-NII-A LysR motif (Fig.…”

The regulatory mechanism of 2,4,6-trichlorophenol catabolic operon expression by HadR in Ralstonia pickettii DTP0602

“…The TrpIdependent DNA bending angle was not affected with or without indole glycerol phosphate (InGP). However, the protection region of TrpI extended to 232 in the presence of InGP (Gao & Gussin, 1991;Piñeiro et al, 1997). The protection region of MetR was different from that of HadR (Fig.…”