Identification of amino acid residues of the Agrobacterium tumefaciens quorum-sensing regulator TraR that are critical for positive control of transcription.
The LuxR-type quorum-sensing transcription factor TraR regulates replication and conjugal transfer of the tumour-inducing (Ti) plasmid in the plant pathogen Agrobacterium tumefaciens. TraR is a two-domain protein with an N-terminal domain that binds to the quorum-sensing signal N-3-oxooctanoyl- l-homoserine lactone (OOHL) and a C-terminal domain that binds to specific DNA sequences called tra boxes. TraR-OOHL complexes form homodimers that activate transcription of at least seven promoters on the Ti plasmid. At five promoters, a tra box overlaps the binding site of core RNA polymerase (class II promoters), while in the other two promoters, this site is located farther upstream (class I promoters). In this study, we performed saturating point mutagenesis of the surface residues of the TraR C-terminal domain. Each mutant was tested for proteolytic stability and transcription activity in vivo, and for DNA binding activity in vitro. Mutants of TraR with single substitutions at positions W184, V187, K189, E193Q, V197 and D217 have wild-type levels of accumulation and DNA binding, but are defective in transcription of both types of promoters. These residues constitute a patch on the surface of the DNA-binding domain. We propose that this patch is an activating region that recruits RNA polymerase to TraR-dependent promoters through direct contact. As residues of this patch are critical for activation at both a class I and a class II promoter, we predict that these residues may contact the C-terminal domain of the RNA polymerase alpha-subunit.
White
Catharine E
CE
Department of Microbiology, Cornell University, Ithaca, NY 14853, USA.
Winans
Stephen C
SC
D016960:Agrobacterium tumefaciens / Q000235:genetics* / Q000502:physiology; D000596:Amino Acids / Q000502:physiology; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005809:Genes, Regulator / Q000235:genetics; D006714:Homoserine / Q000031:analogs & derivatives / Q000378:metabolism / Q000502:physiology*; D009154:Mutation / Q000235:genetics / Q000502:physiology; D010957:Plasmids / Q000235:genetics / Q000502:physiology; D011401:Promoter Regions, Genetic; D015398:Signal Transduction; D015534:Trans-Activators
D000596:Amino Acids; D001426:Bacterial Proteins; C080512:TraR protein, Agrobacterium tumefaciens; D015534:Trans-Activators; D006714:Homoserine