Domains formed within the N-terminal region of the quorum-sensing activator TraR are required for transcriptional activation and direct interaction with RpoA from agrobacterium.
TraR, a quorum-sensing activator, induces transcription from its binding site, the tra-box, located upstream of Ti plasmid target promoters. TraR activated expression of a lacZ reporter in Escherichia coli only when RpoAAt from Agrobacterium tumefaciens was co-expressed. As assessed by gel retardation assays RpoAAt, but not RpoAEc, formed a ternary complex with TraR and a tra-box probe in vitro. TraR formed similar ternary complexes with alphaCTDAt but not with NTDAt, the C- and N-terminal segments of RpoAAt. As measured by surface plasmon resonance refractometry, TraR interacted directly with RpoAAt with an affinity about five times greater than that observed for its interaction with RpoAEc. The activator interacted with alphaCTDAt with kinetics and affinities similar to those of the full-sized -subunit. Positive control (PC) mutations at Asp-10 and Gly-123 of TraR did not affect DNA binding but greatly decreased the TraR-RpoAAt interaction. These two residues combine to form two patches on the activator, one of which may be involved in interaction with RpoA. When co-expressed, mutants of TraR with substitutions at Asp-10 complementing mutants with substitutions at Gly-123 for gene activation in an allele-specific manner. Co-expression studies with TraR and its PC mutants, and also with complementary PC alleles of TraR, coupled with three-dimensional structure are consistent with a hypothesis that both Asp-10/Gly-123 patches are required for activator function.
Qin
Yinping
Y
Department of Crop Sciences, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
Luo
Zhao-Qing
ZQ
Farrand
Stephen K
SK
D016960:Agrobacterium tumefaciens / Q000378:metabolism; D000483:Alleles; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000378:metabolism; D019281:Dimerization; D004926:Escherichia coli / Q000378:metabolism; D005799:Genes, Dominant; D017930:Genes, Reporter; D005816:Genetic Complementation Test; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012231:Rhizobium / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D013997:Time Factors; D015533:Transcriptional Activation
D001426:Bacterial Proteins; C080512:TraR protein, Agrobacterium tumefaciens; D004247:DNA; D012321:DNA-Directed RNA Polymerases; C113275:RNA polymerase alpha subunit