Hybrid Ptr2-like activators of archaeal transcription.
Methanocaldococcus jannaschii Ptr2, a member of the Lrp/AsnC family of bacterial DNA-binding proteins, is an activator of its eukaryal-type core transcription apparatus. In Lrp-family proteins, an N-terminal helix-turn-helix DNA-binding and dimerizing domain is joined to a C-terminal effector and multimerizing domain. A cysteine-scanning surface mutagenesis shows that the C-terminal domain of Ptr2 is responsible for transcriptional activation; two types of DNA binding-positive but activation-defective mutants are found: those unable to recruit the TBP and TFB initiation factors to the promoter, and those failing at a post-recruitment step. Transcriptional activation through the C-terminal Ptr2 effector domain is exploited in a screen of other Lrp effector domains for activation capability by constructing hybrid proteins with the N-terminal DNA-binding domain of Ptr2. Two hybrid proteins are effective activators: Ptr-H10, fusing the effector domain of Pyrococcus furiosus LrpA, and Ptr-H16, fusing the P. furiosus ORF1231 effector domain. Both new activators exhibit distinguishing characteristics: unlike octameric Ptr2, Ptr-H10 is a dimer; unlike Ptr2, the octameric Ptr-H16 poorly recruits TBP to the promoter, but more effectively co-recruits TFB with TBP. In contrast, the effector domain of Ptr1, the M. jannaschii Ptr2 paralogue, yields only very weak activation.
Pritchett
Matthew A
MA
Division of Biological Sciences and Center for Molecular Genetics, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0634, USA.
Wilkinson
Steven P
SP
Geiduschek
E Peter
EP
Ouhammouch
Mohamed
M
D000818:Animals; D001105:Archaea / Q000235:genetics / Q000378:metabolism; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D017124:Conserved Sequence / Q000235:genetics; D019641:DNA, Archaeal / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019848:Gene Expression Regulation, Archaeal; D019077:Helix-Turn-Helix Motifs / Q000235:genetics; D051756:Leucine-Responsive Regulatory Protein / Q000737:chemistry / Q000235:genetics; D017016:Methanococcaceae / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D020027:Pyrococcus furiosus / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation
D019843:Archaeal Proteins; D019641:DNA, Archaeal; D004268:DNA-Binding Proteins; C418107:LrpA protein, Pyrococcus furiosus; C423032:Ptr2 protein, Methanococcus jannaschii; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D051756:Leucine-Responsive Regulatory Protein