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.
Division of Biological Sciences and Center for Molecular Genetics, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0634, USA.
Matthew A
MA
Pritchett
Steven P
SP
Wilkinson
E Peter
EP
Geiduschek
Mohamed
M
Ouhammouch
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