title: Flexibility in the second half-site sequence recognised by the c-Myb R2 domain--in vitro and in vivo analysis.
abstract: The oncoprotein c-Myb is a transcription factor that recognises its specific target sequences through two subdomains. The R3-domain binds the first half-site, YAAC, and plays a dominant role in sequence recognition, while the homologous R2-domain interacts with a more loosely defined sequence in the second half-site. The difficulty in precisely defining a preferred second half-site sequence might reflect the flexible nature of R2 which only attains its fully folded structure upon binding to DNA, a process that might allow the protein to adapt to different half-site sequences. Here we report that shifting the most conserved base in the second half-site, the G6, into position 5 resulted only in a minor reduction of complex stability in vitro. From an analysis of a series of second half-site variants by EMSA and DMS-interference, we conclude that the preferred recognition sequence should be revised to read [YAACNG or YAACGN]. Modeling the structure of c-Myb R2R3 in complex with a GT half-site variant revealed specific interactions with G5. When second half-site variants were tested in vivo using a sensitive yeast effector-reporter system, both the TG and GT half-site variants were functional mediating c-Myb-dependent transactivation. Unexpectedly, we observed large differences between the best second half-site variants at low levels of c-Myb-effector, the GG variant being five- to fifteen-fold more active in vivo than the single-G half-sites, the GH or HG variants.
authors.affiliation: Department of Biochemistry, University of Oslo, Norway.
authors.forename: E
authors.initials: E
authors.lastname: Ording
authors.forename: S
authors.initials: S
authors.lastname: Bergholtz
authors.forename: E M
authors.initials: EM
authors.lastname: Brendeford
authors.forename: N
authors.initials: N
authors.lastname: Jamin
authors.forename: O S
authors.initials: OS
authors.lastname: Gabrielsen
mesh_terms: D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D004586:Electrophoresis / Q000379:methods; D017930:Genes, Reporter; D006151:Guanosine / Q000378:metabolism; D008745:Methylation; D008958:Models, Molecular; D009154:Mutation; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020598:Proto-Oncogene Proteins c-myb; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D015003:Yeasts / Q000235:genetics
chemical_list: D004268:DNA-Binding Proteins; D011518:Proto-Oncogene Proteins; D020598:Proto-Oncogene Proteins c-myb; D015534:Trans-Activators; D006151:Guanosine; D004247:DNA