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. 2007 Oct 8;27(24):8492–8501. doi: 10.1128/MCB.01173-07

FIG. 2.

FIG. 2.

DdTFIIIB subunits interact with TRE5-A ORF1p. (A) Model for initiation of tRNA gene transcription in D. discoideum. DdTFIIIC binds to the tRNA gene-internal B box sequence and then recruits DdTFIIIB. The latter consists of the three subunits DdTBP, DdBrf1, and DdBdp1 and occupies space at the 5′ end of the tRNA gene close to the TRE5-A integration site. (B) DdTFIIIB subunits were tested for interaction with TRE5-A-derived ORF1p and ORF2p proteins. Selection plates contained minimal medium without histidine that were supplemented with either 5 mM 3-AT (left) or 6 mM 3-AT (right). Combinations of two-hybrid vectors are listed; the left column represents proteins expressed from pBT vectors, and the right column lists proteins cloned into the pTRG vector. (C) Schematic presentation of full-length TRE5-A.1 and the derived proteins tested in two-hybrid assays. The ORF2 protein was cloned in three parts according to the predicted ENp and RTp domains and the HCp domain of unknown function. Modules A, B, and C represent 5′ and 3′ untranslated regions of TRE5-A.1. (D) Expression of pBT-derived fusion proteins in the bacterial two-hybrid reporter strain. Western blot showing expression levels of DdTFIIIB subunits and TRE5-A-encoded ORF proteins. The corresponding DNA fragments, cloned in vector pBT, were transformed into the BacterioMatch II reporter strain. Bacteria were grown at 30°C. Extracts were prepared from whole cells and subjected to SDS-PAGE on 10% polyacrylamide gels. After electrophoretic transfer onto nitrocellulose membranes, proteins were stained with a commercial polyclonal antiserum directed against the λcI repressor protein encoded in the pBT vector (Stratagene). Lanes 1 and 2, DdTBP; lanes 3 and 4, DdBrf1-270; lanes 5 and 6, DdBrf271-706; lanes 7 and 8, DdBdp1; lanes 9 and 10, ORF1p; lanes 11 and 12, ENp; lanes 13 and 14, RTp; lanes 15 and 16, HCp. Lanes 1, 3, 5, 7, 9, 11, 13, and 15 show extracts of bacteria without induction of recombinant protein expression. In lanes 2, 4, 6, 8, 10, 12, 14, and 16, expression of recombinant proteins was induced with 0.5 mM IPTG for 3 h at 30°C. Asterisks denote the bands corresponding to the molecular masses of the full-length λcI fusion proteins. (E) Pull-down experiment. 35S-labeled DdTBP or full-length DdBrf1 was incubated with bacterially expressed MBP or an MBP-ORF1p fusion protein. Bound DdTBP or DdBrf1 was subjected to SDS-PAGE followed by autoradiography. Input refers to 10% of a labeling reaction.