FIG 2.

Recombinant YlMhb1p binds DNA in vitro. (A) Schematic representation of four versions of YlMhb1p produced in E. coli and used for in vitro experiments. YlMhb1p+MTS, full-length protein containing a mitochondrial targeting sequence (MTS); YlMhb1p-WT, wild-type protein lacking MTS; YlMhb1p-N-term and YlMhb1p-C-term, truncation mutants containing amino acids 15 to 156 and amino acids 157 to 238, respectively. (B) Analysis by EMSA indicates that increasing concentrations of YlMhb1p bind to DNA fragments in vitro. For comparison, purified mitochondrial HMG box-containing proteins from S. cerevisiae (ScAbf2p) and C. albicans (CaGcf1p) were subjected to the same assay. Purified glutathione S-transferase (GST) was used as a negative control. (C) EMSAs of various forms of YlMhb1p bound to HindIII fragments of bacteriophage λ DNA. Purified proteins were added at the same molar concentrations to compensate for the differences in molecular weight. (D) mtDNA was compacted in vitro as described in Materials and Methods. The reaction mixtures contained 50 ng of purified mtDNA and either 0.2 μg of YlMhb1p-WT, 0.16 μg of YlMhb1p-N-term, or 0.1 μg of YlMhb1p-C-term.