Both HMG boxes in Hmo1 are essential for DNA binding in vitro and in vivo

Abstract

Hmo1, a member of the high mobility group B family proteins in Saccharomyces cerevisiae, associates with the promoters of ribosomal protein genes (RPGs) to direct accurate transcriptional initiation. Here, to identify factors involved in the binding of Hmo1 to its targets and the mechanism of Hmo1-dependent transcriptional initiation, we developed a novel reporter system using the promoter of the RPG RPS5. A genetic screen did not identify any factors that influence Hmo1 binding, but did identify a number of mutations in Hmo1 that impair its DNA binding activity in vivo and in vitro. These results suggest that Hmo1 binds to its target promoters autonomously without any aid of additional factors. Furthermore, characterization of Hmo1 mutants showed that the box A domain plays a pivotal role in DNA binding and may be required for the recognition of structural properties of target promoters that occur in native chromatin.

Oligomerization of Hmo1 mediated by box A is essential for DNA binding in vitro and in vivo

“…Based on this result, we proposed that box A, in addition to box B, is essential for DNA‐binding activity of Hmo1 (Higashino et al . ). However, another previous study carried out by others suggested that although box B is responsible for most of the DNA‐binding activity of Hmo1, box A has only a slight, if any, influence on DNA binding (Kamau et al .…”

“…For this purpose, we conducted the TSS (Transcription Start Site) shift assay, using a reporter plasmid consisting of the modified RPS5 promoter and HIS3 gene, developed in our previous study to test the DNA binding of Hmo1 in vivo (Higashino et al . ). Hmo1 binds to the intervening region between the UAS and core promoter in the modified RPS5 promoter, thereby inhibiting transcriptional initiation of ectopic mRNAs that can produce functional His3 protein.…”

“…In addition to in vivo analyses, DNA‐binding activity of Hmo1 mutant proteins was tested in vitro using an immobilized template assay, as described previously (Higashino et al . ). As expected, the DNA‐binding activity of Hmo1 mutants lacking either box A or box B was virtually abolished (Fig.…”

“…In a previous study (Higashino et al . ), we identified various mutations that significantly reduced the DNA‐binding activity of Hmo1 within aa 27–51 (e.g., T30R, A31V, S33P, V35A, F37S and N39D). Hence, it is puzzling that deletion of aa 27–51 (i.e., Δ27–51) did not significantly affect DNA binding by Hmo1.…”

“…Although we did not identify any such factors, we identified many mutations in box A, as well as in box B, that significantly decreased the DNA binding of Hmo1 (Higashino et al . ). Based on these findings, we proposed that box A is essential for DNA binding by Hmo1.…”