In vitro analysis on the assembly mechanisms of the Aspergillus CCAAT-box binding factor.

Masashi Kato and Tetsuo Kobayashi.

Author address: 

Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.

Abstract: 

The CCAAT-box is one of the most common cis-elements present in the promoter regions in eukaryotes. It has been shown to be important in the high-level expression of many genes in Aspergillus species. The factor which binds to the CCAAT-box, so-called the Hap complex, belongs to the NF-Y family and consists of three subunits, HapB, HapC and HapE. Previously we have shown that the number of HapE is strictly dependent on the number of HapE, suggesting the number of HapC could adjust that of HapE by forming stable heterodimers prior to assembly of the Hap complex1). In this study, we performed a reconstitution study with the recombinant subunits and 35S-labeled in vitro translated subunits. Significant amounts of the translated HapE were recovered in the insoluble fraction while the other two subunits were in the soluble fraction. However, HapE was recovered in the soluble fraction when the recombinant HapC was added in the translation reaction. These results suggest that the HapC subunit plays a chaperon-like role specific to the HapE subunit. Furthermore, pull-down assays of the labeled subunits with recombinant GST or MBP fusion subunits were also carried out to investigate the mode of subunit assembly of the Aspergillus Hap complex. 1) M. Kato et al. FEBS Lett. 512, 227-229 (2002)
2005

abstract No: 

410

Full conference title: 

23rd Fungal Genetics Conference
    • Fungal Genetics Conference 23rd (2002)