In vivo and in vitro approaches for studying the yeast mitochondrial RNA degradosome complex.
The mitochondrial degradosome (mtEXO) of S. cerevisiae is the main exoribonuclease of yeast mitochondria. It is involved in many pathways of mitochondrial RNA metabolism, including RNA degradation, surveillance, and processing, and its activity is essential for mitochondrial gene function. The mitochondrial degradosome is a very simple example of a 3' to 5'-exoribonucleolytic complex. It is composed of only two subunits: Dss1p, which is an RNR (RNase II-like) family exoribonuclease, and Suv3p, which is a DExH/D-box RNA helicase. The two subunits form a tight complex and their activities are highly interdependent, with the RNase activity greatly enhanced in the presence of the helicase subunit, and the helicase activity entirely dependent on the presence of the ribonuclease subunit. In this chapter, we present methods for studying the function of the yeast mitochondrial degradosome in vivo, through the analysis of degradosome-deficient mutant yeast strains, and in vitro, through heterologous expression in E. coli and purification of the degradosome subunits and reconstitution of a functional complex. We provide the protocols for studying ribonuclease, ATPase, and helicase activities and for measuring the RNA binding capacity of the complex and its subunits.
Malecki
Michal
M
Department of Genetics and Biotechnology, Warsaw University, Warsaw, Poland.
Jedrzejczak
Robert
R
Puchta
Olga
O
Stepien
Piotr P
PP
Golik
Pawel
P
D000251:Adenosine Triphosphatases / Q000378:metabolism; D001483:Base Sequence; D015152:Blotting, Northern; D002855:Chromatography, Thin Layer; D017931:DNA Primers; D006868:Hydrolysis; D007700:Kinetics; D008928:Mitochondria / Q000235:genetics*; D012331:RNA, Fungal / Q000378:metabolism*; D012260:Ribonucleases / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D014158:Transcription, Genetic
D017931:DNA Primers; D012331:RNA, Fungal; D012260:Ribonucleases; D000251:Adenosine Triphosphatases