Gel shift assay

The 5' end FAM-labeled oligoribonucleotides were chemically synthesized by Hokkaido System Science Co., Ltd, Japan. Binding mixture containing this oligonucleotide and the protein pools expressed from P. furiosus genome were incubated at either 75$^\circ$ C or under various temperatures for 15 minutes in 20 $\mu$l of RNA binding buffer (10mM Tris/HCl (pH7.5), 50mM NaCl, 0.5mM EDTA, 0.5mM MnCl2, 5% glycerol and 1mM dithiotheitol). The RNA-protein complexes were analyzed by electrophoresis on non-denaturing 6% (w/v) polyacrylamide gels. Results were evaluated by scanning the radioactive image using a computerized image analyzer (Bio-Rad Laboratories, Hercules, CA, USA).