The RNase E/G homologue from the thermophilic eubacterium Aquifex aeolicus has been overexpressed in Escherichia coli, purified, and characterized in vitro. We show that A. aeolicus RNase E/G has a temperature-dependent, endoribonucleolytic activity. The enzyme site-specifically cleaves oligonucleotides and structured RNAs at locations that are partly overlapping or completely different when compared to the positions of E coli RNase E and RNase G cleavage sites. The efficiency of cleavage by A. aeolicus RNase E/G is dependent on the 5'-phosphorylation status of RNA suggesting differential susceptibility of primary transcripts and their degradative intermediates to the nuclease activity of this enzyme in vivo. Similar to E. coli RNase E, A. aeolicus RNase E/G is able to selectively cleave internucleotide bonds in the 3'-5' direction, and to cut in intercistronic regions of putative tRNA precursors, thus suggesting a common function for RNase E/G homologues in eubacteria. (C) 2004 Elsevier Inc. All rights reserved.