Low temperature regulated DEAD-box RNA helicase from the Antarctic archaeon, Methanococcoides burtonii

DEAD-box RNA helicases, by unwinding duplex RNA in bacteria and eukaryotes, are involved in essential cellular processes, including translation initia tion and ribosome biogenesis, and have recently been implicated in enabling bacteria to survive cold-shock and grow at low temperature. Despite these critical physiological roles, they have not been characterized in archaea. Due to their presumed importance in removing cold-stabilised secondary stru ctures in mRNA, we have characterised a putative DEAD-box RNA helicase gene (deaD) from the Antarctic methanogen, Methanococcoides burtonii. The encod ed protein, DeaD is predicted to contain a core element involved in ATP hyd rolysis and RNA-binding, and an unusual C-terminal domain that contains sev en perfect, tridecapeptide, direct repeats that may be involved in RNA bind ing. Alignment and phylogenetic analyses were performed on the core regions of the M. burtonii and other DEAD-box RNA helicases. These revealed a loos e but consistent clustering of archaeal and bacterial sequences and enabled the generation of a prokaryotic-specific consensus sequence. The consensus highlights the importance of residues other than the eight motifs that are often associated with DEAD-box RNA helicases, as well as de-emphasising th e importance of the "A" residue within the "DEAD" motif. Cells growing at 4 degrees C contained abundant levels of deaD mRNA, however no mRNA was dete cted in cells growing at 23 degrees C (the optimal temperature for growth). The transcription initiation site was mapped downstream from an archaeal b ox-A element (TATA box), which preceded a long (113 nucleotides) 5'-untrans lated region (5'-UTR). Within the 5'-UTR was an 11 bp sequence that closely matches (nine out of 11) cold-box elements that are present in the 5'-UTRs of cold-shock induced genes from bacteria. To determine if the archaeal 5' -UTR performs an analagous function to the bacterial 5'-UTRs, the archaeal deaD 5'-UTR was transcribed in E. coli under the control of the cspA promot er and transcriptional terminator. It has previously been reported that ove rexpression of the cspA 5'-UTR leads to an extended cold-shock response due to the 5'-UTR titrating cellular levels of a cold-shock repressor protein( s). Tn our hands, the cold-shock protein profiles resulting from overexpres sion of Escherichia coli cspA and M. burtonii deaD 5'-UTRs were similar, ho wever they did not differ from those for the overexpression of a control pl asmid lacking a 5'-UTR. In association with other recent data from E. coli, our results indicate that the role of the 5'-UTR in gene regulation is pre sently unclear. Irrespective of the mechanisms, it is striking that highly similar 5'-UTRs with cold-box elements are present in cold induced genes fr om E. coli, Anabaena and M. burtonii. This is the first study examining low temperature regulation in archaea and provides initial evidence that gene expression from a-cold adapted archaeon involves a bacterial-like transcrip tional regulatory mechanism. In addition, it provides the foundation for fu rther studies into the function and regulation of DEAD-box RNA helicases in archaea, and in particular, their roles in low temperature adaptation. (C) 2000 Academic Press.