Cloning, sequencing and transcript analysis of the gene encoding formylmethanofuran: tetrahydromethanopterin formyltransferase from the hyperthermophilic Methanothermus fervidus.
The formylmethanofuran:tetrahydromethanopterin formyltransferase (FTR) from Methanothermus fervidus was partially purified and its N-terminal amino acid sequence determined. Using as probe a mixture of oligonucleotides derived from the FTR N-terminus, the corresponding gene (ftr) was cloned and sequenced. The ftr gene codes for 297 amino acids, corresponding to a molecular mass of 31,836 daltons, in contrast to the 41,000 daltons estimated for the protein by sodium dodecylsulphate-polyacrylamide gel electrophoresis. The deduced amino acid sequence of the hyperthermophilic FTR from M. fervidus is 76% identical to the thermophilic FTR from Methanobacterium thermoautotrophicum and has a larger number of lysine residues. A putative ATP-binding site of the FTR is reported. The size of the ftr mRNA was estimated as 1000 nucleotides indicating monocistronic transcription of the 891 bp gene. The ftr mRNA starts 27 bp downstream of the centre of a putative archaeal box A motif and terminates at an oligo-dT stretch. In vitro transcription of the ftr gene, utilizing a transcription system developed for the distantly related Sulfolobus shibatae, is discussed with respect to the functional conservation of the basal transcription apparatus of Archaea.