Transcription signals for stable RNA genes in Methanococcus

A previous survey of upstream sequences of tRNA genes from the archaebacterium Methanococcus vannielii has revealed that there are two boxes of sequence homology: A box "A" of about 20 conserved nucleotides at a distance of 30 to 49 basepairs upstream from the gene and a box "B" 18 to 19 nucleotides downstream from box "A" (Wich, G., Sibold, L., and Böck, A. (1985) System. Appl. Microbiol. (in press). Nuclease S1 mapping experiments were carried out with two of these tRNA transcriptional units and with a ribosomal RNA operon, to determine whether these consensus sequences have a function in the initiation of transcription. Use was made of the fact that cells from Methanococcus accumulate primary transcript and processing intermediates of ribosomal RNA under conditions of protein synthesis inhibition. The following results were obtained: (i) Transcription in all three systems starts at the G within the conserved trinucleotide TGC of box "B". Since the box "B" motif, 5'TGCaagT3', also occurs at the site of transcription initiation of protein encoding genes, both in methanogenic and halophilic organisms, it appears to constitute a frequently used transcription start signal within these archaebacterial groups. (ii) The box "A" motif occurs with constant spacing, relative to box "B", in all 10 tRNA and ribosomal RNA transcriptional units investigated from Methanococcus. Since it is not present in the leader region of genes coding for proteins, it seems to function as a specific element which is required for the expression of genes for stable RNA. (iii) Termination of transcription of the ribosomal RNA operon from Methanococcus occurs at a distinct T within an oligo-T stretch immediately downstream from the 3'-terminal 5S RNA gene. This signal occurs in all 3'-flanking regions of transcriptional units for stable RNA from the Methanococcus strains studied. Termination signals for stable RNA genes in Methanococcus appear to be similar with those of stable RNA genes in eukaryotes. (iv) By nuclease S1 mapping a recognition site was identified for a processing enzyme involved in the maturation of preribosomal RNA.