Sequence, Organization and Transcription of the Ribosomal RNA Operon and the Downstream tRNA and Protein Genes in the Archaebacterium Thermofilum pendents

Abstract

Summary The single ribosomal RNA (rRNA) operon from the extremely thermophilic archaebacterium Thermofilum pendens was sequenced together with the immediate downstream tRNA genes and open reading frames on both DNA strands. The genes for 16S and 23S RNA were separated by a short spacer sequence and were not followed by a 5S RNA gene. Sites of initiation and termination of the rRNA transcript, and its processing sites, were localized by S1 or mung bean nuclease mapping and by primer-directed reverse transcriptase analysis. Initiation occurred primarily 187 nucleotides upstream from the 16S RNA gene, after an archaebacterial promoter and this was confirmed by a guanyltransferase capping experiment. The transcript terminated inefficiently before a polypyrimidine sequence 45 nucleotides downstream from the 23S RNA gene. The 16S RNA leader sequence, the spacer region and the sequence downstream from the 23S RNA can generate extensive secondary structure, including the processing stems for the two rRNAs. Moreover, much of this structure is supported phylogenetically by coordinated base changes. We propose that some of these double helical structures are involved in transcriptional regulation. The 16S and 23S RNA sequences were aligned with those of other organisms. Secondary structures were generated from the alignments which are characteristic of the extreme thermophiles. Moreover, phylogenetic trees were derived which placed T. pendens close to Thermoproteus tenax. The downstream tRNA genes and open reading frames each exhibited an archaebacterial promoter-like motif and a putative primary initiation site. Incomplete termination also occurred at polypyrimidine sequences. A 919 bp sequence between the two tRNA genes, which are located on opposing DNA strands, was rich in polypyrimidine sequences on both strands. Transcript mapping suggested that this constitutes a major termination region.