Genes for stable RNA in the extreme thermophile Thermoproteus tenax: introns and transcription signals

Abstract

To investigate gene organization and expression signals in extreme thermophilic archaebacteria, tRNA genes were cloned from Thermoproteus tenax. Clones for five tRNA species were obtained, namely for tRNAAla (TGC), tRNAAla (CGC), tRNALeu (CAG), tRNALeu (CAA) and tRNAMet (CAT). Three of the respective genes were located singly in the chromosome, the two others (tRNAAla and tRNAMet) were clustered but in a head to head position. Four of the genes contained intervening sequences, either in the classical position 3' to the anticodon (tRNAMet), or within the anticodon sequence (tRNALeuCAG), or in the hitherto unique position 5' to the anticodon within the anticodon stem region (tRNAAla). Existence of a transcript containing the intervening sequence was demonstrated by nuclease S1 mapping. All tRNA genes were extremely rich in G-C basepairs of helical regions, a feature which may contribute to thermostability of the secondary structure. The start site of transcription of the 16S/23S rRNA operon and of two tRNA genes of Thermoproteus was determined by nuclease S1 mapping. Transcription of the tRNA genes initiates close to or immediately at the 5' end of the structural gene, that of the rRNA operon 175 bp upstream of the coding region. About 18 bp upstream of the transcription initiation site a conserved AT-rich sequence motif occurs within a fairly GC-rich intercistronic spacer. Its putative instability at the high growth temperature of Thermoproteus suggests a function as entry site for RNA polymerase.