Günter Wich

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

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 (tRNALeu CAG), 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.

The Nucleotide Sequence of the 16S rRNA Gene and Flanking Regions from Methanobacterium formicicum: The Phylogenetic Relationship between Methanogenic and Halophilic Archaebacteria

Summary By blot hybridization experiments it was demonstrated that Methanobacterium formicicum possesses two putative rDNA transcriptional units of the eubacterial type (5′-16S-tRNA Ala -23S-5S-3′). Fragments of each unit which contain the 16S rRNA gene plus flanking regions were cloned and their nucleotide sequences determined. The 16S rRNA encoding sequence was compared to the respective sequences of Methanococcus vannielii, Halobacterium volcanii, Halobacterium cutirubrum and Halococcus morrhuae . The following results were obtained: (i) The three halophiles share an equal evolutionary distance; (ii) the Methanococcus and Methanobacterium sequences are more closely related than previously indicated by the S AB values; the evolutionary distance between Methanobacterium and the halophile sequences is less than the one exhibited by the Methanococcus structure; (iii) the evolutionary rates of the sequences are quite different; the Methanobacterium formicium sequence exhibits an evolutionary rate somewhat lower than the Methanococcus structure and is drastically different from the high genetic drift of the halophile small subunit rRNAs.

Genes for tRNA and their putative expression signals in Methanococcus

Summary Genes for tRNA are suitable systems for the search for the consensus signal sequences involved in gene expression in archaebacteria, because they are subject to the same regulatory mechanisms and their products display identical functions. To this end, a number of tRNA genes from Methanococcus vannielii were cloned and sequenced. They belong to a total of six putative transcriptional units comprising 11 tRNA genes. Together with the tRNA gene sequences previously reported, this brings the total of Methanococcus tRNA genes now analyzed to 19, organized in seven putative transcriptional units. In two of the tRNA gene clusters (one comprising two genes, the other six) one of the genes possesses opposite transcriptional polarity and is separated from the remaining gene(s) by a spacer of 146 and 115 nucleotides respectively. Comparision of the region flanking the seven transcriptional units at the 5′ end yielded a consensus sequence between −25 and −50 bases upstream. In the tRNA gene clusters with opposite transcriptional polarity this sequence occurred twice and also in inverse polarity. This observation and the fact that this sequence was the only detectable motif of homologous primary structures in 5′ upstream regions of tRNA genes indicates that it may be involved in transcription initiation. Common motifs at the 3′ flanking regions, which may possibly be involved in transcription termination, are also presented.

Divergent Evolution of 5 S rRNA Genes in Methanococcus

Abstract The organization of genes for 5S rRNA in the methanogenic archaebacterium Methanococcus (M .) voltae and their nucleotide sequences have been determined. M. voltae possesses three 5S rRNA genes, one of them is organized in an rRNA transcriptional unit coding for 16S-23S-5S rRNA. The other two are associated with seven tRNA genes in a putative transcriptional unit composed of 5′-tRNAThr-tRNAPro-tRNATyr-tRNALys - 5S rRNA-tRNAAsp-tRNALys - 5S rRNA-tRNAAsp-3′. Coding regions plus spacers of the tRNALys-5S rRNA-tRNAAsp block of this gene cluster occur twice with identical sequence. The 5S rRNA from this cluster displays considerable sequence divergence to the rRNA operon-linked 5S rRNA gene. Comparison of the M. voltae 5S rRNA sequences with those from M. vannielii revealed that the operon-linked genes on one hand and the tRNA-linked 5S genes on the other share a greater sequence homology than the two types of genes within each of the two organisms. This indicates an independent evolution of the two sets of 5S rRNA genes without selective pressure from other ribosomal components or, alternatively, lateral gene transfer.