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Leaderless transcripts of the crenarchaeal hyperthermophile Pyrobaculum aerophilum1

https://doi.org/10.1006/jmbi.2001.4669Get rights and content

Abstract

We mapped transcription start sites for ten unrelated protein-encoding Pyrobaculum aerophilum genes by primer extension and S1 nuclease mapping. All of the mapped transcripts start at the computationally predicted translation start codons, two of which were supported by N-terminal protein sequencing. A whole genome computational analysis of the regions from −50 to +50 nt around the predicted translation starts codons revealed a clear upstream pattern matching the consensus sequence of the archaeal TATA box located unusually close to the translation starts. For genes with the TATA boxes that best matched the consensus sequence, the distance between the TATA box and the translation start codon appears to be shorter than 30 nt. Two other promoter elements distinguished were also found unusually close to the translation start codons: a transcription initiator element with significant elevation of C and T frequencies at the −1 position and a BRE element with more frequent A bases at position −29 to −32 (counting from the translation start site). We also show that one of the mapped genes is transcribed as the first gene of an operon. For a set of genes likely to be internal in operons the upstream signal extracted by computer analysis was a Shine-Dalgarno pattern matching the complementary sequence of P. aerophilum 16 S rRNA. Together these results suggest that the translation of proteins encoded by single genes or genes that are first in operons in the hyperthermophilic crenarchaeon P. aerophilum proceeds mostly, if not exclusively, through leaderless transcripts. Internal genes in operons are likely to undergo translation via a mechanism that is facilitated by ribosome binding to the Shine-Dalgarno sequence.

Introduction

Archaea (archaebacteria) constitute one of the three major evolutionary lineages of life on Earth. They are globally widespread, populating soil1 and other ecological niches; it has been found that Archaea may constitute up to 30% of the oceanic pikoplankton in coastal Antarctic surface water.2 Hyperthermophilic archaea represent the organisms at the upper temperature border of life, with upper growth limits between 80°C-113°C.3 They are an interesting source for the study of phylogeny and organization of ancestral life. Archaea are currently divided into three kingdoms: Euryarchaeota, Crenarchaeota and Korarchaeota.4

Pyrobaculum aerophilum is a rod-shaped crenarchaeon that grows optimally at 100°C. The species was isolated by Karl Stetter and co-workers from a boiling marine water hole.5 It is facultatively aerobic and has a plating efficiency of up to 100%.5 These features make it a good candidate for development as a model hyperthermophilic organism. The entire genome of P. aerophilum has recently been sequenced. We have reported the results from sampling small cDNA and genomic DNA libraries,6 and described the development of a genome map and identification of 489 genes.7 Here we present the next step in our study of P. aerophilum. In order to get some information on the mechanisms of P. aerophilum transcription and translation we mapped the transcription initiation sites by primer extension and in some cases additionally by S1 nuclease mapping of ten randomly chosen P. aerophilum genes. Surprisingly, all mapped transcripts start just before the computationally predicted translation start codons, two of which were confirmed by N-terminal sequencing. By Northern blot analysis we have shown that one of the mapped genes is transcribed as the first gene in an operon. We also performed whole genome analysis and found that the predominant start context signal, located unusually close to the translation start codons, was similar to an archaeal promoter sequence with consensus TTTA{A,T}A. We did not detect a Shine-Dalgarno pattern while dealing with the whole set of genes from P. aerophilum, but found this pattern for a set of genes likely to be integral in operons. The experimental and computational results together suggest that the translation of proteins encoded by single or operon-leading genes in P. aerophilum proceeds mostly through leaderless transcripts, whereas genes that are inside operons are likely to utilize Shine-Dalgarno sequences for initiation of translation. We discuss this phenomenon in light of the available data on leaderless transcripts and possible translation mechanisms.

Section snippets

Genes chosen for analysis

We chose a few unrelated P. aerophilum genes with high BLAST scores to sequences from a variety of organisms and obtained BESTFIT8 pairwise alignment of the encoded protein sequences with their homologs. Genes chosen for the study, their accession numbers, and homology to known genes are shown in Table 1. Initially we designated the AUG or GUG near the 5′ end of an open reading frame, and close to the sequence resembling the consensus sequence for the archaeal TATA box, as the putative

Discussion

Results presented here suggest that transcription of single genes and polycistronic mRNA in P. aerophilum proceeds mostly with the formation of leaderless transcripts, which do not contain 5′ untranslated sequences (5′-UTR). Two lines of evidence support this conclusion: (i) the experimental mapping of the transcripts of randomly picked genes; and (ii) results of computational analysis of the genomic DNA sequence.

The experimental mapping of ten randomly picked genes, with primer extension and S1

Strains and media

P. aerophilum strain IM2 growth conditions were as described5 except that 5% oxygen was used for anaerobic growth.

DNA synthesis and sequencing

Oligonucleotides were synthesized on a Beckman oligo 1000 DNA synthesizer by using solid-phase cyanoethyl phosphoramidite chemistry. All oligonucleotides were deprotected in ammonium hydroxide and used without further purification.

DNA sequencing was done by using [α-32P]dATP and the SequiTherm Cycle Sequencing Kit (Epicentre Technologies, Madison, WI) with reagents supplied by the

Acknowledgements

The authors thank Claudia Baikalov and Ju-Huei Chiang for help with DNA sequencing, Dr Jorg Soppa for helpful references and Alex Lomsadze his help and advice on using GeneMark.hmm 2.0.

This work was supported by the grant from the National Institutes of Health (GM 57917 to J.H.M.) and by the grant from the National Institutes of Health (HG 00783 to M.B.).

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    1

    Edited by M. Gottesman

    2

    Present address: A. G. King, Department of Pathology and Developmental Biology, B259 Beckman Center, Stanford University School of Medicine, Stanford, CA 94305, USA.

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