Haloferax volcanii, a Prokaryotic Species that Does Not Use the Shine Dalgarno Mechanism for Translation Initiation at 5′-UTRs

Abstract

It was long assumed that translation initiation in prokaryotes generally occurs via the so-called Shine Dalgarno (SD) mechanism. Recently, it became clear that translation initiation in prokaryotes is more heterogeneous. In the haloarchaeon Haloferax volcanii, the majority of transcripts is leaderless and most transcripts with a 5′-UTR lack a SD motif. Nevertheless, a bioinformatic analysis predicted that 20–30% of all genes are preceded by a SD motif in haloarchaea. To analyze the importance of the SD mechanism for translation initiation in haloarchaea experimentally the monocistronic sod gene was chosen, which contains a 5′-UTR with an extensive SD motif of seven nucleotides and a length of 19 nt, the average length of 5′UTRs in this organism. A translational fusion of part of the sod gene with the dhfr reporter gene was constructed. A mutant series was generated that matched the SD motif from zero to eight positions, respectively. Surprisingly, there was no correlation between the base pairing ability between transcripts and 16S rRNA and translational efficiency in vivo under several different growth conditions. Furthermore, complete replacement of the SD motif by three unrelated sequences did not reduce translational efficiency. The results indicate that H. volcanii does not make use of the SD mechanism for translation initiation in 5′-UTRs. A genome analysis revealed that while the number of SD motifs in 5′-UTRs is rare, their fraction within open reading frames is high. Possible biological functions for intragenic SD motifs are discussed, including re-initiation of translation at distal genes in operons.

Internal RNAs overlapping coding sequences can drive the production of alternative proteins in archaea

“…This figure is similar to the estimated rate of 20% in 5ʹ UTRs from annotated CDS [43,44] and above the 10% rate found for random 20-mer sequences inside CDS. Unsupervised search for novel motifs did not yield any significant pattern (data not shown).…”

Alternative Translation Initiation of a Haloarchaeal Serine Protease Transcript Containing Two In-Frame Start Codons

“…Sequence analysis of this region showed that it contains a transcription factor B recognition element (BRE) motif, a TATA box, a WW motif, and a pyrimidine at position Ϫ1 (Fig. 1A), which are highly conserved cis-acting elements of haloarchaeal promoters (7,9). Therefore, it is very likely that the sequence between ATG 0 and ATG 1 acts as a promoter and that the transcription of sptA is initiated downstream of ATG 0 .…”

“…Some archaea, such as Sulfolobus solfataricus, also utilize SD-dependent translation initiation (6). Recent studies on translation initiation in haloarchaea revealed the existence of an SD-less mechanism that operates on mRNAs with a 5= UTR that lacks a SD motif or IRES and does not involve ribosomal scanning (7)(8)(9). In contrast to the aforementioned mechanisms, the leaderless mechanism operates on mRNAs that lack or have very short 5= UTRs and contain no substantial ribosomal recruitment signals except the start codon (2).…”

“…Translation initiation of leaderless mRNAs occurs in all three domains of life and, because of its simplicity, is hypothesized to be the ancestral translation initiation mechanism (10,11). Compared with the canonical SD-dependent and scanning mechanisms, the mechanistic and physiologic details of SD-less and leaderless initiation are poorly understood (8,9,12).…”

“…Moreover, the haloarchaeon Haloferax volcanii does not use the SD-dependent translation initiation mechanism, despite the presence of an SD motif in the 5= UTR of some transcripts, indicating that an SD-less mechanism for translation initiation operates at least in haloarchaea (8,9,22). Experimental studies of alternative translation initiation of single haloarchaeal transcripts are limited.…”