Abstract
Translation is an important step in gene expression. The initiation of translation is phylogenetically diverse, since currently five different initiation mechanisms are known. For bacteria the three initiation factors IF1 – IF3 are described in contrast to archaea and eukaryotes, which contain a considerably higher number of initiation factor genes. As eukaryotes and archaea use a non-overlapping set of initiation mechanisms, orthologous proteins of both domains do not necessarily fulfill the same function. The genome of Haloferax volcanii contains 14 annotated genes that encode (subunits of) initiation factors. To gain a comprehensive overview of the importance of these genes, it was attempted to construct single gene deletion mutants of all genes. In 9 cases single deletion mutants were successfully constructed, showing that the respective genes are not essential. In contrast, the genes encoding initiation factors aIF1, aIF2γ, aIF5A, aIF5B, and aIF6 were found to be essential. Factors aIF1A and aIF2β are encoded by two orthologous genes in H. volcanii. Attempts to generate double mutants failed in both cases, indicating that also these factors are essential. A translatome analysis of one of the single aIF2β deletion mutants revealed that the translational efficiency of the second ortholog was enhanced tenfold and thus the two proteins can replace one another. The phenotypes of the single deletion mutants also revealed that the two aIF1As and aIF2βs have redundant but not identical functions. Remarkably, the gene encoding aIF2α, a subunit of aIF2 involved in initiator tRNA binding, could be deleted. However, the mutant had a severe growth defect under all tested conditions. Conditional depletion mutants were generated for the five essential genes. The phenotypes of deletion mutants and conditional depletion mutants were compared to that of the wild-type under various conditions, and growth characteristics are discussed.
Highlights
Translation is a very important step in the expression of genetic information in all three domains of life
In two cases it was shown that this was due to the presence of two orthologous genes, which cannot be deleted simultaneously, and that the functions of aIF1A and aIF2b are essential
The gene encoding the a subunit of the heterotrimeric factor aIF2 could be deleted, which was unexpected because aIF2a of Sulfolobus is involved in initiator tRNA binding
Summary
Translation is a very important step in the expression of genetic information in all three domains of life. The first mechanism involves the so-called Shine Dalgarno (SD) sequence, a motif of 4–8 nt that is localized in the 59-UTR of transcripts 5–7 nt upstream of the start codon. It interacts via base-pairing with the 39-end of the 16S rRNA of the small ribosomal subunit and thereby determines the localization of the small subunit on the transcript. This mechanism has been thoroughly characterized in Escherichia coli and operates in many bacteria and some archaea. A mutational analysis indicated that the SD mechanism does not operate at all in the species used in this study, Haloferax volcanii (Kramer et al, submitted)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
