A Comprehensive Analysis of the Importance of Translation Initiation Factors for Haloferax volcanii Applying Deletion and Conditional Depletion Mutants

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.

Stall no more at polyproline stretches with the translation elongation factors EF‐P and IF‐5A

“…In addition, plants also contain EF‐P with N‐terminal extensions, which presumably target to the chloroplasts, whereas there is no evidence for a mitochondrial EF‐P homolog. In contrast to archaea and eukaryotes where a/eIF‐5A is essential for viability (Gabel et al ., ; Dever et al ., ), the efp gene can be deleted in many bacteria, including Bacillus subtilis (Ohashi et al ., ; Kearns et al ., ), E. coli (Baba et al ., ), S. enterica (Bearson et al ., 2006; 2011; Navarre et al ., ; Zou et al ., ), Brucella abortus (Iannino et al ., ), Agrobacterium tumefaciens (Peng et al ., ), Pseudomonas aeruginosa and Shewanella oneidensis (Lassak et al ., ).…”

Deciphering the Translation Initiation Factor 5A Modification Pathway in Halophilic Archaea

“…The HVO_2297 gene is physically clustered with the gene encoding aIF5a ( HVO_2300 ) (Figure 2). To our knowledge, the activity of HVO_2300 (as aIF5A) has never been experimentally validated, the gene is essential [12]. Hence, HVO_2297 is a potential candidate for the canonical DHS enzyme and HVO_B0182 could be involved in homospermidine synthesis as proposed by [37] (Figure 1(a)).…”

“…volcanii gene predicted to encode aIF5a ( HVO_2300 ) is essential [12], but little else is known about this gene/protein in this organism. To obtain a better understanding of aIF5A in Archaea and specifically in halophiles, aIF5A levels at different growth phases were monitored by Western blot using Hfx.…”

“…First, both eIF5A and aIF5A are essential [11, 12] whereas deletion of bacterial efp can be viable and leads to a range of phenotypes depending on the organism [13–16]. Second, the posttranslational modification of a strictly conserved lysine (K50, Human eIF5A) into pN ε -(4-amino-2hydroxybutyl)-lysine or hypusine is required for eIF5A activity and the hypusine modification pathway is conserved in Eukaryotes [11].…”

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

“…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.…”