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

Gäbel, Katrin; Schmitt, Jessica; Schulz, Sebastian; Näther, Daniela J.; Soppa, Jörg

doi:10.1371/journal.pone.0077188

Cited by 18 publications

(48 citation statements)

References 48 publications

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“…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 ., ).…”

Section: Evolution Of Aif‐5a Eif‐5a and Ef‐pmentioning

confidence: 98%

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

Lassak

Wilson

Jung

2015

Molecular Microbiology

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SummarySynthesis of polyproline proteins leads to translation arrest. To overcome this ribosome stalling effect, bacteria depend on a specialized translation elongation factor P (EF-P), being orthologous and functionally identical to eukaryotic/archaeal elongation factor e/aIF-5A (recently renamed 'EF5'). EF-P binds to the stalled ribosome between the peptidyl-tRNA binding and tRNA-exiting sites, and stimulates peptidyltransferase activity, thus allowing translation to resume. In their active form, both EF-P and e/aIF-5A are post-translationally modified at a positively charged residue, which protrudes toward the peptidyltransferase center when bound to the ribosome. While archaeal and eukaryotic IF-5A strictly depend on (deoxy-) hypusination (hypusinylation) of a conserved lysine, bacteria have evolved diverse analogous modification strategies to activate EF-P. In Escherichia coli and Salmonella enterica a lysine is extended by β-lysinylation and subsequently hydroxylated, whereas in Pseudomonas aeruginosa and Shewanella oneidensis an arginine in the equivalent position is rhamnosylated. Inactivation of EF-P, or the corresponding modification systems, reduces not only bacterial fitness, but also impairs virulence. Here, we review the function of EF-P and IF-5A and their unusual posttranslational protein modifications.

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Section: Evolution Of Aif‐5a Eif‐5a and Ef‐pmentioning

confidence: 98%

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

Lassak

Wilson

Jung

2015

Molecular Microbiology

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“…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)).…”

Section: Resultsmentioning

confidence: 99%

“…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.…”

Section: Resultsmentioning

confidence: 99%

“…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].…”

Section: Introductionmentioning

confidence: 99%

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

Prunetti

Graf

Blaby

et al. 2016

Archaea

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Translation initiation factor 5A (IF5A) is essential and highly conserved in Eukarya (eIF5A) and Archaea (aIF5A). The activity of IF5A requires hypusine, a posttranslational modification synthesized in Eukarya from the polyamine precursor spermidine. Intracellular polyamine analyses revealed that agmatine and cadaverine were the main polyamines produced in Haloferax volcanii in minimal medium, raising the question of how hypusine is synthesized in this halophilic Archaea. Metabolic reconstruction led to a tentative picture of polyamine metabolism and aIF5A modification in Hfx. volcanii that was experimentally tested. Analysis of aIF5A from Hfx. volcanii by LC-MS/MS revealed it was exclusively deoxyhypusinylated. Genetic studies confirmed the role of the predicted arginine decarboxylase gene (HVO_1958) in agmatine synthesis. The agmatinase-like gene (HVO_2299) was found to be essential, consistent with a role in aIF5A modification predicted by physical clustering evidence. Recombinant deoxyhypusine synthase (DHS) from S. cerevisiae was shown to transfer 4-aminobutyl moiety from spermidine to aIF5A from Hfx. volcanii in vitro. However, at least under conditions tested, this transfer was not observed with the Hfx. volcanii DHS. Furthermore, the growth of Hfx. volcanii was not inhibited by the classical DHS inhibitor GC7. We propose a model of deoxyhypusine synthesis in Hfx. volcanii that differs from the canonical eukaryotic pathway, paving the way for further studies.

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“…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.…”

mentioning

confidence: 99%

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

Tang

et al. 2016

J Bacteriol

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Recent studies have shown that haloarchaea employ leaderless and Shine-Dalgarno (SD)-less mechanisms for translation initiation of leaderless transcripts with a 5= untranslated region (5= UTR) of <10 nucleotides (nt) and leadered transcripts with a 5= UTR of >10 nt, respectively. However, whether the two mechanisms can operate on the same naturally occurring haloarchaeal transcript carrying multiple potential start codons is unknown. In this study, the transcript of the sptA gene (encoding an extracellular serine protease of Natrinema sp. strain J7-2) was experimentally determined and found to contain two potential in-frame AUG codons (AUG 1 and AUG 2 ) located 5 and 29 nt, respectively, downstream of the transcription start site. Mutational analysis revealed that both AUGs can function as the translation start codon for production of active SptA, although AUG 1 is more efficient than AUG 2 for translation initiation. Insertion of a stable stem-loop structure between the two AUGs completely abolished initiation at AUG 1 but did not affect initiation at AUG 2 , indicating that AUG 2 -initiated translation does not involve ribosome scanning from the 5= end of the transcript. Furthermore, the efficiency of AUG 2 -initiated translation was not influenced by an upstream SD-like sequence. In addition, both AUG 1 and AUG 2 contribute to transcript stability, probably by recruiting ribosomes to protect the transcript against degradation. These data suggest that depending on which of two in-frame start codons is used, the sptA transcript can act as either a leaderless or a leadered transcript for SptA production in haloarchaea. IMPORTANCEIn eukaryotes and bacteria, alternative translation start sites contribute to proteome complexity and can be used as a functional mechanism to increase translation efficiency. However, little is known about alternative translation initiation in archaea. Our results demonstrate that leaderless and SD-less mechanisms can be used for translation initiation of the sptA transcript from two in-frame start codons, raising the possibility that in haloarchaea, alternative translation initiation on one transcript functions to increase translation efficiency and/or contribute to proteome complexity.

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

Cited by 18 publications

References 48 publications

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

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

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

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

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