(R)-β-Lysine-modified Elongation Factor P Functions in Translation Elongation

Bullwinkle, Tammy J.; Zou, Shengwei; Rajkovic, Andrei; Hersch, Steven J.; Elgamal, Sara; Robinson, Nathaniel; Smil, D.; Bolshan, Yuri; Navarre, William Wiley; Ibba, Michael

doi:10.1074/jbc.m112.438879

Cited by 49 publications

(68 citation statements)

References 33 publications

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“…Although, the remainder of the PPX motifs may not be pausing translation, the observed increase in ribosome density likely reflects that many PPX motifs can still slow translation. This might offer some explanation for the increased polysome retention previously observed for E. coli Δ efp strains [17]. Similar effects are also observed after addition of chloramphenicol to E. coli cultures [17] or depletion of eIF5A (EF-P paralog) in eukaryotic cells [38]–[40].…”

Section: Discussionsupporting

confidence: 70%

EF-P Dependent Pauses Integrate Proximal and Distal Signals during Translation

et al. 2014

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Elongation factor P (EF-P) is required for the efficient synthesis of proteins with stretches of consecutive prolines and other motifs that would otherwise lead to ribosome pausing. However, previous reports also demonstrated that levels of most diprolyl-containing proteins are not altered by the deletion of efp. To define the particular sequences that trigger ribosome stalling at diprolyl (PPX) motifs, we used ribosome profiling to monitor global ribosome occupancy in Escherichia coli strains lacking EF-P. Only 2.8% of PPX motifs caused significant ribosomal pausing in the Δefp strain, with up to a 45-fold increase in ribosome density observed at the pausing site. The unexpectedly low fraction of PPX motifs that produce a pause in translation led us to investigate the possible role of sequences upstream of PPX. Our data indicate that EF-P dependent pauses are strongly affected by sequences upstream of the PPX pattern. We found that residues as far as 3 codons upstream of the ribosomal peptidyl-tRNA site had a dramatic effect on whether or not a particular PPX motif triggered a ribosomal pause, while internal Shine Dalgarno sequences upstream of the motif had no effect on EF-P dependent translation efficiency. Increased ribosome occupancy at particular stall sites did not reliably correlate with a decrease in total protein levels, suggesting that in many cases other factors compensate for the potentially deleterious effects of stalling on protein synthesis. These findings indicate that the ability of a given PPX motif to initiate an EF-P-alleviated stall is strongly influenced by its local context, and that other indirect post-transcriptional effects determine the influence of such stalls on protein levels within the cell.

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Section: Discussionsupporting

confidence: 70%

EF-P Dependent Pauses Integrate Proximal and Distal Signals during Translation

et al. 2014

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“…The other two codons that also fall into this category are AGA (Arg) and CCC (Pro). While the lower efficiency of AGA (Arg) has been discussed above, lower initiation from CCC (Pro) could be due to its lower efficiency of peptide bond formation with the incoming amino acid in the A site (29)(30)(31). Other codons tested in this study are in fact decoded by tRNAs requiring wobble pairing with the third nucleotide of the codon, which are not normally favored for initiation (32).…”

Section: Discussionmentioning

confidence: 71%

Initiation with Elongator tRNAs

Samhita

Virumäe

Rèmme

et al. 2013

Journal of Bacteriology

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In all domains of life, initiator tRNA functions exclusively at the first step of protein synthesis while elongator tRNAs extend the polypeptide chain. Unique features of initiator tRNA enable it to preferentially bind the ribosomal P site and initiate translation. Recently, we showed that the abundance of initiator tRNA also contributes to its specialized role. This motivates the question, can a cell also use elongator tRNA to initiate translation under certain conditions? To address this, we introduced non-AUG initiation codons CCC (Pro), GAG (Glu), GGU (Gly), UCU (Ser), UGU (Cys), ACG (Thr), AAU (Asn), and AGA (Arg) into the uracil DNA glycosylase gene (ung) used as a reporter gene. Enzyme assays from log-phase cells revealed initiation from non-AUG codons when intracellular initiator tRNA levels were reduced. The activity increased significantly in stationary phase. Further increases in initiation from non-AUG codons occurred in both growth phases upon introduction of plasmid-borne genes of cognate elongator tRNAs. Since purine-rich Shine-Dalgarno sequences occur frequently on mRNAs (in places other than the canonical AUG codon initiation contexts), initiation with elongator tRNAs from the alternate contexts may generate proteome diversity under stress without compromising genomic integrity. Thus, by changing the relative amounts of initiator and elongator tRNAs within the cell, we have blurred the distinction between the two classes of tRNAs thought to be frozen through years of evolution. Most organisms possess two kinds of tRNAs, the initiator tRNA, which decodes the initiation codon, and the elongator tRNAs, which decode the subsequent codons within the open reading frame (ORF). In Escherichia coli, there are four copies of the initiator tRNA genes; three of these, metZ, metW, and metV, are present at 63.5 min, whereas the fourth one, metY, is found at 71.5 min in the genome (1). It is also known that either (but not both) of these loci may be deleted from E. coli. The strain deleted for metZWV becomes cold sensitive. However, at temperatures of 30°C or higher the strain grows normally (2, 3).Initiator and elongator tRNAs are believed to perform their respective decoding functions without functional interference from one another. Although they share the same cloverleaf-like backbone structure, years of evolution have led to their separate and distinct functions, now held in place by an intricate support system comprising translation factors, quality control mechanisms, and the ribosome itself. Elongator tRNAs are excluded from participation at the start of protein synthesis by sequence features specific to the initiator tRNA that are recognized exclusively at the P site of the ribosome (4-6). Various other details, such as the fact that the initiation and elongation factors bind specifically to their cognate species of tRNAs, further aid the specificity of their functions.However, earlier studies have enabled initiation by elongator tRNAs, by transplanting the critical features of initiator tRNA onto the...

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“…Purification of Recombinant and Native B. subtilis EF-PNative B. subtilis EF-P purified for top-down mass spectrometry was adapted from a prior paper with the following changes (25). Wild-type B. subtilis 3610 cells were grown in 40 liters of LB medium.…”

Section: Methodsmentioning

confidence: 99%

“…Therefore, FLAG-tagged B. subtilis EF-P was chromosomally integrated into a ⌬efp B. subtilis strain and purified with anti-FLAG M2 magnetic beads (Sigma-Aldrich) following the instructions of the manufacturer. Recombinant intein-tagged B. subtilis EF-P was expressed in XJB BL21(DE3) cells in an autoinduction medium as described before, with slight modifications (25). Lysate expressing recombinant EF-P was applied to an in-house packed intein column (New England Biolabs) and allowed to sit overnight at room temperature to increase cleaving efficiency.…”

Section: Methodsmentioning

confidence: 99%

Translation Control of Swarming Proficiency in Bacillus subtilis by 5-Amino-pentanolylated Elongation Factor P

Rajkovic

Hummels

Witzky

et al. 2016

Journal of Biological Chemistry

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Elongation factor P (EF-P) accelerates diprolyl synthesis and requires a posttranslational modification to maintain proteostasis. Two phylogenetically distinct EF-P modification pathways have been described and are encoded in the majority of Gramnegative bacteria, but neither is present in Gram-positive bacteria. Prior work suggested that the EF-P-encoding gene (efp) primarily supports Bacillus subtilis swarming differentiation, whereas EF-P in Gram-negative bacteria has a more global housekeeping role, prompting our investigation to determine whether EF-P is modified and how it impacts gene expression in motile cells. We identified a 5-aminopentanol moiety attached to Lys 32 of B. subtilis EF-P that is required for swarming motility. A fluorescent in vivo B. subtilis reporter system identified peptide motifs whose efficient synthesis was most dependent on 5-aminopentanol EF-P. Examination of the B. subtilis genome sequence showed that these EF-P-dependent peptide motifs were represented in flagellar genes. Taken together, these data show that, in B. subtilis, a previously uncharacterized posttranslational modification of EF-P can modulate the synthesis of specific diprolyl motifs present in proteins required for swarming motility.

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(R)-β-Lysine-modified Elongation Factor P Functions in Translation Elongation

Cited by 49 publications

References 33 publications

EF-P Dependent Pauses Integrate Proximal and Distal Signals during Translation

EF-P Dependent Pauses Integrate Proximal and Distal Signals during Translation

Initiation with Elongator tRNAs

Translation Control of Swarming Proficiency in Bacillus subtilis by 5-Amino-pentanolylated Elongation Factor P

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