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Promoting Bacterial Synthesis of Oligo-prolines by Modifying Elongation Factor P Post-translationally

Rajkovic, Andrei
http://orcid.org/0000-0002-9293-0437
http://rave.ohiolink.edu/etdc/view?acc_num=osu1469123846

Abstract Details

2016, Doctor of Philosophy, Ohio State University, Molecular, Cellular and Developmental Biology.
Elongation factor P (EF-P) binds to ribosomes requiring assistance with the formation of oligo-prolines. In addition, post-translational modification of EF-P in Escherichia coli with (R)-ß-lysine is required for maximal activity. Polysome analysis indicated that post-translationally modified EF-P functions in translation elongation, rather than in initiation as previously proposed. This was further supported by the inability of EF-P to enhance the rate of formation of fMet-Lys or fMet-Phe, indicating that the role of EF-P is not to specifically stimulate formation of the first peptide bond. Three proteins, Yjek, PoxA, and YfcM comprise the Escherichia coli EF-P modification pathway that perform a two-step reaction, initially yielding a (R)-ß-lysylated EF-P. (R)-ß-lysylated EF-P receives a hydroxylation on Lys34 by the hydroxylase YfcM that marginally increases EF-P activity. However, deletion of yfcM had no phenotypic consequences in Salmonella, suggesting EF-P’s in vivo role is critically dependent on post-translational ß-lysylation but not hydroxylation. Even though the role of EF-P’s hydroxylation is still poorly understood, the crystal structure of YfcM was solved and the mechanism of hydroxylation was revealed. According to its crystal structure, YfcM has a 2-His-1-carboxylate motif that coordinates a Fe(II) ion and forms the catalytic site of non-heme iron enzymes. The 2-His-1-carboxylate motif was mutated and characterized using hydroxylation reactions with ß-lysylated EF-P. Semi-quantitative mass spectrometry revealed the hydroxylation capacity was diminished for these mutants. These findings redemonstrate that YfcM hydroxylates EF-P and they additionally show that it happens by a mechanism that is reminiscent of non-heme iron enzymes. While (R)-ß-lysine is critical for E. coli EF-P’s function, the distribution of the (R)-ß-lysine modification pathway is mainly confined to gammaproteobacteria. To address whether alternative EF-P modification pathways exist, we purified EF-P from organisms lacking poxA and employed structural mass spectrometry to investigate the presence of different modifications. We determined Pseudomonas aeruginosa EF-P to be post-translationally modified with L-rhamnose on a conserved arginine residue. Bioinformatics analysis identified a conserved gene consistently adjacent to efp that was a predicted glycosyltransferase. A series of gene knockouts confirmed the adjacent gene glycosylates P. aeruginosa EF-P using dTDP-L-rhamnose as the substrate. Furthermore, an in vivo reporter encoding four consecutive prolines had decreased expression in the absence of modified EF-P. These experiments show an amino-linked (N-linked) glycosylation on EF-P’s conserved Arg32 as a means to enhance the function of EF-P. The previously mentioned phylogenetically distinct EF-P modification pathways are encoded strictly in Gram-negative bacteria, while absent from any Gram-positive bacteria. Prior work suggested efp primarily supports Bacillus subtilis swarming differentiation, in contrast with the global housekeeping role observed in Gram-negatives, hence prompting our investigation to determine whether EF-P is modified and how it impacts gene expression in these cells. We identified a 5-aminopentanol moiety attached to Lys32 of B. subtilis EF-P that is required for swarming motility. Bioinformatics studies indicated that EF-P dependent polyproline motifs are represented in swarming motility associated genes and use of a fluorescent in vivo reporter in B. subtilis confirmed fully modified EF-P is required for the expression of such motifs. Taken together, these findings support a widespread convergence for the evolution of EF-P modifications.
Michael Ibba, Dr. (Advisor)
214 p.
Biochemistry; Bioinformatics; Microbiology; Molecular Biology
Elongation factor P; Ribosomes; Translational pausing; Protein synthesis; EF-P

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Citations

  • Rajkovic, A. (2016). Promoting Bacterial Synthesis of Oligo-prolines by Modifying Elongation Factor P Post-translationally [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1469123846

    APA Style (7th edition)

  • Rajkovic, Andrei. Promoting Bacterial Synthesis of Oligo-prolines by Modifying Elongation Factor P Post-translationally . 2016. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1469123846.

    MLA Style (8th edition)

  • Rajkovic, Andrei. "Promoting Bacterial Synthesis of Oligo-prolines by Modifying Elongation Factor P Post-translationally ." Doctoral dissertation, Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1469123846

    Chicago Manual of Style (17th edition)

osu1469123846
42
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This open access ETD is published by The Ohio State University and OhioLINK.