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

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

Bibliographic Details
Main Author: Rajkovic, Andrei
Language:English
Published: The Ohio State University / OhioLINK 2016
Subjects:
Microbiology
Biochemistry
Bioinformatics
Molecular Biology
Elongation factor P
Ribosomes
Translational pausing
Protein synthesis
EF-P
Online Access:http://rave.ohiolink.edu/etdc/view?acc_num=osu1469123846
id ndltd-OhioLink-oai-etd.ohiolink.edu-osu1469123846
record_format oai_dc
collection NDLTD
language English
sources NDLTD
topic Microbiology
Biochemistry
Bioinformatics
Molecular Biology
Elongation factor P
Ribosomes
Translational pausing
Protein synthesis
EF-P
spellingShingle Microbiology
Biochemistry
Bioinformatics
Molecular Biology
Elongation factor P
Ribosomes
Translational pausing
Protein synthesis
EF-P
Rajkovic, Andrei
Promoting Bacterial Synthesis of Oligo-prolines by Modifying Elongation Factor P Post-translationally
author Rajkovic, Andrei
author_facet Rajkovic, Andrei
author_sort Rajkovic, Andrei
title Promoting Bacterial Synthesis of Oligo-prolines by Modifying Elongation Factor P Post-translationally
title_short Promoting Bacterial Synthesis of Oligo-prolines by Modifying Elongation Factor P Post-translationally
title_full Promoting Bacterial Synthesis of Oligo-prolines by Modifying Elongation Factor P Post-translationally
title_fullStr Promoting Bacterial Synthesis of Oligo-prolines by Modifying Elongation Factor P Post-translationally
title_full_unstemmed Promoting Bacterial Synthesis of Oligo-prolines by Modifying Elongation Factor P Post-translationally
title_sort promoting bacterial synthesis of oligo-prolines by modifying elongation factor p post-translationally
publisher The Ohio State University / OhioLINK
publishDate 2016
url http://rave.ohiolink.edu/etdc/view?acc_num=osu1469123846
work_keys_str_mv AT rajkovicandrei promotingbacterialsynthesisofoligoprolinesbymodifyingelongationfactorpposttranslationally
_version_ 1719459223003201536
spelling ndltd-OhioLink-oai-etd.ohiolink.edu-osu14691238462021-08-09T05:10:25Z Promoting Bacterial Synthesis of Oligo-prolines by Modifying Elongation Factor P Post-translationally Rajkovic, Andrei Microbiology Biochemistry Bioinformatics Molecular Biology Elongation factor P Ribosomes Translational pausing Protein synthesis EF-P 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. 2016 English text The Ohio State University / OhioLINK http://rave.ohiolink.edu/etdc/view?acc_num=osu1469123846 http://rave.ohiolink.edu/etdc/view?acc_num=osu1469123846 unrestricted This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws.

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