| Popis: |
In order for life to function properly, proteins must be correctly translated. One instance that poses as a threat to translation is stretches of poly prolines in the transcript. Because of the pyrrolidine ring in proline, proline is a poor imino acceptor and donor, and a string of three or more prolines in a row can cause the ribosome to pause. The translation factor Elongation Factor-P (EF-P) binds to the ribosome and stimulates bond formation between the prolines, alleviating the pause and allowing translation to continue. EF-P is post translationally modified on a conserved residue, and while EF-P or an EF-P homolog is found in all domains of life, there is variety in the post-translational modifications from one bacterial organism to another. Thus far, it has been discovered that EF-P in E. coli is modified with (R)-ß-Lysine, with dTDP-Rhamnose in P. aeruginosa, and a hypusine in the eukaryotic homolog, eIF5A. These modifications currently known in bacteria only represent a small amount of bacteria, and the question of what other modifications are remains unanswered. It has been confirmed by mass spectrometry that Bacillus subtilis EF-P is post-translationally modified. The identity of this modification however, still remains elusive. In this work, methods including mass spectrometry, isoelectric focusing, and in vitro reactions are used to probe what the modification on B. subtilis EF-P is. Besides getting closer to understanding what the modification on EF-P is, these techniques are also used to reveal the potential regulation of EF-P. Finally, this work addresses some of the experiments performed in identifying the modification on P. aeruginosa EF-P, including the synthesis of the substrate for modification of EF-P, dTDP-rhamnose. |
