Function of Elongation Factor P in Translation
Dörfel, Lili Klara
Dissertation
Angenommen am:
2015-11-16
Erschienen:
2016-04-25
Betreuer:
Rodnina, Marina Prof. Dr.
Gutachter:
Rodnina, Marina Prof. Dr.
Gutachter:
Neumann, Heinz Prof. Dr.
Gutachter:
Stark, Holger Prof. Dr.
Gutachter:
Ficner, Ralf Prof. Dr.
Gutachter:
Konrad, Manfred Dr.
Gutachter:
Bohnsack, Markus Prof. Dr.
Zum Verlinken/Zitieren: http://hdl.handle.net/11858/00-1735-0000-0028-873A-3
Dateien
Name:
Doerfel_LK.pdf
Größe:
4,5 MB
Format:
PDF
Beschreibung:
Dissertation
Lizenzbestimmungen:
Zusammenfassung
Englisch
The ribosome translates the genetic information encoded in the mRNA into amino acid sequence of a peptide chain. The catalytic site of the ribosome where peptide bonds are made, the peptidyl transferase center, consists of RNA and usually does not accessory proteins for function. By applying systematic sequence permutations in several in-vitro translation assays we discovered that the ribosome – while proficient in making peptide bonds between most amino acids – is surprisingly slow when synthesizing polyproline and PPG motifs. The combination of poor A- and P-site substrates led to the inhibition of peptide bond formation after incorporation of the second proline into the nascent chain resulting in robust ribosomal stalling. Linear-free-energy relationships (LFER) of peptidyl transfer using different proline analogs as well as the investigation of sequence-context effects in the nascent peptidyl chain revealed that the poor reactivity of peptidyl-Pro-tRNAPro in the P site of the ribosome originates from stereo-electronic properties of proline which induce an unfavorable orientation of the peptidyl-tRNA in the peptidyl transferase center. We showed that the functionally uncharacterized elongation factor P (EF-P) is a specialized translation elongation factor which augments the peptidyl transferase activity of the ribosome by alleviating proline-induced stalling. The identified function of EF-P as facilitator of polyproline synthesis explains the pleiotropic phenotypes observed in EF-P knockout strains in vivo. EF-P catalyzes peptidyl transfer entropically, presumably by positioning of the peptidyl-tRNA, its CCA end and/or the attached amino acid in a more active conformation. In Escherichia coli EF-P is lysylated and hydroxylated at Lys34 a residue that points to the peptidyl transferase center. Kinetically we show that this unique modification is crucial for the catalytic activity of EF-P. Because the modifications are specific for bacteria, these findings may pave the way for the discovery of new antibiotics.
Keywords: Elongation factor P; ribosome; proline
