Lecture 6

Regulation of Elongation Michael Altmann FS 2015

Institut für Biochemie und Molekulare Medizin

Regulation of polypeptide chain elongation

Factor modification Phosphorylation of eEF2 / eEF2 kinase

ADP-ribosylation

Diphthamide

Transit time Elongation rate

Selenocysteine

0 min:

2.5 min:

7.5 min:

25 min:

Ribosome transit time

1

2

3

4

5

time (min)5 10 15 20 25

1

2

3

4

5

time (min)5 10 15 20 25

transit time transit time

fast elongation slow elongation

total

free

eEF2 kinase

•  Single polypeptide, 725 aa long

•  Activation requires Ca2+/calmodulin-binding (but not for activity after

•  Phosphorylation of eEF2 at Thr 56 leads to reduced ribosome-binding

•  Activated by PKA (phosphorylation of Ser500)

•  Inactivated by S6K1 in response to insulin (phosphorylation of Ser366)

•  Physiological relevance: reduced translation during mitosis, in active

activation) and self-phosphorylation

activity and reduced eEF2 activity

nerve cells (?), in contracting muscle (?)

Regulation of the eEF2 kinase

Reference: GJ Browne & CG Proud Eur. J. Biochem. 269: 5360-5368, 2002

Elongation factors have unusual posttranslational modifications

Reference: Greganova et al (2011) FEBS-Journal 278, 2613-2624

• eIF5A/EF-P become hypusinated (spermidine) at a specific lysine residue. • eEF2 becomes dipthamide-modified at a specific histidine residue (not in bacteria). • eEF1A becomes EPG (ethanolamine phosphoglycerol)-modified at one or two glutamate residues of eukaryotic factors. • Though eEF1A, eEF2 and eIF5A are essential, none of these modifications (which attachment requires the activity of several enzymes) is essential (remnants of an ancient system?).

ADP-ribosylation

Reference: W. Müller-Esterl Biochemie, Spektrum 2004

• ADP ribosylation is one (out of many) protein modification which is introduced by ADP ribosylases at certain arginine or histidine residues e.g. on histones. Normally, it is a reversible reaction (can be removed by specific hydrolases). • The cholera bacteria (Vibrium cholerae) produces a toxin ADP-ribosylase which is taken up by the intestine epithelium of infected persons. • The toxin catalyzes the transfer of a ADP-ribosyl residue to the alpha subunit of a G-protein. Thereby the GTPase-activity is inhibited and high levels of cAMP are constitutively produced. • This leads to an enhanced Na+- and water loss through a channel into the intestinal lumen = strong diarrhea.

ADP-ribosylated diphthamide inactivates eEF2

Reference:

• Diphtheria was until the middle of the 20th Century one of the main causes for children‘s deaths (today it is efficiently controlled thanks to vaccination). Mechanism • The bacteria (Corynebacterium diphtheriae) produces a toxin protein, which gives rise to ADP-ribosylation of a diphthamid-histidine residue in eEF2. • This modification leads to a complete inhibition of eEF2 translocase-activity. • High toxicity: A single bacterial ADP-ribosylase molecule can completely inhibit translation elongation in an infected cell!

Reference: PR Hoffmann& MJ Berry Thyroid 15: 769-775, 2005

Selenocysteine: the 21st amino acid

Seminar 2

-  Where comes the energy for peptide bond formation from? -  What are the implications of the finding that peptidyltransferase is a ribozyme for our view

of evolution? -  Aminoglycosides: what are they and how could they affect eukaryotic cells? -  Compare polyribosome profiles from cells with inhibited initiation with those of cells with

inhibited elongation! -  What are the signals that activate or inactivate the EF2 kinase?