Seminar 1 Components and Regulation of Initiation of Translation Michael Altmann FS 2011

Institut für Biochemie und Molekulare Medizin

Seminar 1 (2012)

- What are the biol. consequences of mRNA transport and localized translation?

- Inform yourself about CCA adding enzyme (tRNA)!

- How many rRNA genes does a cell need to make 106 ribosomes in 6 hours?

- What are the most prominent differences in the mechanism of initiation between prokaryotes and eukaryotes?

- Why should a cell need so many helicases?

- What‘s about translation in mitochondria?

- Are there orthologs of eIF‘s?

- What do you know about the roles of small RNAs in gene expression?

- Why are 10-20% of eIF2α-P sufficient to block translation?

- Physiological consequences of translational regulation of transcription factors?

- Viruses fight against eIF2 kinases! How?

- How are protein-protein interactions measured?

- What do you know about G-proteins (function, subunits, mechanism)?

What are the biol. consequences of mRNA transport and localized translation?

• Synthesis of protein in place where it is needed • Example: nerve cells. Synapse. • Programming of cells • Example: oocytes and maternal mRNA. Yeast: buds

Inform yourself about the CCA adding enzyme (tRNA)!

• CCA is essential for aminoacylation and ribosome-binding of tRNA. Archaea and eukaryotes have structurally different enzymes. • Nucleotidyltransferase (CTP, ATP), template-independent. One single catalytic center. • Mechanism: unknown. 3D structures solved.

- How many rRNA genes does a cell need to make 106 ribosomes in 6 hours?

• Number of nucleotides per ribosome? About 5400 nt.

• Speed of transcription? About 50 nt per second or 108 sec / rRNA.

• About 50 DΝΑ-polymerase I molecules synthetize in tandem per rDNA

gene (1 new polymerase molecule jumps on rDNA gene each 2 seconds).

• G1 phase? 6 hr (21600 sec) -> 200 molecules rRNA / RNA-polymerase I

molecule. About 10'000 rRNAs per rDNA gene.

• For 1 million rRNAs: 100 rDNA genes required.

Die Transkription von hintereinander angeordneten rRNA Genen!

Velocity and fidelity of macromolecule synthesis

Macromolecule synthesis rate fidelity

DNA 50-100 nucl./sec 10-10

RNA 50 nucl./sec 10-4

Protein 20 aa/sec 10-3 - 10-4

What are the most prominent differences in the mechanism of initiation between prokaryotes and eukaryotes?

• coupling of transcription and translation • only 3 initiation factors • tRNA-Meti is not formylated • Shine/Dalgarno sequence • internal initiation

Translation initiation in bacteria

Reference: Simonetti et al (2009) Cell. Mol. Life Sci. 66, 423-436

Why should a cell need so many helicases?

• Base-pairing and melting • Reactions depending on RNA-RNA interactions • RNA chaperone • Protein-RNA interactions and their resolution

What’s about translation in mitochondria? Translation system: unique but more related to prokaryotes than eukaryotes Ribosomes: only 2 rRNAs (rRNA encoded in mitochondria, rproteins encoded in nucleus, except 1) mRNA: uncapped and lacking poly(A), no S/D but perhaps octanucleotide UAUAAAUA recognized by ribosomes.

Are there orthologs of eIF’s?

Eukarotes Prokaryotes Archaea

eIF1 IF3 (CTD) aIF1

eIF1A IF1 aIF1A

eIF2: α, β, γ aIF2: α, β, γ

eIF4A IF4A/W2 aIF4A

eIF5B IF2

How are protein-protein interactions measured? • 2-hybrid • tagged proteins on solid matrix • Co-precipitation (antibodies) • complexes on sizing columns • Surface Plasmon Resonance

In complex mixtures:

• FRET

• Co-localization

Why are 10-20% of eIF2α-P sufficient to block translation?

• eIF2B is 10-20% as abundant as eIF2 • stable complex leads to sequesteration

Physiological consequences of translational regulation of transcription factors?

• Translation controls transcription • Programming of cells • Multi-level control

Viruses fight against eIF2 kinases! How? Inhibition of the kinases:

• Production of high concentrations of dsRNA which inhibit kinase (Adeno) • Production of dsRNA-binding proteins (HSV, Vaccinia, Reo, Influenza) • Production of pseudo-substrate (K3L of Vaccinia) • Activation of cellular kinase-inhibitor (p58 by Influenza)

Activation of eIF2:

• Activation of PP1alpha (dephosphorylation of eIF2)

What do you know about the roles of small RNAs in gene expression?

• snRNP and splicing • RNA modification: snoRNA • mRNA translation and half-life