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