Mechanism of Protein Biosynthesis
1. Overview of protein biosynthesis in prokaryotes and eukaryotes. Types of protein synthesis in three subcellular compatments of plant cells (cytoplasm, plastids and mitochondria) and differences in protein synthesis machinery. The genetic code and codon usage in organelles. Crick’s adaptor hypothesis. Wobble pairing in the translation of genetic code.
2. Attachment of amino acids to tRNAs, the process of amino acid activation reaction. Multiple tRNAs for individual amino acids - existence of degenerate and redundant isoacceptor tRNAs. The two subclasses of aminoacyl-tRNA-synthetases and their stuctural characteristics. Role of identity elements and antideterminants in the idiosyncratic way of tRNA - synthetase recognition. Exeptions from the rule of „one amino-acyl-tRNA per one enzyme”.
3. The structure of cytoplasmic, plastid and mitochondrial ribosomes. RNA and protein components of large and small ribosomal subunits of subcellular compartments, their structure, functional domains and biochemical activities. The active sites of fully assembled ribosome (Peptidyl-tRNA-binding-site, aminoacyl-tRNA-binding site, exit site, aminoacyl-transferase centre, tunnel). The ribosome cycle.
4. Overview of the three main steps (initiation, elongation and termination) of translation. Vectorial movements on ribosomes. Hybrid sites. Prokaryotic initiation factors, their structures and roles in translation initiation. Process of initiation (mRNA binding, start codon selection and initiator tRNA binding, association of ribosomal subunits, release of initiation factors).
5. Similarities of chloroplast protein synthesis to bacterial protein synthesis. Differences due to the special characteristics of plastid mRNAs. Selection of start codon of mRNS without Shine-Dalgarno sequence. Role of secondary structures in specifying the initiation codon. Initiation factors of protein synthesis in plastids.
6. Steps of elongation process in prokaryotic protein synthesis (binding of aminoacyl-tRNA to the A site, formation of peptide bond, translocation of peptidyl-tRNA to the P site, release of deacylated tRNA). Structure and function of elongation factors. Mechanism of aminoacyl tRNA selection. Special characteristics of elongation in the plastids and mitochondria.
7. Steps of prokaryotic termination process (termination factor binding, peptide release, dissociation of termination complex). Structure and role of release factors. Special features of termination in chloroplasts and mitochondria.
8. Overview of the process of eukaryotic initiation, families of participating protein factors (eIFs). Major differencies in the initiation factor families of plant and animal protein synthesis machinery functioning in the cell cytosol. Characteristics of eukaryotic type initiation phase, the „closed loop” model. Role of 5’ cap, process of scanning, initiation codon recognition and release of eIFs.
9. Alternative possibilities for the formation of active initiation complex. Shunting, leaky scanning, start at initiation codon dependent and AUG-independent entry sites (IRES).
10. Mechanism of the elongation of the polypeptide chain in eukaryotes. Characteristics and participation of a special interphase factor in the formation of the first peptide bond. Elongation factors and their roles in the elongation phase of translation. Proofreading. Special roles of elongation factors unrelated to protein synthesis.
11. Steps of termination phase of protein synthesis in the cytosol. Structure and function of termination and related factors. Supression of termination in eukaryotes. Translational redefinition, frameshifting, bypassing.
12. Regulation of the process of translation in the plastids, the different levels in regulation (gene specific, group specific and general regulation). Regulation by light, redox control in regulation processes. Interaction of nucleus and plastid in regulation.
13. Regulation mechanisms controlling protein synthesis in the cytosol of eukaryotic cells. Multitude regulatory elements in 3’ and 5’ untranslated regions of mRNAs. Regulation by affecting stability of mRNA (protein/enzyme- and miRNA-dependent processes), by inhibition of transcript circularization and by ribosome scanning.
14. Regulation of protein synthesis by modifying ribosomal proteins and translation factors. Specific inhibitors of different steps of translation in prokaryotes and eukaryotes (antibiotics, komplex macromolecules, toxins of plants and microbes). Mode of action of inhibitors.
15. Special possibilities of protein synthesis with mechanisms independent of ribosomes and mRNA. Stepwise reactions and the participating molecules in the biosynthesis of bacterial cyclic peptides and antibiotics.
Current reviews in Annual Review of Plant Sciences, - Biochemistry, - Cell Biology, - Genetics and Progress in Nucleic Acid Research and Molecular Biology