1. From RNA to protein Kanokporn Boonsirichai

2. The coding problem  How is the information in a linear sequence of nucleotides in mRNAs translated into the linear sequence of amino acids in proteins? DNADeoxynucleotides RNARibonucleotides ProteinAmino acids 4 kinds 20 kinds MoleculesMonomersMonomer variations

3. The Genetic Code  mRNA sequence is decoded in a set of three nucleotides  For a sequence, there exists three possible reading frames.

4. tRNA The code is translated as a triplet of three nucleotides into the corresponding amino acid via a transfer RNA. Codon Anticodon

5. Structure of a tRNA

6. Redundancy of the code  More than one tRNAs for some amino acids  Some tRNAs can base pair with more than one codon

7. tRNA Biosynthesis  Synthesized by RNA polymerase III  Synthesized as a larger precursor - trimmed - spliced - base modifications

8. tRNA Splicing  Some precursor-tRNAs are with introns  tRNAs introns are cut and pasted together by endonuclease and tRNA ligase Cut within the tRNA Paste the tRNA pieces together

9. Coupling of amino acid to tRNAs  Achieved by aminoacyl-tRNA synthetases

11. Aminoacyl-t-RNA synthethase  How is accuracy achieved?  The correct amino acid has the highest affinity for the active site.  Hydrolytic editing  Nucleotide binding pockets/ acceptor stem recognition  1 mistake in 40,000 tRNA couplings

12. tRNA Gln

13. Synthesis of Proteins  Formation of a peptide bond between the carboxyl group at the end of a growing polypeptide chain and a free amino group on an incoming amino acid  The synthesis is stepwise from the N-terminal end to the C-terminal end.  The growing carboxyl end of the polypeptide chain remains activated by its covalent attachment to a tRNA molecule (a peptidyl-tRNA molecule).

14. Peptidyltransferase reaction

15. Decoding mRNAs  1 mistakes in 10,000 amino acid polymerized  2 amino acids are polymerized per second in eukaryotes  20 amino acids are polymerized per second in prokaryotes  So, where is it done?

16. What do you on this micrograph?

17. Ribosome

18.  Ribosome subunits and their rRNAs are assembled in the nucleolus and exported to the cytoplasm where translation occurs.

19. Translation Elongation  A ribosome contains four binding sites for RNA molecules: 3 for tRNAs and 1 for mRNA.  tRNA anticodon interacts with the codons on the mRNA in the small subunit of the ribosome.  The large subunit catalyzes the peptidyl transferase reaction.

20. A-site: Aminoacyl-tRNA P-site: Peptidyl-tRNA E-site: Exit site

21. Translation Elongation

22. Elongation factors and translation accuracy

23. Ribosomes  Contain 2/3 RNAs and 1/3 proteins  rRNAs are folded into a highly compact three dimensional structure responsible for the overall shape of the ribosome  23S rRNA forms the catalytic site

24. Translation Initiation  Initiator tRNAs always carry the amino acid methionine.  The small ribosomal subunit scans the mRNA in the 5’ to 3’ direction.  Nucleotides immediately surrounding the start site in eucaryotic mRNAs influence the efficiency of AUG recognition.

26. Bacterial mRNAs are polycistronic  Specific sequence for ribosome binding:  Shine-Dalgarno sequence  Located 8 bp upstream of the start codon.

27. Translation Termination  Stop codons (UAA, UGA, UAG) signal the ribosome to stop translation  Release factors bind to the ribosome at the A-site causing H 2 O to be added to the growing peptide chain, releasing it from attachment with the tRNA.

28. eRF1 and tRNA structure comparison

29. Polysomes: multiple ribosomes on a single mRNA  Protein synthesis takes 20 seconds to several minutes  Ribosomes are spaced ~80 nt apart on an mRNA

30. Translation Accuracy  1 mistake per 10,000 amino acids joined  20 amino acids are incorporated per second in bacteria.  Translation is a compromise between accuracy and speed.  If too slow, cannot produce enough proteins to survive.

31. Price of Protein Synthesis  Four high-energy phosphate bonds must be split per peptide bond.  Two to charge the tRNA with an amino acid  Two to drive the synthesis itself on the ribosome  Extra energy is consumed for incorrectly charged tRNA and for entrance of an incorrect tRNA into the ribosome.

32. Quality control features  Both the 5’ cap and the poly-A tail must be recognized before an mRNA enters the ribosome.  Why?