1. Topic 24 - Translation of mRNA to polypeptide • - a 3 stage process to translate mRNA code to amino acids • Recognition of mRNA & its start codon by ribosomes • Reading the mRNA codons and translating this to amino acids: - transfer RNAs (tRNA) in conjunction with ribosomes • Terminating translation once a stop codon is encountered

2. Translation overview ribosome subunits

3. Translation – the gist of it: Ribosome assembles mRNA together with tRNAs tRNAs translate mRNA Codon to an amino acid. Fig. 17.14

4. tRNA: each has an anti-codon that compliments codon(s) of mRNA This tRNA compliments the mRNA codon UUC UUC codes for the amino acid phenylalanine which would attach to this specific tRNA 3 Amino acidattachmentsite Figure 17.15 5 Amino acidattachmentsite 5 3 Hydrogenbonds Hydrogenbonds A A G 3 5 Anticodon Anticodon Anticodon

5. tRNA features • Each tRNA is approx. 80 ribo-nucleotides long • 3D shape is provided by H-bonds in double stranded RNA (dsRNA) • Each tRNA has 2 functional sites: • Anti-codon that complements mRNA codon • AA attachment site - AA that will join growing polypeptide • There are 45 different tRNAs which are used to translate the 61 codons • into 20 amino acids. • Therefore some tRNAs must recognize more than one codon!!

6. Some of 45 tRNA's can recognize more than one of the 61 codons This is because one tRNA anti-codon nucleotide can flex or 'wobble' See Fig 17.5

7. The structure of the tRNA is such that the 5' anti-codon nucleotide can conform (wobble) to H-bond degenerate nucleotides in the codon 3 Amino acidattachmentsite 5 45 tRNA versions Amino acidattachmentsite Figure 17.15 5 3 A A G 3 5 wobble site Anticodon Anticodon Anticodon UUC codon

8. some tRNA anti-codons recognize more than one codon A Uracil in the tRNA wobble position can bind Adenine or Guanine A special nucleotide called Inosine (I) may also be used in anti-codons. Inosine in the wobble position can bind Uracil, Cytosine or Adenine .e.g. the anti-codon 3’-UAI-5’ would complement all 3 isoleucine codons: 5’-AUU-3’ 5’-AUC-3’ 5’-AUA-3’

9. A wobble Inosine can base pair with each of adenine, uracil & cytosine I:A I:U I:C R – sugar-phosphate of nucleotide

10. Joining of an amino acid to a tRNA by aminoacyl-tRNAsynthetase A different synthetase for each AA. tRNAs differ at variable loops enzyme activated AA amino acid (AA) P - P - P -ATP P -AMP P - P (based on fig 17.16) tRNA charged tRNA P -AMP

11. model of the interaction of aminoacyl tRNA synthetase (blue) with tRNA (yellow & red) and an amino acid (purple) fig 17.16

12. Differences in the variable loop nucleotides (red dots) distinguish the 45 tRNAs. These differences are what the synthetases recognize. 3 5 Figure 17.15 variable loops . . . . . . . . . . . A A G

13. Functional sites in an assembled ribosome see fig 17.18

14. Ribosomes - ribosomal RNA + polypeptides rRNA synthesized & assembled with proteins in nucleolus (Eukaryote) Small & Large Ribosome sub-units are exported to cytoplasm (Euk) Small sub-unit has mRNA binding site Large sub-unit has 3 tRNA binding sites - A-site, P-site & E-site These sites hold tRNAs, AAs and polypeptide in close proximity prokaryote & eukaryote ribosomes are similar but distinct

15. Initiation of Translation by a small ribosomal sub-unit & initiator tRNA 1) small ribosome subunit attaches to 5’ methylguanine cap (Eukaryote) or "leader nucleotides" (a sequence motif in prokaryote mRNA) 2) subunit moves along mRNA until AUG start codon encountered 3) Initiator Met-tRNA binds codon Initiation factors are also needed, as well as GTP - for energy fig 17.18

16. Translation Initiation - con't 4) After initiator tRNA is added the large Ribosome sub-unit joins the small subunit & completes initiation. The initiation complex is complete and can allow polypeptide elongation. fig 17.18

17. Elongation is a 3 step process fig. 17.18 V Codon recognition (requires energy - GTP) N 2) Peptide bond formation N N V V 3) tRNA translocation (requires energy - GTP) N V "N" and "V" are just two different tRNAs

18. Termination elongation continues until a stop codon (UAA, UAG or UGA) reaches the A-site Release Factor recognizes & binds stop codon - it adds a -OH instead of AA, causing the translation complex to disassemble. fig 17-20

19. Polyribosomes simultaneous translation of mRNA by multiple successive ribosomes occurs in cytoplasm or on rough ER of eukaryotic cells fig 17.21

20. In prokaryotes there is simultaneous transcription & translation • no compartments • to prevent this • - maximal use of • short lived mRNA fig 17-25

21. Polypeptide Trafficking - using Signal Peptides (aka Leader peptides) to direct polypeptides to the endoplasmic reticulum (or to non-endomembrane organelles) 1 2 3 4 5 6 Ribosome mRNA Signalpeptide ERmembrane Signalpeptideremoved SRP Protein SRPreceptorprotein CYTOSOL ERLUMEN Translocationcomplex fig 17.22 SRP – signal recognition particle

22. Polypeptides are often modified in preparation for their final function These modifications are called Post-translational modifications Complexing with lipids --> proteolipids

23. Differences in prokaryotic & eukaryotic transcription & translation provide a strategy for halting bacterial infections - antibiotics RNA polymerase(s) Targets for antibiotics Ribosomes Other transcription/translation proteins Antibiotics target components of the transcription & translation pathways that are unique to prokaryotes

24. Antibiotics: - mostly target the unique bacterial ribosomes (translation) Kanamycin, Streptomycin & Tetracyclin interfere with bacterial ribosomes & bacterial translation - Rifamycin targets & interferes with bacterial RNA polymerases

25. Translation animations of possible interest: www.whfreeman.com/lodish4e Chapter 4 animations Note – only know this to the extent covered in lecture