1. ©2001 Timothy G. Standish 1 Corinthians 1:10 10 Now I beseech you, brethren, by the name of our Lord Jesus Christ, that ye all speak the same thing, and that there be no divisions among you; but that ye be perfectly joined together in the same mind and in the same judgement.

2. ©2001 Timothy G. Standish Polypeptides: Protein Synthesis Timothy G. Standish, Ph. D.

3. ©2001 Timothy G. Standish Ad Hominem “The phrase arguments ad hominem translates literally as ‘argument directed to the man.’.... It is committed when, instead of trying to disprove what is asserted one attacks the person who made the assertion.... This argument is fallacious, because the personal character of an individual is logically irrelevant to the truth or falsehood of what that individual says or the correctness or incorrectness of that individual's argument.... The way in which this irrelevant argument may sometimes persuade is through the psychological process of transference. Where an attitude of disapproval toward a person can be evoked, it may possibly tend to overflow the strictly emotional field and become disagreement with what that person says. But this connection is only psychological, not logical. Even the most wicked of men may sometimes tell the truth or argue correctly.” Copi I.M. 1953. Introduction to Logic," Macmillan Publishing Co: New York NY, Seventh Edition, 1986, p 92.

4. mRNA Transcription Introduction The Central Dogma of Molecular Biology Cell Polypeptide (protein) Translation Ribosome Reverse transcription DNA

5. ©2001 Timothy G. Standish ANYTHING ACID AMINE Protein Synthesis C H O OHCN H H H C HOH Serine C H O OHCN H H H C HH Alanine H C O OHC R N H H Amino Acid H2OH2O C O OHCN H H H C HOH C H O CN H H H C HH

6. ©2001 Timothy G. Standish Requirements for Translation Ribosomes - rRNA and Proteins mRNA - Nucleotides tRNA –The RNA world theory might explain these three components Aminoacyl-tRNA Synthetase, –A protein, thus a product of translation and cannot be explained away by the RNA world theory L Amino Acids ATP - For energy This appears to be an irreducibly complex system

7. ©2001 Timothy G. Standish 3’ 5’ 3’ Transcription And Translation In Prokaryotes Ribosome 5’ mRNA RNA Pol.

8. ©2001 Timothy G. Standish DNA Cytoplasm Nucleus G AAAAAA Export Degradation etc. G AAAAAA Eukaryotic Gene Expression G AAAAAA RNA Processing mRNA RNA Transcription Nuclear pores Ribosome Translation Packaging Modification Transportation Degradation

9. ©2001 Timothy G. Standish Ribosomes The Protein Factories Ribosomes are the organelles in which the mRNA nucleotide language is translated into the protein language The two ribosome subunits are made up of ribosomal RNA (rRNA) and proteins Ribosomes in eukaryotes follow the same basic plan as those in prokaryotes although they are slightly larger

10. ©2001 Timothy G. Standish Small subunit E AP Large subunit GAG...C-AGGAGG-NNNNNNNNNN-AUG---NNN---NNN---NNN---NNN--- 5’ mRNA 3’ Ribosome Structure Aminoacyl-tRNA binding site Exit site Peptidyl-tRNA binding site

11. ©2001 Timothy G. Standish Ribosome Structure E. coli ribosome at 25 A resolution from Frank et al. 1995. Biochem. Cell Biol. 73:757-767. (see also Frank et al. 1995. Nature 376:441-444.) Yellow: 30S subunit, blue: 50S subunit

12. ©2001 Timothy G. Standish E. Coli Ribosome In 4 D

13. ©2001 Timothy G. Standish A E Large subunit P Small subunit Translation - Initiation fMet UAC GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA 5’ mRNA 3’

14. ©2001 Timothy G. Standish A E Ribosome P UCU Arg Aminoacyl tRNA Phe Leu Met Ser Gly Polypeptide CCA Translation - Elongation GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA 5’ mRNA 3’

15. ©2001 Timothy G. Standish A E Ribosome P Phe Leu Met Ser Gly Polypeptide Arg Aminoacyl tRNA UCUCCA Translation - Elongation GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA 5’ mRNA 3’

16. ©2001 Timothy G. Standish A E Ribosome P CCA Arg UCU Phe Leu Met Ser Gly Polypeptide Translation - Elongation GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA 5’ mRNA 3’

17. ©2001 Timothy G. Standish A E Ribosome P Translation - Elongation Aminoacyl tRNA CGA Ala CCA Arg UCU Phe Leu Met Ser Gly Polypeptide GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA 5’ mRNA 3’

18. ©2001 Timothy G. Standish A E Ribosome P Translation - Elongation CCA Arg UCU Phe Leu Met Ser Gly Polypeptide CGA Ala GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA 5’ mRNA 3’

19. ©2001 Timothy G. Standish A E Ribosome P Translation - Termination CGA Phe Leu Met Ser Gly Polypeptide Ala Arg Val CGA GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA 5’ mRNA 3’ STOP

20. ©2001 Timothy G. Standish A E P Translation - Termination CGA Phe Leu Met Ser Gly Polypeptide Ala Arg Val CGA GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA 5’ mRNA 3’ STOP

21. ©2001 Timothy G. StandishInitiation The small ribosome subunit binds to the 5’ untranslated region of mRNA The small ribosomal subunit slides along the mRNA 5’ to 3’ until it finds a start codon (AUG) The initiator tRNA with methionine binds to the start codon The large ribosomal subunit binds with the initiator tRNA in the P site

22. ©2001 Timothy G. Standish Prokaryotic Initiation Prokaryotic initiation involves the recognition of a conserved sequence 10 bases upstream from the start codon on mRNA This conserved sequence is called the Shine- Dalgarno sequence - 5’…AGGAGG…3’ This sequence is complimentary to a highly conserved sequence near the 16S rRNA 3’ end - 3’…UCCUCC…5’ The start codon is usually AUG, but less often GUG and (least often) UUG are used

23. ©2001 Timothy G. Standish Small subunit Prokaryote Initiation Shine- Dalgarno sequence Start Codon (May also be GUG and UUG) GAG...C-AGGAGG-NNNNNNNNNN-AUG---NNN---NNN---NNN---NNN--- 5’ mRNA 3’ Initiation Factor 3 is needed to allow specific binding between the small subunit and the mRNA translation initiation site. IF3

24. ©2001 Timothy G. Standish Initiation Factor 1 may stabilize the initiation complex IF1 Small subunit Prokaryote Initiation GAG...C-AGGAGG-NNNNNNNNNN-AUG---NNN---NNN---NNN---NNN--- 5’ mRNA 3’ IF3

25. ©2001 Timothy G. Standish IF1 IF2 Small subunit Prokaryote Initiation fMet UAC GAG...C-AGGAGG-NNNNNNNNNN-AUG---NNN---NNN---NNN---NNN--- 5’ mRNA 3’ IF3 Initiation Factor 2 binds to and mediates the insertion of initiator tRNA into the initiation complex Formyl Methionine is modified with a formyl group on the amine group so that a peptide bond can only be formed at the carboxyl group OH H H O H H H2NH2N C C H H C H H C S Methionine H H H H H C H O C H H C H H C S Formyl Methionine N OH O C

26. ©2001 Timothy G. Standish E A P Large subunit IF1 IF2 Small subunit fMet UAC GAG...C-AGGAGG-NNNNNNNNNN-AUG---NNN---NNN---NNN---NNN--- 5’ mRNA 3’ IF3 Prokaryote Initiation

27. ©2001 Timothy G. Standish Small subunit E AP Large subunit fMet UAC GAG...C-AGGAGG-NNNNNNNNNN-AUG---NNN---NNN---NNN---NNN--- 5’ mRNA 3’ IF1 IF2 IF3 Prokaryote Initiation

28. ©2001 Timothy G. Standish Small subunit E AP Large subunit fMet UAC GAG...C-AGGAGG-NNNNNNNNNN-AUG---NNN---NNN---NNN---NNN--- 5’ mRNA 3’ Prokaryote Initiation

29. ©2001 Timothy G. StandishMet-tRNA C C G G CG AC X Signals for entry into the P site Necessary for formylation

30. ©2001 Timothy G. Standish Aminoacyl-tRNA Synthetase Aminoacyl-tRNA Synthetase enzymes attach the correct amino acids to the correct tRNA This is an energy-consuming process Aminoacyl-tRNA Synthetases recognize tRNAs on the basis of their looped structure, not by direct recognition of the anticodon

31. Gly Amino- acyl-tRNA Synthetase Gly Amino- acyl-tRNA Synthetase A P Making Aminoacyl- tRNA P P Pyrophosphate A P P P ATP Amino- acyl-tRNA Synthetase A P Gly CCA

32. Amino- acyl-tRNA Synthetase Making Aminoacyl- tRNA Gly CCA Aminoacyl- tRNA Note that the amino acid is not paired with the tRNA on the basis of the anticodon. The correct tRNA for a given amino acid is recognized on the basis of other parts of the molecule. ©1998 Timothy G. Standish Gly P P Pyrophosphate A P P P ATP Amino- acyl-tRNA Synthetase Gly Amino- acyl-tRNA Synthetase A P A P AMP Amino- acyl-tRNA Synthetase Gly CCA

33. ©2001 Timothy G. Standish Aminoacylation of tRNA H H C O C N C O N C C C C C HHO H H O P O H O N N C C O HH H N H H 3’ 5’ H CN C O H R H HO H OH

34. ©2001 Timothy G. Standish Amino acid tRNA Aminoacylation of tRNA 3’ 5’ H H C O C N C O N C C C C C H HO H H O P O H O N N C C O H H N H H H CN C O H R H H OH Class I Aminoacyl tRNA Synthetases attach amino acids to the 2’ carbon while Class II attach to the 3’carbon

35. ©2001 Timothy G. Standish Classification of Aminoacyl- tRNA Synthetases Class I - 2’ OH Glu (  ) Gln (  ) Arg (  ) Val (  ) Ile (  ) Leu (  ) Met (    Tyr (      (     Class II - 3’ OH Gly (    2  Ala (  4  Pro (    Ser (    Thr (    Asp (   ?? Asn (    His (    Lys (    Aminoacyl-tRNA Synthetases (ARS) may be mono or multimeric. Two types of polypeptide chains are recognized:  and .

36. ©2001 Timothy G. Standish

37. 3’5’ Exon 2Exon 3 Int. 2 Exon 1 Int. 1 Processing Eukaryotic mRNA Protein Coding Region 3’ Untranslated Region5’ Untranslated Region 3’ AAAAA 3’ Poly A Tail 5’ G 5’ Cap Exon 2 Exon 3Exon 1 Int. 2Int. 1 RNA processing achieves three things:  Removal of introns  Addition of a 5’ cap  Addition of a 3’ tail l This signals the mRNA is ready to move out of the nucleus and may control its lifespan in the cytoplasm

38. Gly Amino- acyl-tRNA Synthetase Gly CCA Amino- acyl-tRNA Synthetase Gly Amino- acyl-tRNA Synthetase A P Amino- acyl-tRNA Synthetase Making Aminoacyl- tRNA P P Pyrophosphate A P AMP Gly CCA Aminoacyl- tRNA Note that the amino acid is not paired with the tRNA on the basis of the anticodon. The correct tRNA for a given amino acid is recognized on the basis of other parts of the molecule. A P P P ATP ©1998 Timothy G. Standish