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.

Protein Synthesis Timothy G. Standish, Ph. D.

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

Protein Synthesis Amino Acid H2O H C O OH R N C H O OH N C H O OH N HO AMINE H C O OH R N Amino Acid ACID C H O OH N Alanine C H O OH N HO Serine ANYTHING C O OH N H HO H2O

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

Transcription And Translation In Prokaryotes 3’ 5’ 5’ mRNA RNA Pol. Ribosome Ribosome

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

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

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

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

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

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

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

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

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

Initiation 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

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

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

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

Prokaryote Initiation OH H O H2N C S Methionine H C O S Formyl Methionine N OH 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 IF2 fMet UAC Initiation Factor 2 binds to and mediates the insertion of initiator tRNA into the initiation complex IF1 GAG...C-AGGAGG-NNNNNNNNNN-AUG---NNN---NNN---NNN---NNN--- 5’ mRNA 3’ Small subunit IF3

Prokaryote Initiation Large subunit Prokaryote Initiation IF2 Small subunit fMet UAC GAG...C-AGGAGG-NNNNNNNNNN-AUG---NNN---NNN---NNN---NNN--- 5’ mRNA 3’ IF3 IF1

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

Prokaryote Initiation Large subunit fMet UAC GAG...C-AGGAGG-NNNNNNNNNN-AUG---NNN---NNN---NNN---NNN--- 5’ mRNA 3’ Small subunit

Met-tRNA Necessary for formylation Signals for entry into the P site 9 U* 9 26 22 23 Pu 16 12 Py 10 25 20:1 G* 17:1 A 20:2 17 13 20 G 50 51 65 64 63 62 52 C 59 y A* T 49 39 41 42 31 29 28 Pu* 43 1 27 U 35 38 36 Py* 34 40 30 47:1 47:15 46 47:16 45 44 47 73 70 71 72 66 67 68 69 3 2 7 6 5 4 Necessary for formylation A C X Signals for entry into the P site C G

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

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

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

Aminoacylation of tRNA H C N O R H C O N P H O 3’ 5’

Aminoacylation of tRNA Class I Aminoacyl tRNA Synthetases attach amino acids to the 2’ carbon while Class II attach to the 3’carbon Amino acid H C O N P R tRNA H O 3’ 5’

Classification of Aminoacyl-tRNA Synthetases Aminoacyl-tRNA Synthetases (ARS) may be mono or multimeric. Two types of polypeptide chains are recognized: a and b. Class I - 2’ OH Glu (a) Gln (a) Arg (a) Val (a) Ile (a) Leu (a) Met (a Tyr (a  (a Class II - 3’ OH Gly (ab2 Ala (a4 Pro (a Ser (a Thr (a Asp (a?? Asn (a His (a Lys (a After Lodish et al., 1995. Molecular Cell Biology 3rd edtion. Scientific American Books, W. H. Freeman and Co., New York. Quoting G. Eriani et al., 1990, Nature 347:203

The End

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