1. Protein Synthesis: Translation of the Genetic Message Mar. 8, 2016 CHEM 281

2. Translating the Genetic Message  Protein biosynthesis is a complex process requiring ribosomes, mRNA, tRNA, and protein factors  Several steps are involved  Before being incorporated into growing protein chain, a.a. must be activated by tRNA and aminoacyl-tRNA synthetases

3. The Genetic Code  Salient features of the genetic code  triplet: a sequence of three bases (a codon) is needed to specify one amino acid  nonoverlapping: no bases are shared between consecutive codons  commaless: no intervening bases between codons  degenerate: more than one triplet can code for the same amino acid; Leu, Ser, and Arg, for example, are each coded for by six triplets  universal: the same in viruses, prokaryotes, and eukaryotes; the only exceptions are some codons in mitochondria

4. The Genetic Code (Cont’d)  The ribosome moves along the mRNA three bases at a time rather than one or two at a time

5. The Genetic Code (Cont’d)  All 64 codons (4 3 ; 4 bases in a triplet) have assigned meanings  61 code for amino acids  3 (UAA, UAG, and UGA) serve as termination signals  only Trp and Met have one codon each  the third base is irrelevant for Leu, Val, Ser, Pro, Thr, Ala, Gly, and Arg  the second base is important for the type of amino acid; for example, if the second base is U, the amino acids coded for are hydrophobic  for the 15 amino acids coded for by 2, 3, or 4 triplets, it is only the third letter of the codon that varies. Gly, for example, is coded for by GGA, GGG, GGC, and GGU

6. The Genetic Code (Cont’d)

7.  Assignments of triplets in genetic code based on several different experiments  synthetic mRNA: if mRNA is polyU, polyPhe is formed; if mRNA is poly ---ACACACACACACACACACACA---, poly(Thr-His) is formed  binding assay: aminoacyl-tRNAs bind to ribosomes in the presence of trinucleotides synthesize trinucleotides by chemical means carry out a binding assay for each type of trinucleotide aminoacyl-tRNAs are tested for their ability to bind in the presence of a given trinucleotide

8. The Filter-Binding Assay Helps Elucidate the Genetic Code

9. Wobble Base Pairing  Some tRNAs bond to one codon exclusively, but many tRNAs can recognize more than one codon because of variations in allowed patterns of hydrogen bonding  the variation is called “wobble”  wobble is in the first base of the anticodon

10. Base Pairing Combination in the Wobble Scheme

11. Wobble Base Pairing Alternatives

12. Wobble Base Pairing Hypothesis  The wobble hypothesis provides insight into some aspects of the degeneracy of the code  in many cases, the degenerate codons for a given amino acid differ only in the third base; therefore fewer different tRNAs are needed because a given tRNA can base-pair with several codons  the existence of wobble minimizes the damage that can be caused by a misreading of the code; for example, if the Leu codon CUU were misread CUC or CUA or CUG during transcription of mRNA, the codon would still be translated as Leu during protein synthesis

13. Amino Acid Activation  Amino acid activation and formation of the aminoacyl- tRNA take place in two separate steps  Both catalyzed by aminoacyl-tRNA synthetase  Free energy of hydrolysis of ATP provides energy for bond formation

16. Amino Acid Activation (Cont’d)  This two-stage reaction allows selectivity at two levels  the amino acid: the aminoacyl-AMP remains bound to the enzyme and binding of the correct amino acid is verified by an editing site in the tRNA synthetase  tRNA: there are specific binding sites on tRNAs that are recognized by aminoacyl-tRNA synthetases.

17. tRNA Tertiary Structure  There are several recognition sites for various amino acids on the tRNA

18. Chain Initiation  In all organisms, synthesis of polypeptide chain starts at the N-terminal end, and grows from N-terminus to C- terminus  In E. coli Initiation requires:  tRNA fmet  initiation codon (AUG) of mRNA  30S ribosomal subunit  50S ribosomal subunit  initiation factors IF-1, IF-2, and IF-3  GTP, Mg 2+  Forms the initiation complex

19. The Initiation Complex

20. Chain Initiation  tRNA met and tRNA fmet contain the triplet 3’-UAC-5’  Triplet base pairs with 5’-AUG-3’ in mRNA  3’-UAC-5’ triplet on tRNA fmet recognizes the AUG triplet (the start signal) when it occurs at the beginning of the mRNA sequence that directs polypeptide synthesis  3’-UAC-5’ triplet on tRNA met recognizes the AUG triplet when it is found in an internal position in the mRNA sequence  Start signal is preceded by a Shine-Dalgarno purine-rich leader segment, 5’-GGAGGU-3’, which usually lies about 10 nucleotides upstream of the AUG start signal and acts as a ribosomal binding site

21. Shine-Dalgarno Sequence Recognized by E. Coli Ribosomes

22. Chain Elongation  Uses three binding sites for tRNA present on the 50S subunit of the 70S ribosome: P (peptidyl) site, A (aminoacyl) site, E (exit) site.  Requires  70S ribosome  codons of mRNA  aminoacyl-tRNAs  elongation factors EF-Tu (Elongation factor temperature- unstable), EF-Ts (Elongation factor temperature-stable), and EF-G (Elongation factor-GTP)  GTP, and Mg 2+

23. Elongation Steps  Step 1  an aminoacyl-tRNA is bound to the A site  the P site is already occupied  2nd amino acid bound to 70S initiation complex. Defined by the mRNA  Step 2  EF-Tu is released in a reaction requiring EF-Ts  Step 3  the peptide bond is formed, the P site is uncharged  Step 4  the uncharged tRNA is released  the peptidyl-tRNA is translocated to the P site  EF-G and GTP are required  the next aminoacyl-tRNA occupies the empty A site

24. Chain Elongation Fig. 12.12 pg 335

25. Chain Termination  Chain termination requires  stop codons (UAA, UAG, or UGA) of mRNA  RF-1 (Release factor-1) which binds to UAA and UAG or RF- 2 (Release factor-2) which binds to UAA and UGA  RF-3 which does not bind to any termination codon, but facilitates the binding of RF-1 and RF-2  GTP which is bound to RF-3  The entire complex dissociates setting free the completed polypeptide, the release factors, tRNA, mRNA, and the 30S and 50S ribosomal subunits

26. Chain Termination

27. Components of Protein Synthesis

28. Protein Synthesis  In prokaryotes, translation begins very soon after mRNA transcription  It is possible to have several molecules of RNA polymerase bound to a single DNA gene, each in a different stage of transcription  It is also possible to have several ribosomes bound to a single mRNA, each in a different stage of translation  Polysome: mRNA bound to several ribosomes  Coupled translation: the process in which a prokaryotic gene is being simultaneously transcribed and translated

29. Simultaneous Protein Synthesis on Polysomes  A single mRNA molecule is translated by several ribosomes simultaneously  Each ribosome produces a copy of the polypeptide chain specified by the mRNA  When protein has been completed, the ribosome dissociates into subunits that are used again in protein synthesis

30. Simultaneous Protein Synthesis on Polysomes (Cont’d)

31. Eukaryotic Translation  Chain Initiation: the most different from process in prokaryotes 13 more initiation factors are given the designation eIF (eukaryotic initiation factor) (Table 12.4)

32. Eukaryotic Translation (Cont’d)  Chain elongation  uses the same mechanism of peptidyl transferase and ribosome translocation as prokaryotes  there is no E site on eukaryotic ribosomes, only A and P sites  there are two elongation factors, eEF-1 and eEF-2  eEF2 is the counterpart to EF-G, which causes translocation  Chain termination  stop codons are the same: UAG, UAA, and UGA  only one release factor that binds to all three stop codons

33. Posttranslational Modification  Newly synthesized polypeptides are frequently modified before they reach their final form where they exhibit biological activity  N-formylmethionine in prokaryotes is cleaved  specific bonds in precursors are cleaved, as for example, preproinsulin to proinsulin to insulin  leader sequences are removed by specific proteases of the endoplasmic reticulum; the Golgi apparatus then directs the finished protein to its final destination  factors such as heme groups may be attached  disulfide bonds may be formed  amino acids may be modified, as for example, conversion of proline to hydroxyproline  other covalent modifications; e.g., addition of carbohydrates

35. Protein Degradation  Proteins are in a dynamic state and are often turned over  Degradative pathways are restricted to  subcellular organelles such as lysosomes  macromolecular structures called proteosomes  In eukaryotes, ubiquitinylation (becoming bonded to ubiquitin) targets a protein for destruction  protein must have an N-terminus  those with an N-terminus of Met, Ser, Ala, Thr, Val, Gly, and Cys are resistant  those with an N-terminus of Arg, Lys, His, Phe, Tyr, Trp, Leu, Asn, Gln, Asp, Glu have short half-lives

36. Ubiquitin-Proteosome Degradation

37. Acidic N-termini Induced Protein Degradation