1. PowerPoint Presentation Materials to accompany Genetics: Analysis and Principles Robert J. Brooker Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display CHAPTER 13 TRANSLATION OF mRNA

2. 13-13

3. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Special codons: AUG (which specifies methionine) = start codon AUG specifies additional methionines within the coding sequence UAA, UAG and UGA = termination, or stop, codons The code is degenerate More than one codon can specify the same amino acid For example: GGU, GGC, GGA and GGG all code for lysine In most instances, the third base is the degenerate base It is sometime referred to as the wobble base The code is nearly universal Only a few rare exceptions have been noted Refer to Table 13.3 13-14

4. 13-15

5. 13-16 Figure 13.2 Figure 13.2 provides an overview of gene expression Note that the start codon sets the reading frame for all remaining codons

6. 13-30 Figure 13.5 Carboxyl groupAmino group Condensation reaction releasing a water molecule Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

7. 13-31 Figure 13.5 N terminalC terminal Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

8. 13-32 Figure 13.6 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display There are 20 amino acids that may be found in polypeptides Each contains a different side chain, or R group Nonpolar amino acids are hydrophobic They are often buried within the interior of a folded protein

9. 13-33 Figure 13.6 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Nonpolar and charged amino acids are hydrophilic They are more likely to be on the surface of the protein

10. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display There are four levels of structures in proteins 1. Primary 2. Secondary 3. Tertiary 4. Quaternary A protein’s primary structure is its amino acid sequence Refer to Figure 13.7 Levels of Structures in Proteins 13-34

11. 13-35 Figure 13.7 The amino acid sequence of the enzyme lysozyme 129 amino acids long Within the cell, the protein will not be found in this linear state Rather, it will adapt a compact 3-D structure Indeed, this folding can begin during translation The progression from the primary to the 3-D structure is dictated by the amino acid sequence within the polypeptide Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

12. The primary structure of a protein folds to form regular, repeating shapes known as secondary structures There are two types of secondary structures  helix  sheet Certain amino acids are good candidates for each structure These are stabilized by the formation of hydrogen bonds Refer to Figure 13.8 Levels of Structures in Proteins 13-36

13. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The short regions of secondary structure in a protein fold into a three-dimensional tertiary structure Refer to Figure 13.8 This is the final conformation of proteins that are composed of a single polypeptide Structure determined by hydrophobic and ionic interactions as well as hydrogen bonds and Van der Waals interactions Proteins made up of two or more polypeptides have a quaternary structure This is formed when the various polypeptides associate together to make a functional protein Refer to Figure 13.8 Levels of Structures in Proteins 13-37

14. 13-38 Figure 13.8 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

15. In the 1950s, Francis Crick and Mahon Hoagland proposed the adaptor hypothesis tRNAs play a direct role in the recognition of codons in the mRNA In particular, the hypothesis proposed that tRNA has two functions 1. Recognizing a 3-base codon in mRNA 2. Carrying an amino acid that is specific for that codon Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 13.2 STRUCTURE AND FUNCTION OF tRNA 13-42

16. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display During mRNA-tRNA recognition, the anticodon in tRNA binds to a complementary codon in mRNA Recognition Between tRNA and mRNA 13-43 Figure 13.10 tRNAs are named according to the amino acid they bear The anticodon is anti-parallel to the codon

17. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The secondary structure of tRNAs exhibits a cloverleaf pattern It contains Three stem-loop structures; Variable region An acceptor stem and 3’ single strand region The actual three-dimensional or tertiary structure involves additional folding In addition to the normal A, U, G and C nucleotides, tRNAs commonly contain modified nucleotides More than 60 of these can occur tRNAs Share Common Structural Features 13-51

18. 13-52 Structure of tRNA Figure 13.12 Found in all tRNAs Not found in all tRNAs Other variable sites are shown in blue as well The modified bases are: I = inosine mI = methylinosine T = ribothymidine UH 2 = dihydrouridine m 2 G = dimethylguanosine   = pseudouridine

20. 1.The acceptor stem is a 7-base pair (bp) stem made by the base pairing of the 5'- terminal nucleotide with the 3'-terminal nucleotide (which contains the CCA 3'- terminal group used to attach the amino acid). The CCA tail This sequence is important for the recognition of tRNA by enzymes and critical in translation. In prokaryotes, In most prokaryotic tRNAs and eukaryotic tRNAs, the CCA sequence is added during processing and therefore does not appear in the tRNA gene The D arm is a 4 bp stem ending in a loop that often contains dihydrouridine. The anticodon arm is a 5-bp stem whose loop contains the anticodon. The T arm is a 5 bp stem containing the sequence TΨC where Ψ is a pseudouridine.

23. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The enzymes that attach amino acids to tRNAs are known as aminoacyl-tRNA synthetases There are 20 types One for each amino acid Aminoacyl-tRNA synthetases catalyze a two-step reaction involving three different molecules Amino acid, tRNA and ATP Refer to Figure 13.13 Charging of tRNAs 13-53

24. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The aminoacyl-tRNA synthetases are responsible for the “second genetic code” The selection of the correct amino acid must be highly accurate or the polypeptides may be nonfunctional Error rate is less than one in every 100,000 Sequences throughout the tRNA including but not limited to the anticodon are used as recognition sites Many modified bases are used as markers Charging of tRNAs 13-54

27. 13-55 Figure 13.13 The amino acid is attached to the 3’ end by an ester bond

28. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display As mentioned earlier, the genetic code is degenerate With the exception of serine, arginine and leucine, this degeneracy always occurs at the codon’s third position To explain this pattern of degeneracy, Francis Crick proposed in 1966 the wobble hypothesis In the codon-anticodon recognition process, the first two positions pair strictly according to the A – U /G – C rule However, the third position can actually “wobble” or move a bit Thus tolerating certain types of mismatches tRNAs and the Wobble Rule 13-56

29. 13-57 Wobble position and base pairing rules Figure 13.14 tRNAs that can recognize the same codon are termed isoacceptor tRNAs Recognized very poorly by the tRNA 5-methyl-2-thiouridine inosine 5-methyl-2’-O-methyluridine 5-methyluridine lysidine 2’-O-methyluridine 5-hydroxyuridine Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

30. Translation occurs on the surface of a large macromolecular complex termed the ribosome Bacterial cells have one type of ribosome Found in their cytoplasm Eukaryotic cells have two types of ribosomes One type is found in the cytoplasm The other is found in organelles Mitochondria ; Chloroplasts Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 13.3 RIBOSOME STRUCTURE AND ASSEMBLY 13-58

31. Unless otherwise noted the term eukaryotic ribosome refers to the ribosomes in the cytosol A ribosome is composed of structures called the large and small subunits Each subunit is formed from the assembly of Proteins rRNA Figure 13.15 presents the composition of bacterial and eukaryotic ribosomes Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 13.3 RIBOSOME STRUCTURE AND ASSEMBLY 13-59

32. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 13-60 Figure 13.15 Note: S or Svedberg units are not additive Synthesis and assembly of all ribosome components occurs in the cytoplasm

33. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 13-61 Figure 13.15 The 40S and 60S subunits are assembled in the nucleolus Then exported to the cytoplasm Synthesized in the nucleus Produced in the cytosol Formed in the cytoplasm during translation

34. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display During bacterial translation, the mRNA lies on the surface of the 30S subunit As a polypeptide is being synthesized, it exits through a hole within the 50S subunit Ribosomes contain three discrete sites Peptidyl site (P site) Aminoacyl site (A site) Exit site (E site) Ribosomal structure is shown in Figure 13.16 Functional Sites of Ribosomes 13-62

35. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 13-63 Figure 13.16

36. Translation can be viewed as occurring in three stages Initiation Elongation Termination Refer to 13.17 for an overview of translation Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 13.4 STAGES OF TRANSLATION 13-64

37. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 13-65 Figure 13.17 Initiator tRNA

38. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The mRNA, initiator tRNA, and ribosomal subunits associate to form an initiation complex This process requires three Initiation Factors The initiator tRNA recognizes the start codon in mRNA In bacteria, this tRNA is designated tRNA fmet It carries a methionine that has been covalently modified to N-formylmethionine The start codon is AUG, but in some cases GUG or UUG In all three cases, the first amino acid is N-formylmethionine The Translation Initiation Stage 13-66

39. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The binding of mRNA to the 30S subunit is facilitated by a ribosomal-binding site or Shine-Dalgarno sequence This is complementary to a sequence in the 16S rRNA 13-67 Figure 13.18 outlines the steps that occur during translational initiation in bacteria Figure 13.19 Hydrogen bonding Component of the 30S subunit

40. 13-68 Figure 13.18 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

41. 13-69 Figure 13.18 70S initiation complex This marks the end of the first stage The only charged tRNA that enters through the P site All others enter through the A site Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

42. In eukaryotes, the assembly of the initiation complex is similar to that in bacteria However, additional factors are required Note that eukaryotic Initiation Factors are denoted eIF Refer to Table 13.7 The initiator tRNA is designated tRNA met It carries a methionine rather than a formylmethionine The Translation Initiation Stage 13-70

43. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The start codon for eukaryotic translation is AUG It is usually the first AUG after the 5’ Cap The consensus sequence for optimal start codon recognition is show here 13-71 Start codon G C C (A/G) C C A U G G -6 -5 -4 -3 -2 -1 +1 +2 +3 +4 Most important positions for codon selection These rules are called Kozak’s rules After Marilyn Kozak who first proposed them With that in mind, the start codon for eukaryotic translation is usually the first AUG after the 5’ Cap!

44. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Translational initiation in eukaryotes can be summarized as such: A number of initiation factors bind to the 5’ cap in mRNA These are joined by a complex consisting of the 40S subunit, tRNA met, and other initiation factors The entire assembly moves along the mRNA scanning for the right start codon Once it finds this AUG, the 40S subunit binds to it The 60S subunit joins This forms the 80S initiation complex 13-72

45. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display During this stage, the amino acids are added to the polypeptide chain, one at a time In bacteria  15-18 amino acids per second In eukaryotes  6 amino acids per second The Translation Elongation Stage 13-73

46. 13-74 Figure 13.20 The 23S rRNA (a component of the large subunit) is the actual peptidyl transferase Thus, the ribosome is a ribozyme! Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

47. 13-75 Figure 13.20 tRNAs at the P and A sites move into the E and P sites, respectively Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

48. 16S rRNA (a part of the 30S ribosomal subunit) plays a key role in codon-anticodon recognition It can detect an incorrect tRNA bound at the A site It will prevent elongation until the mispaired tRNA is released This phenomenon is termed the decoding function of the ribosome It is important in maintaining the high fidelity in mRNA translation Error rate: 1 mistake per 10,000 amino acids added The Translation Elongation Stage 13-76

49. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The final stage occurs when a stop codon is reached in the mRNA In most species there are three stop or nonsense codons UAG UAA UGA These codons are not recognized by tRNAs, but by proteins called release factors Indeed, the 3-D structure of release factors mimics that of tRNAs The Translation Termination Stage 13-77

50. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Bacteria have three release factors RF1, which recognizes UAA and UAG RF2, which recognizes UAA and UGA RF3, which does not recognize any of the three codons It binds GTP and helps facilitate the termination process Eukaryotes only have one release factor eRF, which recognizes all three stop codons The Translation Termination Stage 13-78

51. 13-79 Figure 13.21

52. 13-80