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PowerPoint Presentation Materials to accompany Genetics: Analysis and Principles Robert J. Brooker CHAPTER 13 Part 2 TRANSLATION OF mRNA Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 13.2 STRUCTURE AND FUNCTION OF tRNA 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 13-38 Recognition Between tRNA and mRNA During mRNA-tRNA recognition, the anticodon in tRNA binds to a complementary codon in mRNA tRNAs are named according to the amino acid they bear Proline anticodon Figure 13.8 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 13-39 Experiment 13B: The Adaptor Hypothesis Put to the Test 1962, François Chapeville Hypothesis: the amino acid attached to tRNA is not directly involved in codon recognition Therefore, the alteration of an amino acid already attached to tRNA should cause that altered amino acid to be incorporated into the polypeptide instead of the normal amino acid Example: Cysteine on a tRNAcys is changed to alanine cys will add alanine instead of the Therefore, the tRNA usual cysteine 13-40 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Raney nickel converts cysteine to alanine The experiment made use of a cell-free translation system similar to the one used by Nirenberg Refer to Figure 13.3 Chapeville used an mRNA template that contained only U and G Therefore, it could only contain the following eight codons UUU = phenylalanine GUU = valine UUG = leucine GUG = valine UGU = cysteine GGU = glycine UGG =tryptophan GGG = glycine Note: One cysteine codon and no alanine codons Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 13-41 The Hypothesis Codon recognition is dictated only by the tRNA The chemical structure of the amino acid attached to tRNA does not play a role Testing the Hypothesis Refer to Figure 13.9 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 13-42 Figure 13.9 13-43 Figure 13.9 13-44 The Data Relative Amount of Radiolabeled Amino Acid Incorporated into Polypeptide (cpm)* Conditions Control, untreated tRNA Raney nickel-treated tRNA Cysteine Alanine Total 2,835 83 2,918 990 2,020 3,010 *Cpm is counts per minute of radioactivity in the sample Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 13-45 Interpreting the Data Relative Amount of Radiolabeled Amino Acid Incorporated into Polypeptide (cpm)* Conditions Control, untreated tRNA Raney nickel-treated tRNA Cysteine Alanine Total 2,835 83 2,918 990 2,020 3,010 Expected result since only radiolabeled cysteine was added Probably a result of cysteine contamination *Cpm is counts per minute of radioactivity in the sample About a third of the tRNAcys did not react with the Raney nickel Large amount of incorporated alanine even though template mRNA lacks alanine codons Overall, these results support the adaptor hypothesis tRNAs act as adaptors to carry the correct amino acid to the ribosome based on their anticodon sequence Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 13-46 tRNAs Share Common Structural Features 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 In addition to the normal A, U, G and C nucleotides, tRNAs commonly contain modified nucleotides More than 60 of these can occur Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 13-47 Found in all tRNAs Not found in all tRNAs Other variable sites are shown in blue as well Figure 13.10 Structure of tRNA The modified bases are: I = inosine mI = methylinosine T = ribothymidine UH2 = dihydrouridine m2G = dimethylguanosine y = pseudouridine 13-48 Transfer RNA • Molecules of tRNA are not all identical – Each carries a specific amino acid on one end – Each has an anticodon on the other end – Fidelity of translation of the genetic code is determined at two levels • Linkage of specific a.a. to specific tRNA • mRNA codon:tRNA anti-codon recognition Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 5 3 Amino acid attachment site Hydrogen bonds A 3 Anticodon (b) Three-dimensional structure Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings A G Anticodon 5 (c) Symbol used in this book Charging of tRNAs The enzymes that attach amino acids to tRNAs are known as aminoacyl-tRNA synthetases There are 20 types One for each amino acid Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 13-49 The amino acid is attached to the 3’ end by an ester bond Figure 13.11 13-50 tRNAs and the Wobble Rule 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 13-51 tRNAs that can recognize the same codon are termed isoacceptor tRNAs inosine 5-methyl-2-thiouridine 5-methyl-2’-O-methyluridine 2’-O-methyluridine 5-methyluridine 5-hydroxyuridine lysidine Recognized very poorly by the tRNA Figure 13.12 Wobble position and base pairing rules 13-52 tRNAs and the Wobble Rule Wobble allows for economizing: 61 codons specifying amino acids Bacteria: 30 to 40 tRNAs Eukaryotes: up to 50 tRNAs 13.3 RIBOSOME STRUCTURE AND ASSEMBLY 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 13-53 13.3 RIBOSOME STRUCTURE AND ASSEMBLY A ribosome is composed of structures called the large and small subunits Each subunit is formed from the assembly of Proteins rRNA Figure 3.13 presents the composition of bacterial and eukaryotic ribosomes Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 13-54 Synthesis and assembly of all ribosome components occurs in the cytoplasm (a) Bacterial cell Note: S or Svedberg units are not additive Figure 13.13 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 13-55 Synthesized in the nucleus Formed in the cytoplasm during translation Produced in the cytosol The 40S and 60S subunits are assembled in the nucleolus Then exported to the cytoplasm Figure 13.13 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 13-56 13.4 STAGES OF TRANSLATION Translation can be viewed as occurring in three stages Initiation Elongation Termination Refer to 13.15 for an overview of translation Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 13-59 Initiator tRNA Release factors Figure 13.15 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 13-60 The Translation Initiation Stage The mRNA, initiator tRNA, and ribosomal subunits associate to form an initiation complex The initiator tRNA recognizes the start codon AUG in mRNA In bacteria, this tRNA is designated tRNAfmet It carries a methionine that has been covalently modified to N-formylmethionine In eukaryotes, the initiator tRNA is designated tRNAmet It carries a methionine rather than a formylmethionine Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 13-61 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 Hydrogen bonding Component of the 30S subunit Figure 13.17 16S rRNA Figure 13.16 outlines the steps that occur during translational initiation in bacteria Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 13-62 (actually 9 nucleotides long) Figure 13.16 13-63 The only charged tRNA that enters through the P site All others enter through the A site 70S initiation complex This marks the end of the first stage Figure 13.16 13-64 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 Most important positions for codon selection C C A U G G -2 -1 +1 +2 +3 +4 These rules are called Kozak’s rules G C C (A/G) -6 -5 -4 -3 Start codon 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! Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 13-66 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, tRNAmet, and other initiation factors The complex 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 13-67 The Translation Termination Stage 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 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 13-72 The ribosomal subunits and mRNA dissociate Figure 13.19 13-74 A Polypeptide Chain Has Directionality Polypeptide synthesis has a directionality that parallels the 5’ to 3’ orientation of mRNA During each cycle of elongation, a peptide bond is formed between the last amino acid in the polypeptide chain and the amino acid being added Refer to Figure 13.20 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 13-76 Carboxyl group Amino group Condensation reaction releasing a water molecule Figure 13.20 13-77 N terminal Figure 13.20 C terminal 13-78