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

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1 13-13 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

2 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

3 13-16 Figure 13.2 Figure 13.2 provides an overview of gene expression

4 In the 1950s, Francis Crick & Mahon Hoagland proposed the adaptor hypothesis tRNAs play a direct role in the recognition of codons in the mRNA Structure and Function of tRNA Proline anticodon

5 Structure of tRNA Figure Found in all tRNAs tRNA 2 º Structure The modified bases are: I = inosine mI = methylinosine T = ribothymidine D= dihydrouridine m 2 G = dimethylguanosine   = pseudouridine D loop D D TψC loop loop

6 3 º Structure of tRNA

7 aminoacyl-tRNA synthetases The enzymes that attach amino acids to tRNAs There are >20 types One for each amino acid Ones for isoacceptor tRNAs put same a.a. on different tRNAs Aminoacyl-tRNA synthetases catalyze a two-step reaction 1- adenylation of amino acid 2- aminoacylation of tRNA Charging of tRNAs

8 Figure Aminoacyl tRNA Synthetase Function The amino acid is attached to the 3’ OH by an ester bond

9 The genetic code is degenerate There are >20 but < 64 tRNAs How does the same tRNA bind to different codons? Francis Crick proposed the wobble hypothesis in 1966 to explain the pattern of degeneracy, 1 st two bases of the codon-anticodon pair strictly by Watson-Crick rules The 3 rd position can wobble This movement allows alternative H-bonding between bases to form non-WC base paring tRNAs and the Wobble Rule

10 Wobble position and base pairing rules Figure tRNAs charged with the same amino acid, but that recognize multiple codons are termed isoacceptor tRNAs

11 Wobble Base-Pairing between anticodon & codon W-C base pairing Wobble pairing

12 Translation occurs on the surface of a large macromolecular complex termed the ribosome Prokaryotic cells 1 type of ribosome located in the cytoplasm Eukaryotic cells 2 types of ribosomes 1 found in the cytoplasm 2 nd found in organelles -Mitochondria; Chloroplasts These are like prokaryotic ribosomes Ribosome Structure and Assembly

13 Figure (a) Bacterial cell Prokaryotic Ribosomes

14 Figure Eukaryotic Ribosomes

15 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 Functional Sites of Ribosomes 13-57

16 Figure 13.14

17 Stages of Translation Initiation Elongation Termination

18 Release factors Initiator tRNA Stages of Translation Figure 13.15

19 Components mRNA, initiator tRNA, Initiation factors ribosomal subunits The initiator tRNA In prokaryotes, this tRNA is designated tRNA i fmet It carries a methionine modified to N-formylmethionine In eukaryotes, this tRNA is designated tRNA i met It carries an unmodified methionine In both cases the initiator tRNA is different from a tRNA met that reads an internal AUG codon Translation Initiation

20 16S rRNA binds to an mRNA at the ribosomal-binding site or Shine-Dalgarno box 16S rRNA Figure Prokaryotic Ribosome-mRNA Recognition 7 nt

21 (actually 9 nucleotides long) Figure Prokaryotic Translation Initiation

22 Figure The tRNA i Met is positioned in the P site All other tRNAs enter the A site Prokaryotic Translation Initiation

23 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 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 Eukaryotic mRNA-Ribosoime Recognition 13-65

24 The consensus sequence for optimal start codon recognition is show here Start codon G C C (A/G) C C A U G G Most important positions for codon selection This sequence is called Kozak’s consensus after Marilyn Kozak who first determined it Eukaryotic Ribosome Binding

25 Eukaryotic Translation Initiation Initiation factors bind to the 5’ cap in mRNA & to the pA tail These recruit the 40S subunit, tRNA i met The entire assembly scans along the mRNA until reaching a Kozak’s consensus Once right AUG found, the 60S subunit joins Translation intitiates

26 During this stage, the amino acids are added to the polypeptide chain, one at a time The addition of each amino acid occurs via a series of steps outlined in Figure This process, though complex, can occur at a remarkable rate In bacteria  amino acids per second In eukaryotes  6 amino acids per second Translation Elongation

27 Figure Translation Elongation – tRNA Entry A charged tRNA binds to the A site EF-1 facilitates tRNA entry Peptidyl transferase catalyzes peptide bond formation The polypeptide is transferred to the aminoacyl-tRNA in the A site The 23S rRNA (a component of the large subunit) is the actual peptidyl transferase Thus, the ribosome is a ribozyme!

28 Figure The ribosome translocates one codon to the right promoted by EF-G Translation Elongation - Translocation uncharged tRNA released from E site The process is repeated, again and again, until a stop codon is reached

29 Occurs when a stop codon is reached in the mRNA Three stop or nonsense codons UAG UAA UGA Recognized by proteins called release factors – NOT tRNAs Translation Termination

30 Bacteria have three release factors RF1 - recognizes UAA and UAG RF2 - recognizes UAA and UGA RF3 - binds GTP and facilitates termination process Eukaryotes only have one release factor eRF1 - recognizes all three stop codons Translation Termination

31 Ribosomal subunits & mRNA dissociate Figure Translation Termination

32 Translation begins at 5’ end of mRNA 5’  3’ Peptide bonds are formed directionally Peptide bond is formed between the COO - of the previous amino acid in the chain and the NH 2 of the amino acid being added Polypeptides Have Directionality

33 Carboxyl groupAmino group Peptide Bond Formation Figure 13.20

34 N terminalC terminal Colinearity of DNA, mRNA, & Protein Sequence

35 Figure 13.4 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 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

36 Figure 13.6 A protein subunit

37 Molecular Basis of Phenotype

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