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Translation brooker

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Arshad Al-Ghafour

This document summarizes key aspects of protein synthesis and translation. It discusses how genes are transcribed into mRNA, which is then translated into proteins using transfer RNA (tRNA) and ribosomes. The genetic code is explained, where codons in mRNA are translated to specific amino acids. tRNA molecules carry complementary anticodons and the correct amino acid. Ribosomes provide the site for protein synthesis, bringing together mRNA, tRNAs and linking amino acids into polypeptide chains based on the genetic code.

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Genetics: Analysis and Principles Robert J. Brooker Copyright 息The McGraw-Hill Companies, Inc. Permission required for reproduction or display CHAPTER 13 TRANSLATION OF mRNA Protein synthesis
Proteins are the active participants in cell structure and function Genes that encode polypeptides are termed structural genes These are transcribed into messenger RNA (mRNA) The main function of the genetic material is to encode the production of cellular proteins In the correct cell, at the proper time, and in suitable amounts 13.1 THE GENETIC BASIS FOR PROTEIN SYNTHESIS Copyright 息The McGraw-Hill Companies, Inc. Permission required for reproduction or display 13-3
13-16 Figure 13.2 Overview of gene expression Note that the start codon sets the reading frame for all remaining codons
The Genetic Code Copyright 息The McGraw-Hill Companies, Inc. Permission required for reproduction or display Translation involves an interpretation of one language into another In genetics, the nucleotide language of mRNA is translated into the amino acid language of proteins This relies on the genetic code Refer to Table 13.2 The genetic information is coded within mRNA in groups of three nucleotides known as codons 13-12
13-13 Table 13-2
Copyright 息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 13-14
RNA Copolymers Helped to Crack the Genetic Code Copyright 息The McGraw-Hill Companies, Inc. Permission required for reproduction or display In the 1960s, Gobind Khorana and his collaborators developed a novel method to synthesize RNA They first created short RNAs (2 to 4 nucleotide long) that had a defined sequence These were then linked together enzymatically to create long copolymers Refer to Table 13.5 13-26
13-27
A Polypeptide Chain Has Directionality Copyright 息The McGraw-Hill Companies, Inc. Permission required for reproduction or display 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 The first amino acid has an exposed amino group Said to be N-terminal or amino terminal end The last amino acid has an exposed carboxyl group Said to be C-terminal or carboxy terminal end Refer to Figure 13.5 13-29
13-30 Figure 13.5 Condensation reaction releasing a water molecule Copyright 息The McGraw-Hill Companies, Inc. Permission required for reproduction or display Carboxyl group Amino group
13-31 Figure 13.5 N terminal C terminal Copyright 息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 13-32 Figure 13.6 Copyright 息The McGraw-Hill Companies, Inc. Permission required for reproduction or display Nonpolar amino acids are hydrophobic They are often buried within the interior of a folded protein
13-33 Figure 13.6 Copyright 息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
Levels of Structures in Proteins Copyright 息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 proteins primary structure is its amino acid sequence 13-34
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 13-35 Figure 13.7 The amino acid sequence of the enzyme lysozyme 129 amino acids long Copyright 息The McGraw-Hill Companies, Inc. Permission required for reproduction or display
Levels of Structures in Proteins Copyright 息The McGraw-Hill Companies, Inc. Permission required for reproduction or display 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 13-36
Levels of Structures in Proteins Copyright 息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: Also called Protein complex This is formed when the various polypeptides associate together to make a functional protein Refer to Figure 13.8 13-37
13-38 Figure 13.8 Copyright 息The McGraw-Hill Companies, Inc. Permission required for reproduction or display
13-41 Wild type mutant A comparison of phenotype and genotype at the molecular, organismal and cellular levels Figure 13.9
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 13.2 STRUCTURE AND FUNCTION OF tRNA Copyright 息The McGraw-Hill Companies, Inc. Permission required for reproduction or display 13-42
Recognition Between tRNA and mRNA Copyright 息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 13-43 Figure 13.10 tRNAs are named according to the amino acid they bear The anticodon is anti-parallel to the codon
tRNAs Share Common Structural Features Copyright 息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 13-51
The modified bases are: I = inosine mI = methylinosine T = ribothymidine UH 2 = dihydrouridine m 2 G = dimethylguanosine = pseudouridine 13-52 Found in all tRNAs Not found in all tRNAs Other variable sites are shown in blue as well Structure of tRNA Figure 13.12
Charging of tRNAs Copyright 息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 13-53
Charging of tRNAs Copyright 息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 13-54
13-55 Figure 13.13 The amino acid is attached to the 3 end by an ester bond tRNA charging
tRNAs and the Wobble Rule Copyright 息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 codons 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: wobble base / position Thus tolerating certain types of mismatches 13-56
13-57 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 息The McGraw-Hill Companies, Inc. Permission required for reproduction or display Wobble position and base pairing rules Figure 13.14
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 13.3 RIBOSOME STRUCTURE AND ASSEMBLY Copyright 息The McGraw-Hill Companies, Inc. Permission required for reproduction or display 13-58
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 13.3 RIBOSOME STRUCTURE AND ASSEMBLY Copyright 息The McGraw-Hill Companies, Inc. Permission required for reproduction or display 13-59
Copyright 息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
Copyright 息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
Functional Sites of Ribosomes Copyright 息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 13-62
Copyright 息The McGraw-Hill Companies, Inc. Permission required for reproduction or display 13-63 Figure 13.16
Translation can be viewed as occurring in three stages Initiation Elongation Termination Refer to 13.17 for an overview of translation 13.4 STAGES OF TRANSLATION 13-64
Copyright 息The McGraw-Hill Companies, Inc. Permission required for reproduction or display Figure 13.17 Initiator tRNA See animation on your CD
The Translation Initiation Stage Copyright 息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
Copyright 息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
13-68 Figure 13.18 Copyright 息The McGraw-Hill Companies, Inc. Permission required for reproduction or display
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 息The McGraw-Hill Companies, Inc. Permission required for reproduction or display
The Translation Initiation Stage Copyright 息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 e ukaryotic I nitiation F actors are denoted eIF The initiator tRNA is designated tRNA met It carries a methionine rather than a formylmethionine 13-70
Copyright 息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 These rules are called Kozaks 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! 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
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 After the protein is synthesised the first aminoacid is removed! Final protein is 1 aa shorther than the number of aa codons in a mRNA! 13-72
The Translation Elongation Stage Copyright 息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 The addition of each amino acid occurs via a series of steps outlined in Figure 13.20 This process, though complex, can occur at a remarkable rate In bacteria 15-18 amino acids per second In eukaryotes 6 amino acids per second 13-73
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 息The McGraw-Hill Companies, Inc. Permission required for reproduction or display
13-75 Figure 13.20 tRNAs at the P and A sites move into the E and P sites, respectively Copyright 息The McGraw-Hill Companies, Inc. Permission required for reproduction or display Max. 2 tRNAs bound to mRNA in a ribosome ! Peptide bond formation is catalyzed by rRNA, not by one of the proteins in the ribosome!
The Translation Elongation Stage Copyright 息The McGraw-Hill Companies, Inc. Permission required for reproduction or display 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 13-76
The Translation Termination Stage Copyright 息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 13-77
The Translation Termination Stage Copyright 息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 13-78
13-79 Figure 13.21
Bacterial Translation Can Begin Before Transcription Is Completed Copyright 息The McGraw-Hill Companies, Inc. Permission required for reproduction or display Bacteria lack a nucleus Therefore, both transcription and translation occur in the cytoplasm As soon an mRNA strand is long enough, a ribosome will attach to its 5 end So translation begins before transcription ends This phenomenon is termed coupling A polyribosome or polysome is an mRNA transcript that has many bound ribosomes in the act of translation 13-81
Messenger RNA Reading Frame Each mRNA can be read in 3 frames - Sequence of codons is important: start codon indicates in which frames should be read
Sequence of Triplet form the genetic code and the mutations in a gene : Frameshift Mutations: By insertion/deletion of 1 or 2 nucleotiden
Restoration of Frame shift
Mutations: Base changes Very harmful : - deletion / insertion of one base - deletion / insertion of two bases Especially in the beginning of a ORF/gene If at the end of a gene, the shorter (or the to the-C-terminal-end-aberrant) protein can still be active Less harmful : - base substitution (another amino acid; but can also become a stop codon) - deletion / insertion of three bases (loss of an extra amino acid)
End of Chapter 13

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Translation brooker

  • 1. Genetics: Analysis and Principles Robert J. Brooker Copyright 息The McGraw-Hill Companies, Inc. Permission required for reproduction or display CHAPTER 13 TRANSLATION OF mRNA Protein synthesis
  • 2. Proteins are the active participants in cell structure and function Genes that encode polypeptides are termed structural genes These are transcribed into messenger RNA (mRNA) The main function of the genetic material is to encode the production of cellular proteins In the correct cell, at the proper time, and in suitable amounts 13.1 THE GENETIC BASIS FOR PROTEIN SYNTHESIS Copyright 息The McGraw-Hill Companies, Inc. Permission required for reproduction or display 13-3
  • 3. 13-16 Figure 13.2 Overview of gene expression Note that the start codon sets the reading frame for all remaining codons
  • 4. The Genetic Code Copyright 息The McGraw-Hill Companies, Inc. Permission required for reproduction or display Translation involves an interpretation of one language into another In genetics, the nucleotide language of mRNA is translated into the amino acid language of proteins This relies on the genetic code Refer to Table 13.2 The genetic information is coded within mRNA in groups of three nucleotides known as codons 13-12
  • 6. Copyright 息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 13-14
  • 7. RNA Copolymers Helped to Crack the Genetic Code Copyright 息The McGraw-Hill Companies, Inc. Permission required for reproduction or display In the 1960s, Gobind Khorana and his collaborators developed a novel method to synthesize RNA They first created short RNAs (2 to 4 nucleotide long) that had a defined sequence These were then linked together enzymatically to create long copolymers Refer to Table 13.5 13-26
  • 9. A Polypeptide Chain Has Directionality Copyright 息The McGraw-Hill Companies, Inc. Permission required for reproduction or display 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 The first amino acid has an exposed amino group Said to be N-terminal or amino terminal end The last amino acid has an exposed carboxyl group Said to be C-terminal or carboxy terminal end Refer to Figure 13.5 13-29
  • 10. 13-30 Figure 13.5 Condensation reaction releasing a water molecule Copyright 息The McGraw-Hill Companies, Inc. Permission required for reproduction or display Carboxyl group Amino group
  • 11. 13-31 Figure 13.5 N terminal C terminal Copyright 息The McGraw-Hill Companies, Inc. Permission required for reproduction or display
  • 12. There are 20 amino acids that may be found in polypeptides Each contains a different side chain , or R group 13-32 Figure 13.6 Copyright 息The McGraw-Hill Companies, Inc. Permission required for reproduction or display Nonpolar amino acids are hydrophobic They are often buried within the interior of a folded protein
  • 13. 13-33 Figure 13.6 Copyright 息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
  • 14. Levels of Structures in Proteins Copyright 息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 proteins primary structure is its amino acid sequence 13-34
  • 15. 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 13-35 Figure 13.7 The amino acid sequence of the enzyme lysozyme 129 amino acids long Copyright 息The McGraw-Hill Companies, Inc. Permission required for reproduction or display
  • 16. Levels of Structures in Proteins Copyright 息The McGraw-Hill Companies, Inc. Permission required for reproduction or display 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 13-36
  • 17. Levels of Structures in Proteins Copyright 息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: Also called Protein complex This is formed when the various polypeptides associate together to make a functional protein Refer to Figure 13.8 13-37
  • 18. 13-38 Figure 13.8 Copyright 息The McGraw-Hill Companies, Inc. Permission required for reproduction or display
  • 19. 13-41 Wild type mutant A comparison of phenotype and genotype at the molecular, organismal and cellular levels Figure 13.9
  • 20. 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 13.2 STRUCTURE AND FUNCTION OF tRNA Copyright 息The McGraw-Hill Companies, Inc. Permission required for reproduction or display 13-42
  • 21. Recognition Between tRNA and mRNA Copyright 息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 13-43 Figure 13.10 tRNAs are named according to the amino acid they bear The anticodon is anti-parallel to the codon
  • 22. tRNAs Share Common Structural Features Copyright 息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 13-51
  • 23. The modified bases are: I = inosine mI = methylinosine T = ribothymidine UH 2 = dihydrouridine m 2 G = dimethylguanosine = pseudouridine 13-52 Found in all tRNAs Not found in all tRNAs Other variable sites are shown in blue as well Structure of tRNA Figure 13.12
  • 24. Charging of tRNAs Copyright 息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 13-53
  • 25. Charging of tRNAs Copyright 息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 13-54
  • 26. 13-55 Figure 13.13 The amino acid is attached to the 3 end by an ester bond tRNA charging
  • 27. tRNAs and the Wobble Rule Copyright 息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 codons 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: wobble base / position Thus tolerating certain types of mismatches 13-56
  • 28. 13-57 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 息The McGraw-Hill Companies, Inc. Permission required for reproduction or display Wobble position and base pairing rules Figure 13.14
  • 29. 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 13.3 RIBOSOME STRUCTURE AND ASSEMBLY Copyright 息The McGraw-Hill Companies, Inc. Permission required for reproduction or display 13-58
  • 30. 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 13.3 RIBOSOME STRUCTURE AND ASSEMBLY Copyright 息The McGraw-Hill Companies, Inc. Permission required for reproduction or display 13-59
  • 31. Copyright 息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
  • 32. Copyright 息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
  • 33. Functional Sites of Ribosomes Copyright 息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 13-62
  • 34. Copyright 息The McGraw-Hill Companies, Inc. Permission required for reproduction or display 13-63 Figure 13.16
  • 35. Translation can be viewed as occurring in three stages Initiation Elongation Termination Refer to 13.17 for an overview of translation 13.4 STAGES OF TRANSLATION 13-64
  • 36. Copyright 息The McGraw-Hill Companies, Inc. Permission required for reproduction or display Figure 13.17 Initiator tRNA See animation on your CD
  • 37. The Translation Initiation Stage Copyright 息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
  • 38. Copyright 息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
  • 39. 13-68 Figure 13.18 Copyright 息The McGraw-Hill Companies, Inc. Permission required for reproduction or display
  • 40. 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 息The McGraw-Hill Companies, Inc. Permission required for reproduction or display
  • 41. The Translation Initiation Stage Copyright 息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 e ukaryotic I nitiation F actors are denoted eIF The initiator tRNA is designated tRNA met It carries a methionine rather than a formylmethionine 13-70
  • 42. Copyright 息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 These rules are called Kozaks 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! 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
  • 43. 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 After the protein is synthesised the first aminoacid is removed! Final protein is 1 aa shorther than the number of aa codons in a mRNA! 13-72
  • 44. The Translation Elongation Stage Copyright 息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 The addition of each amino acid occurs via a series of steps outlined in Figure 13.20 This process, though complex, can occur at a remarkable rate In bacteria 15-18 amino acids per second In eukaryotes 6 amino acids per second 13-73
  • 45. 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 息The McGraw-Hill Companies, Inc. Permission required for reproduction or display
  • 46. 13-75 Figure 13.20 tRNAs at the P and A sites move into the E and P sites, respectively Copyright 息The McGraw-Hill Companies, Inc. Permission required for reproduction or display Max. 2 tRNAs bound to mRNA in a ribosome ! Peptide bond formation is catalyzed by rRNA, not by one of the proteins in the ribosome!
  • 47. The Translation Elongation Stage Copyright 息The McGraw-Hill Companies, Inc. Permission required for reproduction or display 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 13-76
  • 48. The Translation Termination Stage Copyright 息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 13-77
  • 49. The Translation Termination Stage Copyright 息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 13-78
  • 51. Bacterial Translation Can Begin Before Transcription Is Completed Copyright 息The McGraw-Hill Companies, Inc. Permission required for reproduction or display Bacteria lack a nucleus Therefore, both transcription and translation occur in the cytoplasm As soon an mRNA strand is long enough, a ribosome will attach to its 5 end So translation begins before transcription ends This phenomenon is termed coupling A polyribosome or polysome is an mRNA transcript that has many bound ribosomes in the act of translation 13-81
  • 52. Messenger RNA Reading Frame Each mRNA can be read in 3 frames - Sequence of codons is important: start codon indicates in which frames should be read
  • 53. Sequence of Triplet form the genetic code and the mutations in a gene : Frameshift Mutations: By insertion/deletion of 1 or 2 nucleotiden
  • 55. Mutations: Base changes Very harmful : - deletion / insertion of one base - deletion / insertion of two bases Especially in the beginning of a ORF/gene If at the end of a gene, the shorter (or the to the-C-terminal-end-aberrant) protein can still be active Less harmful : - base substitution (another amino acid; but can also become a stop codon) - deletion / insertion of three bases (loss of an extra amino acid)