1. Protein Synthesis Chapter 17

3. Protein synthesis  DNA  Responsible for hereditary information  DNA divided into genes  Gene:  Sequence of nucleotides  Determines amino acid sequence in proteins  Genes provide information to make proteins

4. Protein synthesis DNA RNA protein

5. Protein Synthesis  Gene Expression:  Process by which DNA directs the synthesis of proteins  2 stages  Transcription  Translation

6. Protein synthesis  Transcription:  DNA sequence is copied into an RNA  Translation:  Information from the RNA is turned into an amino acid sequence

7. Protein synthesis DNA RNA Protein Transcription Translation

10. Protein Synthesis  Central Dogma  Mechanism of reading & expressing genes  Information passes from the genes (DNA) to an RNA copy  Directs sequence of amino acids to make proteins

12. Protein synthesis  Beadle & Tatum  Bread mold  3 enzymes to make arginine  Mutated mold’s DNA  Mutated code for enzymes  Unable to code for arginine

13. Precursor Enzyme A Enzyme B Enzyme C Ornithine Citrulline Arginine No growth: Mutant cells cannot grow and divide Growth: Wild-type cells growing and dividing Control: Minimal medium Results Table Wild type Minimal medium (MM) (control ) MM + ornithin e MM + citrullin e MM + arginine (control ) Summar y of results Can grow with or without any supplements Gene (codes for enzyme) Wild type Precurso r Ornithin e Gene A Gene B Gene C Enzyme A Enzyme B Enzyme C Enzyme A Enzyme B Enzyme C Citrullin e Arginin e Precurso r Ornithin e Citrullin e Arginin e Precurso r Ornithin e Citrullin e Arginin e Precurso r Ornithin e Citrullin e Arginin e Enzyme A Enzyme B Enzyme C Enzyme A Enzyme B Enzyme C Class I mutants (mutation in gene A) Class II mutants (mutation in gene B) Class III mutants (mutation in gene C) Can grow on ornithine, citrulline, or arginine Can grow only on citrulline or arginine Require arginine to grow Class I mutants Class II mutants Class III mutants Classes of Neurospora crassa Condition

14. Protein synthesis  Beadle & Tatum  One gene one enzyme  One gene one protein  One gene one polypeptide

15. An albino racoon

16. Cracking the code  Codons (Triplet code)-mRNA  Each codon corresponds to an aa  20 amino acids  64 triplet codes (codons)  61 code for aa-3 are stop codons  Wobble:  Flexible base pairing in the 3 rd position  3’ end

18. Cracking the code  Reading frame  Reading symbols in correct groupings  1 or 2 deletions or additions  Gene was transcribed incorrectly  3 deletions  Reading frame would shift  Gene was transcribed correctly

19. WHYDIDTHEREDCATEATTHEFATRAT WHYIDTHEREDCATEATTHEFATRAT WHYDTHEREDCATEATTHEFATRAT WHYTHEREDCATEATTHEFATRAT

20. Cracking the code  Universal code  AGA codes for amino acid Arginine  Humans & bacteria  Genes from humans can be transcribed by mRNA from bacteria  Produce human proteins  Insulin

21. RNA  RNA (ribonucleic acid)  Single strand  Sugar –ribose (-OH on 2’ carbon)  Uracil instead of thymine

24. RNA  mRNA:  Messenger RNA  Transcribes information from DNA  Codons  (3 nucleotides) CGU  mRNA  Codes for amino acids  rRNA:  Ribosomal RNA  Polypeptides are assembled

25. RNA  tRNA:  Transfer RNA  Transports aa to build proteins  Positions aa on rRNA  Anticodons  (3 complementary nucleotides) GCA

29. Nuclear envelope CYTOPLASM DNA Pre- mRNA mRNA Ribosom e TRANSLATION (b) Eukaryotic cell NUCLEUS RNA PROCESSING TRANSCRIPTION (a) Bacterial cell Polypeptide DNA mRNA Ribosome CYTOPLASM TRANSCRIPTION TRANSLATION Polypeptide

30. Transcription  Getting the code from DNA  Triplet code  Template strand  Strand of DNA  Provides template or pattern  Transcribed or read  Transcribed RNA is complementary to this DNA strand

32. Transcription  Coding strand  DNA strand not coded  Same sequence of nucleotides as the RNA transcript  Only T instead of U.

33. Figure 17.4 A CC AAA CCG A GT ACTTTTCGGGGT UGGUUUGGCCUA Ser Gly PheTrp Codo n TRANSLATIO N TRANSCRIPTIO N Protei n mRNA 5′5′ 5′5′ 3′3′ Amino acid DNA templat e strand 5′5′ 3′3′ 3′3′

34. Transcription  RNA polymerase  Enzyme  Adds nucleotides to the 3’end  5’to3’ direction  Does not need a primer to start  One polymerase in prokaryotes  Three in eukaryotes  Polymerase II makes mRNA

35. Transcription  Promoters:  Sequence on DNA where transcription starts  TATAAT  TATA box  Sequences are not transcribed

36. Transcription  Stages  Initiation  Elongation  Termination

37. Initiation  RNA polymerase binds promoter  Unwinds DNA  Transcription unit:  RNA polymerase, DNA & growing RNA strand

38. Fig. 17-UN1 Transcription unit Promoter RNA transcript RNA polymerase Template strand of DNA 5 5 53 3 3

39. Initiation  Transcription factors bind first to the promoter in Eukaryotes  RNA pol II binds DNA  Transcription Initiation Complex is formed  Starts to transcribe

40. Promoter Nontemplate strand 1 5′5′ 3′3′ 5′5′ 3′3′ Start point RNA polymerase II Template strand TATA box Transcription factors DNA 3′3′ 5′5′ 3′3′ 5′5′ 3′3′ 5′5′ 2 3 Transcription factors RNA transcript Transcription initiation complex 3′3′ 5′5′ 5′5′ 3′3′ A eukaryotic promoter Several transcription factors bind to DNA. Transcription initiation complex forms. TATA AAA TAATTTT

41. Elongation  RNA polymerase moves along DNA  Untwists DNA  Adds nucleotides to 3’ end

42. Fig. 17-7b Elongation RNA polymerase Nontemplate strand of DNA RNA nucleotides 3 end Direction of transcription (“downstream”) Template strand of DNA Newly made RNA 3 5 5

43. Termination  Prokaryotes  Stop signal  Sequence on DNA  RNA transcript signals polymerase to detach from DNA  RNA strand separates from the DNA

44. Termination  Eurkaryotes  Polyadenylation signal sequence on mRNA  AAUAAA  Recognized by RNA polymerase II  mRNA is released

45. Transcription

46. Promoter Transcription unit RNA polymerase Start point 1 Template strand of DNA RNA transcript Unwound DNA Rewound DNA RNA transcript Direction of transcription (“downstrea m”) Completed RNA transcript Initiation Elongation Termination 2 3 5′5′ 3′3′ 5′5′ 5′5′ 3′3′ 5′5′ 3′3′ 5′5′ 3′3′ 5′5′ 5′5′ 3′3′ 3′3′ 5′5′ 3′3′ 3′3′ 5′5′ 3′3′ 5′5′ 3′3′

47. Eukaryotes  mRNA is modified  Nucleus  RNA processing

48. Eukaryotes  5’ cap  Addition of a GTP  5’ phosphate of the first base of mRNA  Methyl group is added to the GTP  3’poly-A-tail  Several A’s on the end of the mRNA

49. Eukaryotes  Introns:  non-coding sequences of nucleic acids  Exons:  coding sequences of nucleic acids

50. Euraryotes  RNA splicing  Cut out introns  Reconnect exons  snRNP’s (small nuclear RNA’s)  Spliceosome:  Many snRNP’s come together & remove introns

54. Translation  Passing the code to make a polypeptide  mRNA  rRNA  ribosomes  tRNA

55. Translation  Ribosome  Located in the cytoplasm  Site of translation  2 subunits composed of protein & RNA  Small (20 proteins and 1 RNA)  Large (30 proteins and 2 RNA)  3 sites on ribosome surface involved in protein synthesis  E, P, and A sites

56. Ribosome (b) Schematic model showing binding sites Small subunit Large subunit Exit tunnel A site (Aminoacyl- tRNA binding site) P site (Peptidyl-tRNA binding site) E site (Exit site) mRNA binding site EPA

57. Ribosome

58. Ribsome Growing polypeptide Next amino acid to be added to polypeptide chain tRNA 3′3′ 5′5′ mRNA Amino end Codons E (c) Schematic model with mRNA and tRNA

59. Translation  tRNA  Aminoacyl-t-RNA synthetases  Activating enzymes  Link correct tRNA code to correct aa  One for each 20 amino acids  Some read one code, some read several codes  45 tRNA’s

63. Translation  Nonsense codes  UAA, UAG, UGA code to stop  AUG codes for start as well as methionine  Ribosome starts at the first AUG it comes across in the code

64. Translation  mRNA binds to rRNA on the ribosome  mRNA attaches so only one codon is exposed at a time  tRNA (anti-codon)  Complementary sequence  Binds to mRNA  tRNA carries a specific amino acid  Adds to growing polypeptide

65. Translation  1. Initiation  2. Elongation  3. Termination

66. Initiation  Initiation complex  1. tRNA with methionine attached binds to a small ribosome  2. binds at the 5’ cap (Eukayotes)  3. tRNA is positioned on to the mRNA at AUG  4. Initiation factors position the tRNA on the P site  5. Attachment of large ribosomal unit

67. Initiation  Requires energy  GTP  Forms the Initiation complex

68. Initiation 1 2 P site 3′3′ Large ribosomal subunit Translation initiation complex Large ribosomal subunit completes the initiation complex. Small ribosomal subunit binds to mRNA. mRNA binding site Small ribosomal subunit Initiator tRNA Start codon 3′3′ 5′5′ EA 3′3′ 5′5′ GTPGDP P i + 3′3′ 5′5′ 5′5′ U A C G A U Met mRNA

69. Elongation  Elongation factors  Help second tRNA bind to the A-site  Two amino acids bind (peptide bond)  Translocation:  Ribosome moves 3 more nucleotides along mRNA in the 5’to 3’ direction

70. Elongation  Initial tRNA moves to E site  Released  New tRNA moves into A site  Continues to add more aa to form the polypeptide

72. Elongation Amino end of polypeptide Codon recognition 1 3′3′ 5′5′ E PA site E P A mRNA GTP P i 2 3 P i GDP + Translocation E P A Peptide bond formation E P A Ribosome ready for next aminoacyl tRNA

74. Termination  Release factors:  Proteins that release newly made polypeptides  Codon (UAG, UAA, UGA)  Release factor binds to the codon  Polypeptide chain is released from A site

75. Termination 31 2 Release factor 3′3′ 5′5′ 5′5′ 3′3′ Stop codon (UAG, UAA, or UGA) Ribosome reaches a stop codon on mRNA. Release factor promotes hydrolysis. Ribosomal subunits and other components dissociate. Free polypeptid e 3′3′ 5′5′ 2 GTP 2 GDP + 2P i

77. Fig. 17-UN3 mRNA Ribosome Polypeptide

78. Translation

79. Second nucleotide

80. <>

81. Growing polypeptides Completed polypeptide End of mRNA (3 ′ end) Incoming ribosomal subunits Start of mRNA (5 ′ end) Several ribosomes simultaneously translating one mRNA molecule (a) Polyribosome

83. Protein ER membrane Signal peptide removed SRP receptor protein Translocation complex ER LUMEN CYTOSOL SRP Signal peptide mRNA Ribosome Polypeptide synthesis begins. SRP binds to signal peptide. SRP binds to receptor protein. SRP detaches and polypeptide synthesis resumes. Signal- cleaving enzyme cuts off signal peptide. Completed polypeptide folds into final conformation. 1 23 456

84. Similarities DNA RNA Protein Transcription Translation

85. Differences in gene expression  Transcription  1. Prokaryotes one RNA polymerase  Eukaryotes 3 RNA polymerases (poli-II mRNA synthesis)  2. Prokaryotes mRNA contain transcripts of several genes  Eukaryotes only one gene  3. Prokaryotes no nucleus so start translation before transcription is done

86. Differences in gene expression  3. Eukaryotes complete transcription before leaving the nucleus  4. Eukaryotes modify RNA Introns/exons  5. Prokaryotes Polymerase binds promoters  Eukaryotes transcription factors bind first then enzyme  6. Termination

87. Differences in gene expression  Translation  1. Prokaryotes start translation with AUG  Eukaryotes 5’cap initiates translation  2. Prokaryotes smaller ribosomes

88. Nuclear envelope CYTOPLASM DNA Pre- mRNA mRNA Ribosom e TRANSLATION (b) Eukaryotic cell NUCLEUS RNA PROCESSING TRANSCRIPTION (a) Bacterial cell Polypeptide DNA mRNA Ribosome CYTOPLASM TRANSCRIPTION TRANSLATION Polypeptide

90. Mutations  Changes in genetic information  Point mutations:  Change in a single base pair  Sickle cell mutation

91. Point mutation Wild-type β -globinSickle-cell β - globin Mutant β -globin DNA Wild-type β -globin DNA mRNA Normal hemoglobin Sickle-cell hemoglobin Val UGG ACC GGT Glu GGA GGA CCT 3′3′ 5′5′ 5′5′ 5′5′ 5′5′ 5′5′ 3′3′ 3′3′ 3′3′ 3′3′ 3′3′ 5′5′

92. Mutations  Two types  1. Base-pair substitution  2. Insertion or deletion

93. Mutations  1. Base-pair substitution  Exchange one nucleotide and base pair with another  A. Silent mutations  No effect on proteins

94. Silent mutaton Wild type DNA template strand TTTACCAAACCGATT AAAAATGGTTTGGCT 3′3′ 5′5′ 3′3′ 5′5′ AAUCGGUUUGAAGAU 5′5′ 3′3′ mRNA Protein Amino end MetLysPheGly Stop Carboxyl end Nucleotide-pair substitution: silent A instead of G U instead of C 3′3′ 3′3′ 5′5′ Stop MetLysPheGly GGUUUGAAGAUUAAU TTTACCAAACCTTAA AAATGGTTTGGTAAT 5′5′ 5′5′ 3′3′

95. Mutations  B. Missense mutations:  Substitutions that change one aa for another  Little effect

96. Missense Wild type DNA template strand TTTACCAAACCGATT AAAAATGGTTTGGCT 3′3′ 5′5′ 3′3′ 5′5′ AAUCGGUUUGAAGAU 5′5′ 3′3′ mRNA Protein Amino end MetLysPheGly Stop Carboxyl end Nucleotide-pair substitution: missense T instead of C Stop MetLysPheSer A instead of G 5′5′ 3′3′ 5′5′ 5′5′ 3′3′ 3′3′ AUGAAGUUUUAACGA ATGAAAATCGTTTGA TACCCAAAATTTTTG

97. Mutations  C. Nonsense mutations  Point mutation codes for stop codon  Stops translation too soon  Shortens protein  Non-functional proteins

98. Mutations  2. Insertions or deletions  Additions or losses of nucleotides  Frameshift mutations  Improperly grouped codons  Nonfuctional proteins

99. Fig. 17-23 Wild-type 3 DNA template strand 5 5 5 3 3 Stop Carboxyl end Amino end Protein mRNA 3 3 3 5 5 5 A instead of G U instead of C Silent (no effect on amino acid sequence) Stop T instead of C 3 3 3 5 5 5 A instead of G Stop Missense A instead of T U instead of A 3 3 3 5 5 5 Stop Nonsense No frameshift, but one amino acid missing (3 base-pair deletion) Frameshift causing extensive missense (1 base-pair deletion) Frameshift causing immediate nonsense (1 base-pair insertion) 5 5 5 3 3 3 Stop missing 3 3 3 5 5 5 Stop 5 5 5 3 3 3 Extra U Extra A (a) Base-pair substitution(b) Base-pair insertion or deletion

100. Mutagens  Chemical or physical agents  Mutations in DNA