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Chapter 17

Chapter 17. From Gene to Protein (Protein Synthesis). Protein Synthesis. The information content of DNA Is in the form of specific sequences of nucleotides along the DNA strands The DNA inherited by an organism Leads to specific traits by dictating the synthesis of proteins

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Chapter 17

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  1. Chapter 17 From Gene to Protein (Protein Synthesis)

  2. Protein Synthesis • The information content of DNA • Is in the form of specific sequences of nucleotides along the DNA strands • The DNA inherited by an organism • Leads to specific traits by dictating the synthesis of proteins • Gene expression is the process by which DNA directs protein synthesis • Early experiments led researchers to: One Gene—One Polypeptide Hypothesis

  3. Protein Synthesis Overview Gene expression occurs in 2 stages: • Transcription • Is the synthesis of RNA under the direction of DNA • Produces messenger RNA (mRNA)

  4. Protein Synthesis Overview Gene expression occurs in 2 stages: 2. Translation • Is the actual synthesis of a polypeptide, which occurs under the direction of mRNA • Occurs on ribosomes

  5. DNA TRANSCRIPTION mRNA Ribosome TRANSLATION Polypeptide (a) Bacterial cell

  6. Protein Synthesis Overview • In eukaryotes • RNA transcripts are modified before becoming true mRNA • Initial RNA transcript is called primary transcript or pre-mRNA. Cellular chain of command DNA RNA Protein

  7. Nuclearenvelope DNA TRANSCRIPTION Pre-mRNA RNA PROCESSING mRNA TRANSLATION Ribosome Polypeptide (b) Eukaryotic cell

  8. The Genetic Code • Genetic information • Is encoded as a sequence of non-overlapping base triplets, or codons • 4 bases allow for 64 unique codons • Codons must be read in the correct readingframe • For the specified polypeptide to be produced

  9. Different Reading Frames

  10. Transcription & Translation • During transcription • One of the two DNA strands (template strand) provides order of complementary RNA nucleotides for mRNA transcript • Template strand always the same for a given gene • Base pairing rules apply except with uracil (U) replacing thymine (T) on the mRNA strand

  11. Transcription & Translation • During translation • mRNA codons are read in 5’ to 3’ direction • Each codon specifies 1 of the 20 amino acids for correct primary order for that polypeptide • Genetic code is redundant but not ambiguous • 61 codons code for amino acids; 3 are stop codons • No codon specifies more than 1 amino acid!!!

  12. Second mRNA base Genetic Code A U C G UUU UAU UCU UGU U Phe Cys Tyr UUC UCC UAC UGC C U Ser UUA UCA UGA Stop A UAA Stop Leu Trp UUG UCG UGG G UAG Stop CUU CCU U CAU CGU His CUC CAC CGC C CCC C Leu Pro Arg CUA CCA CGA A CAA Gln CUG CCG CGG G CAG First mRNA base (5′ end of codon) Third mRNA base (3′ end of codon) AUU AAU ACU AGU U Ser Asn C AUC Ile AAC ACC AGC A Thr AUA AAA ACA AGA A Lys Arg Met orstart AUG ACG AGG AAG G GUU GCU GAU GGU U Asp GUC GCC C GGC GAC G Val Ala Gly Gly GUA GCA GGA A GAA Glu GUG GCG GGG G GAG

  13. Using the Genetic Code Overview DNAtemplatestrand DNA 5′ 3′ molecule A A A A A T C C C C G G T T T T A G G G G C T C Gene 1 3′ 5′ TRANSCRIPTION Gene 2 U G G U U U G G C U C A 5′ 3′ mRNA Reading Frame Codon TRANSLATION Gly Phe Trp Protein Ser Gene 3 Amino acid

  14. Transcription • RNA synthesis is catalyzed by RNA polymerase • Separates the DNA strands apart, hooks together the RNA nucleotides • Follows the same base-pairing rules as DNA, except that in RNA, uracil substitutes for thymine • DNA sequence where RNA polymerase attaches: Promoter • Stretch of DNA transcribed: Transcription unit

  15. Transcription • 3 stages of transcription are • Initiation • Elongation • Termination

  16. Promoter Transcription unit DNA Start point RNA polymerase Initiation 1 Template strand of DNA RNA transcript Unwound DNA Elongation 2 Rewound DNA RNA transcript Termination 3 Completed RNA transcript

  17. Non-template strand of DNA Elongation RNA nucleotides RNA polymerase T A C C A T A T 3′ U C 3end T G A U G G A A C A U C C C 5′ A A T A G G T T Direction of transcription (“downstream”) Template strand of DNA 5′ Newly made RNA Detailed View of Synthesis of a mRNA transcript

  18. Initiation • Promoters • Signal the transcriptional start point • TATA box: Important promoter in eukaryotes • Transcription factors • Mediate the binding of RNA polymerase • Transcription factors + RNA polymerase = Transcription Initiation Complex

  19. 1 3 2 Initiation A eukaryotic promoter Promoter Nontemplate strand DNA 5′ 3′ T A A A A A T 5′ 3′ A T A T T T T TATA box Template strand Start point Several transcriptionfactors bind to DNA Transcriptionfactors 5′ 3′ 3′ 5′ Transcription initiationcomplex forms RNA polymerase II Transcription factors 5′ 3′ 3′ 5′ 3′ 5′ RNA transcript Transcription initiation complex

  20. Elongation • RNA polymerase moves along the DNA • Untwists the double helix, 10 to 20 bases at a time • A gene can be transcribed simultaneously by several RNA polymerases • Nucleotides are added to the 3’ end of the growing RNA molecule

  21. Processing of pre-mRNA in Eukaryotes • Each end of a pre-mRNA molecule is modified in a particular way • 5′ end receives a modified nucleotide 5’ cap • 3′ end gets a poly-A tail

  22. Processing of pre-mRNA in Eukaryotes • These modifications share several functions • Facilitate the export of mRNA to the cytoplasm • Protect mRNA from hydrolytic enzymes • Help ribosomes attach to the 5’ end

  23. RNA Processing Protein-codingsegment Polyadenylationsignal 5′ 3′ … G P P P AAA AAUAAA AAA Startcodon Stopcodon Cap 5′ UTR 5′ Poly-A tail 3′ UTR

  24. Intron Exon Exon Intron Exon 5′ Cap Poly-A tail Pre-mRNA TRANSCRIPTION DNA 30 31 104 105 146 1 Introns cut out and exons spliced together Pre-mRNA RNA PROCESSING Coding segment mRNA Ribosome TRANSLATION 5′ Cap Poly-A tail mRNA Polypeptide 1 146 3UTR UTR More pre-mRNA Processing • RNA splicing • Removes introns (intervening) and joins exon (expressed)

  25. Translation • A cell translates a mRNA message into protein • With the help of transfer RNA (tRNA)

  26. Translation • Molecules of tRNA are not all identical • Each carries a specific amino acid on one end • Each has an anticodonon the other end • Anticodons base pair with complementary codons on mRNA

  27. Structure of a tRNA molecule 3′ Amino acidattachmentsite Amino acidattachmentsite 5′ 5′ 3′ Hydrogenbonds Hydrogenbonds A A G 3′ 5′ Anticodon Anticodon Anticodon (c) Symbol used (a) Two-dimensional structure in book (b) Three-dimensional structure

  28. Ribosomes • Ribosomes • Facilitate the specific coupling of tRNA anticodons with mRNA codons during protein synthesis • Ribosomes are made of 2 subunits, one large and one small • Constructed of proteins and RNA molecules named ribosomal RNA (rRNA)

  29. Ribosomes • Ribosomes have three binding sites for tRNA • P site holds the tRNA that carries the growing polypeptide chain • A site holds the tRNA that carries the next amino acid to be added to the chain • E site is the exit site, where discharged tRNAs leave the ribosome

  30. Ribosome Structure P site A site E site Largesubunit E A P mRNAbinding site Smallsubunit

  31. DNA TRANSCRIPTION mRNA Ribosome TRANSLATION Amino acids Polypeptide Polypeptide tRNA with amino acid attached Ribosome Trp Phe Gly tRNA C Anticodon C C G G A A A A G G G U G U U U C Codons mRNA Overview of Translation

  32. Largeribosomalsubunit U C A Met Met A G U GDP GTP E A mRNA Start codon mRNA binding site Small ribosomal subunit 2 1 Initiation of Translation • The initiation stage of translation • Brings together mRNA, tRNA bearing the first amino acid of the polypeptide, and two subunits of a ribosome

  33. Amino end ofpolypeptide E 3′ mRNA Ribosome ready fornext tRNA Asite Psite 5′ GTP GDP + P i E E P A A P GDP + P i GTP E A P

  34. Termination of Translation • Termination occurs when a stop codon reaches the A site • A site accepts a protein called a release factor • Reaction releases the polypeptide

  35. Termination of Translation P Releasefactor Freepolypeptide 5′ 3′ 3′ 3′ GTP 2 5′ 5′ + 2 GDP 2 Stop codon i (UAG, UAA, or UGA)

  36. Mutations • Mutations • Are changes in the genetic material (DNA) • Spontaneous mutations can occur during DNA replication, recombination, or repair • Mutagens are physical or chemical agents that can cause mutations

  37. Mutations • Point mutations • Are changes in just one base pair of a gene • Can be divided into two general categories • Base-pair substitutions • Base-pair insertions or deletions

  38. Mutations Summary • Nucleotide-pair substitution: Replaces one nucleotide with a different one • Silent mutations: Has no effect on the amino acid produced because of redundancy • Missense mutations: Still codes for an amino acid, but not the correct one • Nonsense mutations: Changes into a stop codon, nearly always leading to a nonfunctional protein

  39. Wild type DNA template strand G T T T T T A C C A A A C C A 3′ 5′ G G G G T T T T T A A A C A A 5′ 3′ mRNA5′ G G G G C A U A A U U U U A A 3′ Protein Met Lys Phe Gly Stop Amino end Carboxyl end (a) Nucleotide-pair substitution: silent A instead of G A T T T T T A C C A A A C C A 3′ 5′ T G G G G T T T T T A A A A A 5′ 3′ U instead of C 3′ U G G G G 5′ A U A A U U U U A A Met Lys Phe Gly Stop

  40. Wild type DNA template strand G T T T T T A C C A A A C C A 3′ 5′ 5′ G G G G 3′ T T T T C T A A A A A mRNA5′ 3′ G G G G C A U A A U U U U A A Protein Met Lys Phe Gly Stop Amino end Carboxyl end (a) Nucleotide-pair substitution: missense T instead of C G T T T T T A C C A A A T C A 5′ 3′ G G G A C T T T T T A A A A A 5′ 3′ A instead of G G G A G 3′ A U A A U U U C U A A 5′ Met Lys Ser Phe Stop

  41. Wild type DNA template strand G T T T T T A C C A A A C C A 3′ 5′ 5′ G G G G 3′ T T T T C T A A A A A mRNA5′ 3′ G G G G C A U A A U U U U A A Protein Met Lys Phe Gly Stop Amino end Carboxyl end (a) Nucleotide-pair substitution: nonsense T instead of C A instead of T T T T A A A A C G A A C T C C 5′ 3′ A T T A A T C 5′ T T T 3′ G G G G A U instead of A 3′ 5′ G G G G A U A U U U C U A A U Met Stop

  42. Wild type DNA template strand G T T T T T A C C A A A C C A 5′ 3′ G G G G 3′ T T T T C T A A A A A 5′ mRNA5′ 3′ G G G G C A U A A U U U U A A Protein Met Lys Phe Gly Stop Amino end Carboxyl end (b) Nucleotide-pair insertion or deletion: frameshiftcausing immediate nonsense Extra A C A G T T T T A C T C A A A G A 5′ 3′ C T T T G A A G T G T A A T A G 5′ 3′ Extra U G G A U G A A U U C U U A A 5′ G U 3′ Met Stop 1 nucleotide-pair insertion

  43. Wild type C A A A G T T T T T DNA template strand A C C C A 3′ 5′ G G G G T T T T C T A A A A A 5′ 3′ mRNA5′ G G G G C A U A A U U U U A A 3′ Protein Met Gly Lys Phe Stop Carboxyl end Amino end (b) Codon insertion or deletion: no frameshift, but one amino acid missing missing T C T G T A C A A A C C T T A 5′ 3′ G G T T T T C T A G A A 3′ 5′ missing A A G G G G C A A 3′ A U U U U U 5′ Met Phe Gly Stop 3 nucleotide-pair deletion

  44. Wild-type hemoglobin DNA Mutant hemoglobin DNA 3 5 3′ 5′ T T C A T C mRNA mRNA G A A U A G 5′ 3′ 5′ 3′ Normal hemoglobin Sickle-cell hemoglobin Val Glu Consequences of a Point Mutation • The change of a single nucleotide in the DNA’s template strand • Leads to the production of an abnormal protein

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