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Chapter 17

Chapter 17. From Gene to Protein. Figure 17.1 A ribosome, part of the protein synthesis machinery. TRANSCRIPTION. DNA. (a). Prokaryotic cell. In a cell lacking a nucleus, mRNA produced by transcription is immediately translated without additional processing. (b).

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Chapter 17

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  1. Chapter 17 From Gene to Protein

  2. Figure 17.1 A ribosome, part of the protein synthesis machinery

  3. TRANSCRIPTION DNA (a) Prokaryotic cell. In a cell lacking a nucleus, mRNAproduced by transcription is immediately translatedwithout additional processing. (b) Eukaryotic cell. The nucleus provides a separatecompartment for transcription. The original RNAtranscript, called pre-mRNA, is processed in various ways before leaving the nucleus as mRNA. Figure 17.3 Overview: the roles of transcription and translation in the flow of genetic information (layer 1)

  4. TRANSCRIPTION DNA mRNA Ribosome TRANSLATION (a) Prokaryotic cell. In a cell lacking a nucleus, mRNAproduced by transcription is immediately translatedwithout additional processing. Polypeptide (b) Eukaryotic cell. The nucleus provides a separatecompartment for transcription. The original RNAtranscript, called pre-mRNA, is processed in various ways before leaving the nucleus as mRNA. Figure 17.3 Overview: the roles of transcription and translation in the flow of genetic information (layer 2)

  5. TRANSCRIPTION DNA mRNA Ribosome TRANSLATION (a) Prokaryotic cell. In a cell lacking a nucleus, mRNAproduced by transcription is immediately translatedwithout additional processing. Polypeptide Nuclear envelope DNA TRANSCRIPTION (b) Eukaryotic cell. The nucleus provides a separatecompartment for transcription. The original RNAtranscript, called pre-mRNA, is processed in various ways before leaving the nucleus as mRNA. Figure 17.3 Overview: the roles of transcription and translation in the flow of genetic information (layer 3)

  6. TRANSCRIPTION DNA mRNA Ribosome TRANSLATION (a) Prokaryotic cell. In a cell lacking a nucleus, mRNAproduced by transcription is immediately translatedwithout additional processing. Polypeptide Nuclear envelope DNA TRANSCRIPTION Pre-mRNA RNA PROCESSING mRNA (b) Eukaryotic cell. The nucleus provides a separatecompartment for transcription. The original RNAtranscript, called pre-mRNA, is processed in various ways before leaving the nucleus as mRNA. Figure 17.3 Overview: the roles of transcription and translation in the flow of genetic information (layer 4)

  7. TRANSCRIPTION DNA mRNA Ribosome TRANSLATION (a) Prokaryotic cell. In a cell lacking a nucleus, mRNAproduced by transcription is immediately translatedwithout additional processing. Polypeptide Nuclear envelope DNA TRANSCRIPTION Pre-mRNA RNA PROCESSING mRNA Ribosome TRANSLATION (b) Eukaryotic cell. The nucleus provides a separatecompartment for transcription. The original RNAtranscript, called pre-mRNA, is processed in various ways before leaving the nucleus as mRNA. Polypeptide Figure 17.3 Overview: the roles of transcription and translation in the flow of genetic information (layer 5)

  8. DNA molecule Gene 2 Gene 1 Gene 3 DNA strand (template) 5 3 A C C A A A C C G A G T TRANSCRIPTION G U G G U G C A U U U C 5 3 mRNA Codon TRANSLATION Gly Phe Protein Ser Trp Amino acid Figure 17.4 The triplet code

  9. Second mRNA base U C A G U UAU UUU UCU UGU Tyr Cys Phe UAC UUC UCC UGC C U Ser UUA UCA UAA Stop Stop UGA A Leu UAG UUG UCG Stop UGG Trp G CUU CCU U CAU CGU His CUC CCC CAC CGC C C Arg Pro Leu CUA CCA CAA CGA A Gln CUG CCG CAG CGG G Third mRNA base (3 end) First mRNA base (5 end) U AUU ACU AAU AGU Asn Ser C lle AUC ACC AAC AGC A Thr A AUA ACA AAA AGA Lys Arg Met or start G AUG ACG AAG AGG U GUU GCU GAU GGU Asp C GUC GCC GAC GGC G Val Ala Gly GUA GCA GAA GGA A GUG GCG GAG GGG G Figure 17.5 The dictionary of the genetic code Glu

  10. Figure 17.6 A tobacco plant expressing a firefly gene

  11. Promoter Transcription unit 5 3 3 5 Start point RNA polymerase Figure 17.7 The stages of transcription: initiation, elongation, and termination (layer 1) DNA

  12. 1 Promoter Transcription unit 5 3 3 5 Start point RNA polymerase Initiation. After RNA polymerase binds to the promoter, the DNA strands unwind, and the polymerase initiates RNA synthesis at the start point on the template strand. 5 3 3 5 Template strand of DNA Unwound DNA RNA transcript Figure 17.7 The stages of transcription: initiation, elongation, and termination (layer 2) DNA

  13. 2 1 Promoter Transcription unit 5 3 3 5 Start point RNA polymerase Initiation. After RNA polymerase binds to the promoter, the DNA strands unwind, and the polymerase initiates RNA synthesis at the start point on the template strand. 5 3 3 5 Template strand of DNA Unwound DNA RNA transcript Elongation. The polymerase moves downstream, unwinding the DNA and elongating the RNA transcript 5 3 . In the wake of transcription, the DNA strands re-form a double helix. Rewound RNA 5 3 3 5 3 5 RNA transcript Figure 17.7 The stages of transcription: initiation, elongation, and termination (layer 3) DNA

  14. 2 1 3 Promoter Transcription unit 5 3 3 5 DNA Start point RNA polymerase Initiation. After RNA polymerase binds to the promoter, the DNA strands unwind, and the polymerase initiates RNA synthesis at the start point on the template strand. 5 3 3 5 Template strand of DNA Unwound DNA RNA transcript Elongation. The polymerase moves downstream, unwinding the DNA and elongating the RNA transcript 5 3 . In the wake of transcription, the DNA strands re-form a double helix. Rewound RNA 5 3 3 5 3 5 RNA transcript Termination. Eventually, the RNA transcript is released, and the polymerase detaches from the DNA. 5 3 3 5 3 5 Completed RNA transcript Figure 17.7 The stages of transcription: initiation, elongation, and termination (layer 4)

  15. Non-template strand of DNA Elongation RNA nucleotides RNA polymerase T A C C A T A T 3 C U 3 end T G A U G G A G E A C C C A 5 A A T A G G T T Direction of transcription (“downstream) 5 Template strand of DNA Newly made RNA

  16. Eukaryotic promoters 1 2 3 DNA TRANSCRIPTION Pre-mRNA RNA PROCESSING mRNA Ribosome TRANSLATION Polypeptide Promoter 5 3 A T A T A A A 3 5 A T A T T T T TATA box Start point Template DNA strand Several transcription factors Transcription factors 5 3 3 5 Additional transcription factors RNA polymerase II Transcription factors 3 5 5 5 3 RNA transcript Transcription initiation complex Figure 17.8 The initiation of transcription at a eukaryotic promoter

  17. A modified guanine nucleotide added to the 5 end 50 to 250 adenine nucleotides added to the 3 end TRANSCRIPTION DNA Polyadenylation signal Protein-coding segment Pre-mRNA RNA PROCESSING 5 3 mRNA G P P AAA…AAA P AAUAAA Ribosome Start codon Stop codon TRANSLATION 5 Cap 5 UTR Poly-A tail 3 UTR Polypeptide Figure 17.9 RNA processing: addition of the 5 cap and poly-A tail

  18. Intron 5 Exon Exon Intron Exon 3 5 Cap Poly-A tail Pre-mRNA TRANSCRIPTION DNA 30 31 104 105 146 1 Pre-mRNA RNA PROCESSING Introns cut out and exons spliced together Coding segment mRNA Ribosome TRANSLATION 5 Cap Poly-A tail mRNA Polypeptide 1 146 3 UTR 3 UTR Figure 17.10 RNA processing: RNA splicing

  19. 3 2 1 RNA transcript (pre-mRNA) 5 Intron Exon 1 Exon 2 Protein Other proteins snRNA snRNPs Spliceosome 5 Spliceosome components Cut-out intron mRNA 5 Exon 1 Exon 2 Figure 17.11 The roles of snRNPs and spliceosomes in pre-mRNA splicing

  20. Gene DNA Exon 1 Exon 2 Intron Exon 3 Intron Transcription RNA processing Translation Domain 3 Domain 2 Domain 1 Polypeptide Figure 17.12 Correspondence between exons and protein domains

  21. DNA TRANSCRIPTION mRNA Ribosome TRANSLATION Polypeptide Amino acids Polypeptide tRNA with amino acid attached Ribosome Trp Phe Gly tRNA C C C G G Anticodon A A A A G G G U G U U U C 5 Codons 3 mRNA Figure 17.13 Translation: the basic concept

  22. 3 A Amino acid attachment site C C 5 A C G C G C G U G U A A U U A U C G * G U A C A C A * A U C C * G * U G U G G * G A C C G * C * A G U G * * G A G C Hydrogen bonds G C U A G * A * A C * U A G A Anticodon (a) Two-dimensional structure. The four base-paired regions and three loops are characteristic of all tRNAs, as is the base sequence of the amino acid attachment site at the 3 end. The anticodon triplet is unique to each tRNA type. (The asterisks mark bases that have been chemically modified, a characteristic of tRNA.) Figure 17.14 The structure of transfer RNA (tRNA)

  23. Amino acid attachment site 5 3 Hydrogen bonds A A G 3 5 Anticodon Anticodon (c) (b) Three-dimensional structure Symbol used in this book

  24. Amino acid Aminoacyl-tRNA synthetase (enzyme) Active site binds the amino acid and ATP. 1 4 3 2 P P P Adenosine ATP ATP loses two P groups and joins amino acid as AMP. Adenosine P Pyrophosphate P Pi Pi Pi Phosphates tRNA Appropriate tRNA covalently Bonds to amino Acid, displacing AMP. Adenosine P AMP Activated amino acid is released by the enzyme. Aminoacyl tRNA (an “activated amino acid”) Figure 17.15 An aminoacyl-tRNA synthetase joins a specific amino acid to a tRNA

  25. TRANSCRIPTION DNA mRNA Ribosome TRANSLATION Polypeptide Exit tunnel Growing polypeptide tRNA molecules Large subunit E P A Small subunit 5 3 mRNA (a) Computer model of functioning ribosome. This is a model of a bacterial ribosome, showing its overall shape. The eukaryotic ribosome is roughly similar. A ribosomal subunit is an aggregate of ribosomal RNA molecules and proteins. Figure 17.16 The anatomy of a functioning ribosome

  26. P site (Peptidyl-tRNA binding site) A site (Aminoacyl- tRNA binding site) E site (Exit site) Large subunit E P A mRNA binding site Small subunit (b) Schematic model showing binding sites. A ribosome has an mRNA binding site and three tRNA binding sites, known as the A, P, and E sites. This schematic ribosome will appear in later diagrams.

  27. Amino end Growing polypeptide Next amino acid to be added to polypeptide chain tRNA 3 mRNA Codons 5 (c) Schematic model with mRNA and tRNA. A tRNA fits into a binding site when its anticodon base-pairs with an mRNA codon. The P site holds the tRNA attached to the growing polypeptide. The A site holds the tRNA carrying the next amino acid to be added to the polypeptide chain. Discharged tRNA leaves via the E site.

  28. Large ribosomal subunit P site 3 5 U C A Met Met 5 3 A G U Initiator tRNA GDP GTP E A mRNA 5 5 3 3 Start codon mRNA binding site Small ribosomal subunit Translation initiation complex A small ribosomal subunit binds to a molecule of mRNA. In a prokaryotic cell, the mRNA binding site on this subunit recognizes a specific nucleotide sequence on the mRNA just upstream of the start codon. An initiator tRNA, with the anticodon UAC, base-pairs with the start codon, AUG. This tRNA carries the amino acid methionine (Met). The arrival of a large ribosomal subunit completes the initiation complex. Proteins called initiation factors (not shown) are required to bring all the translation components together. GTP provides the energy for the assembly. The initiator tRNA is in the P site; the A site is available to the tRNA bearing the next amino acid. 1 2 Figure 17.17 The initiation of translation

  29. 1 Codon recognition. The anticodon of an incoming aminoacyl tRNA base-pairs with the complementary mRNA codon in the A site. Hydrolysis of GTP increases the accuracy and efficiency of this step. Amino end of polypeptide DNA TRANSCRIPTION mRNA Ribosome TRANSLATION Polypeptide E mRNA 3 Ribosome ready for next aminoacyl tRNA P A site site 5 2 GTP GDP 2 E E P A P A 2 Peptide bond formation. An rRNA molecule of the large Subunit catalyzes the formation of a peptide bond between the new amino acid in the A site and the carboxyl end of the growing polypeptide in the P site. This step attaches the polypeptide to the tRNA in the A site. GDP Translocation. The ribosome translocates the tRNA in the A site to the P site. The empty tRNA in the P site is moved to the E site, where it is released. The mRNA moves along with its bound tRNAs, bringing the next codon to be translated into the A site. 3 GTP E P A Figure 17.18 The elongation cycle of translation

  30. Release factor Free polypeptide 5 3 3 3 5 5 Stop codon (UAG, UAA, or UGA) The two ribosomal subunits and the other components of the assembly dissociate. When a ribosome reaches a stop codon on mRNA, the A site of the ribosome accepts a protein called a release factor instead of tRNA. The release factor hydrolyzes the bond between the tRNA in the P site and the last amino acid of the polypeptide chain. The polypeptide is thus freed from the ribosome. 1 2 3 Figure 17.19 The termination of translation

  31. Completed polypeptide Growing polypeptides Incoming ribosomal subunits Start of mRNA (5 end) Polyribosome End of mRNA (3 end) (a) An mRNA molecule is generally translated simultaneously by several ribosomes in clusters called polyribosomes. Ribosomes mRNA 0.1 µm (b) This micrograph shows a large polyribosome in a prokaryotic cell (TEM). Figure 17.20 Polyribosomes

  32. An SRP binds to the signal peptide, halting synthesis momentarily. The SRP binds to a receptor protein in the ER membrane. This receptor is part of a protein complex (a translocation complex) that has a membrane pore and a signal-cleaving enzyme. The SRP leaves, and the polypeptide resumes growing, meanwhile translocating across the membrane. (The signal peptide stays attached to the membrane.) Polypeptide synthesis begins on a free ribosome in the cytosol. The signal- cleaving enzyme cuts off the signal peptide. 2 3 1 4 5 6 Ribosome mRNA Signal peptide ER membrane Signal peptide removed Signal- recognition particle (SRP) Protein SRP receptor protein CYTOSOL Translocation complex Figure 17.21 The signal mechanism for targeting proteins to the ER The rest of the completed polypeptide leaves the ribosome and folds into its final conformation.

  33. RNA polymerase DNA mRNA Polyribosome Direction of transcription 0.25 m RNA polymerase DNA Polyribosome Polypeptide (amino end) Ribosome mRNA (5 end) Figure 17.22 Coupled transcription and translation in bacteria

  34. Wild-type hemoglobin DNA Mutant hemoglobin DNA In the DNA, the mutant template strand has an A where the wild-type template has a T. 3 5 3 5 T T C A T C mRNA mRNA The mutant mRNA has a U instead of an A in one codon. G A A U A G 5 3 5 3 Normal hemoglobin Sickle-cell hemoglobin The mutant (sickle-cell) hemoglobin has a valine (Val) instead of a glutamic acid (Glu). Val Glu Figure 17.23 The molecular basis of sickle-cell disease: a point mutation

  35. Wild type A U G A A G U U U G G C U A A mRNA 5 3 Protein Lys Met Phe Gly Stop Amino end Carboxyl end Base-pair substitution No effect on amino acid sequence U instead of C A U G A A G U U U G G U U A A Lys Met Phe Gly Stop Missense A instead of G A A A U G A A G U U U A G U U Lys Met Phe Ser Stop Nonsense U instead of A G A U U G G A G U A U U U C A Met Stop Figure 17.24 Base-pair substitution

  36. Wild type G A U A A G U U U G G C U A A mRNA 3 5 Gly Met Lys Phe Protein Stop Amino end Carboxyl end Base-pair insertion or deletion Frameshift causing immediate nonsense Extra U A G U U A A G U U U G G C U A Met Stop Frameshift causing extensive missense Missing U A G U A A G U U G G C U A A Met Lys Ala Leu Insertion or deletion of 3 nucleotides: no frameshift but extra or missing amino acid Missing A A G A G U U U U G G C U A A Met Phe Gly Stop Figure 17.25 Base-pair insertion or deletion

  37. DNA TRANSCRIPTION RNA is transcribed from a DNA template. 5 2 1 3 4 3 Poly-A RNA transcript RNA polymerase 5 Exon RNA PROCESSING In eukaryotes, the RNA transcript (pre- mRNA) is spliced and modified to produce mRNA, which moves from the nucleus to the cytoplasm. RNA transcript (pre-mRNA) Intron Aminoacyl-tRNA synthetase Cap NUCLEUS Amino acid FORMATION OF INITIATION COMPLEX AMINO ACID ACTIVATION tRNA CYTOPLASM After leaving the nucleus, mRNA attaches to the ribosome. Each amino acid attaches to its proper tRNA with the help of a specific enzyme and ATP. Growing polypeptide mRNA Activated amino acid Poly-A Poly-A Ribosomal subunits Cap 5 TRANSLATION C A succession of tRNAs add their amino acids to the polypeptide chain as the mRNA is moved through the ribosome one codon at a time. (When completed, the polypeptide is released from the ribosome.) C A U A E A C Anticodon A A A U G G U G U U U A Codon Ribosome Figure 17.26 A summary of transcription and translation in a eukaryotic cell

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