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Chapter 17: From Gene to Protein Important questions that were answered…. Chapter 13 – “What is the process of making gametes?” – meiosis Chapter 14 – “How are traits passed along to offspring?” – gametes Chapter 15 – “What carries the genetic material?” – chromosomes Chapter 16 – “What is the genetic material?” – DNA Chapter 17 – “How do genes give us our traits?” Chapter 17: From Gene to Protein 1. What is the “Central Dogma of Molecular Biology”? Transcription DNA Nucleotide lang. Translation RNA Nucleotide lang. Protein aa lang. 2. How is this different in prokaryotes & eukaryotes? Figure 17.3 Overview: the roles of transcription and translation in the flow of genetic information TRANSCRIPTION DNA mRNA Ribosome TRANSLATION Polypeptide (a) Prokaryotic cell. In a cell lacking a nucleus, mRNA produced by transcription is immediately translated without additional processing. Nuclear envelope TRANSCRIPTION DNA RNA PROCESSING Pre-mRNA mRNA Ribosome TRANSLATION Polypeptide (b) Eukaryotic cell. The nucleus provides a separate compartment for transcription. The original RNA transcript, called pre-mRNA, is processed in various ways before leaving the nucleus as mRNA. Figure 5.26 Components of nucleic acids Nitrogenous bases Pyrimidines 5’ end 5’C NH2 O Nucleoside O Nitrogenous base O O 5’C O 3’C C CH CH 5’C O P O Purines CH2 O O Phosphate 3’C Pentose group sugar O NH2 HC N C C N N C N H Adenine A (b) Nucleotide OH 3’ end (a) Polynucleotide, or nucleic acid O C C CH3 HN C HN CH C CH C C CH N N O N O O H H H Cytosine Thymine (in DNA) Uracil (in RNA) C U T N 3’C O HC N C N H Guanine G Pentose sugars 5’ HOCH2 O 4 H H H CH N C C NH 3’ 2’ OH 1’ H OH H Deoxyribose (in DNA) C NH2 5’ HOCH2 O OH 4 H H 1’ H 3’ 2’ H OH OH Ribose (in RNA) (c) Nucleoside components Chapter 17: From Gene to Protein 1. What is the “Central Dogma of Molecular Biology? 2. How is this different in prokaryotes & eukaryotes? 3. What are the stages of transcription? - Initiation - Elongation - Termination Chapter 17: From Gene to Protein 1. What is the “Central Dogma of Molecular Biology? 2. How is this different in prokaryotes & eukaryotes? 3. What are the stages of transcription? TRANSCRIPTION RNA PROCESSING At promoter – promotes transcription Transcription factors bind to TATA box RNA polymerase makes RNA Energy from ATP needed to start Pre-mRNA mRNA TRANSLATION - Initiation - 1 Eukaryotic promoters DNA Ribosome Polypeptide Promoter 5 T A T A A AA 3 3 ATAT T T T 5 TATA box Start point Template DNA strand 2 Several transcription factors Transcription factors 5 3 3 5 3 Additional transcription factors RNA polymerase II Transcription factors 3 5 3 5 5 RNA transcript Transcription initiation complex Chapter 17: From Gene to Protein 1. What is the “Central Dogma of Molecular Biology? 2. How is this different in prokaryotes & eukaryotes? 3. What are the stages of transcription? Elongation - Initiation - Elongation - - Bases added to 3’ end (-OH) RNA polymerase – 60 nts/sec opens DNA 10-20 bases at a time NTPs provide energy Non-template strand of DNA RNA nucleotides RNA polymerase 3 A T C C A A 3 end U 5 A U G C A T A G G T 5 T Direction of transcription (“downstream) Newly made RNA Template strand of DNA Chapter 17: From Gene to Protein 1. What is the “Central Dogma of Molecular Biology? 2. How is this different in prokaryotes & eukaryotes? 3. What are the stages of transcription? Promoter Transcription unit - Initiation DNA - Elongation Start point 5 3 - Termination - RNA polymerase 3 5 1 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. AAUAAA – stop sequence 10 – 35 bases later 5 3 3 5 Template strand Unwound RNA of DNA DNA transcript 2 Elongation. The polymerase moves downstream, unwinding the Rewound DNA and elongating the RNA transcript 5 3 . In the wake of transcription, the DNA strands re-form a double helix. RNA 5 3 3 5 3 5 RNA transcript 3 Termination. Eventually, the RNA transcript is released, and the polymerase detaches from the DNA. 5 3 3 5 5 Completed RNA transcript 3 Chapter 17: From Gene to Protein 1. 2. 3. 4. What is the “Central Dogma of Molecular Biology? How is this different in prokaryotes & eukaryotes? What are the stages of transcription? How is the mRNA altered (processed)? - 5’ cap – modified Guanine added with a methyl group (-CH3) - 3’ poly-A tail A modified guanine nucleotide added to the 5 end TRANSCRIPTION RNA PROCESSING 50 to 250 adenine nucleotides added to the 3 end DNA Pre-mRNA 5 mRNA Protein-coding segment Polyadenylation signal 3 G P P P AAUAAA AAA…AAA Ribosome TRANSLATION 5 Cap Polypeptide 5 UTR Start codon Stop codon 3 UTR Poly-A tail Chapter 17: From Gene to Protein 1. 2. 3. 4. What is the “Central Dogma of Molecular Biology? How is this different in prokaryotes & eukaryotes? What are the stages of transcription? How is the mRNA altered (processed)? - 5’ cap - 3’ poly-A tail - Splicing exons together & removing introns TRANSCRIPTION RNA PROCESSING DNA Pre-mRNA 5 Exon Intron Pre-mRNA 5 Cap 30 31 1 Coding segment mRNA Ribosome Intron Exon Exon 3 Poly-A tail 104 105 146 Introns cut out and exons spliced together TRANSLATION Polypeptide mRNA 5 Cap 1 5 UTR - Splicing is done in a spliceosome Poly-A tail 146 3 UTR Figure 17.11 The roles of snRNPs & spliceosomes in pre-mRNA splicing -snRNPs recognize short sequence in intron -Intron is removed & exons joined Chapter 17: From Gene to Protein 1. 2. 3. 4. 5. What is the “Central Dogma of Molecular Biology? How is this different in prokaryotes & eukaryotes? What are the stages of transcription? How is the mRNA altered (processed)? How is the mRNA read to go from NA language to aa language? - Triplet code – codon - 3 bases = 1 aa DNA Gene 2 molecule - Codons do not overlap Gene 1 - Reading frame - The red dog ate the cat. Gene 3 - The red cat ate the dog. - There dc ata tet hed og. DNA strand 3 A C C A A A C C G A - “The Rosetta Stone of (template) Molecular Biology” G T 5 TRANSCRIPTION mRNA 5 U G G U U U G G C U C A Codon TRANSLATION Protein Trp Amino acid Phe Gly Ser 3 Figure 17.5 The dictionary of the genetic code Second mRNA base Let’s translate: - UGG - Trp - ACC - Thr - GAA - Glu - AUG – start - UAA, UAG, UGA – stop UUU UUC U UUA C A A UAU UCU Phe UCC UCA UAC Ser U UGU Tyr UGC Cys C UCG CUU CCU CAU CUC CCC CAC CUA Leu Leu CCA Pro CAA CUG CCG CAG AUU ACU AAU ACC AAC AUC lle AUA ACA Thr AAG GUU GCU GAU GUC GCC GAC GUA GUG Val GCA GCG Ala His Gln Asn AAA AUGMet or ACG start G G UAA Stop UGA Stop A UAG Stop UGG Trp G UUG First mRNA base (5 end) - There is redundancy - Only the 1st 2 bases matter -(a.k.a. “wobble” C GAA GAG Lys U CGU CGC CGA Arg A CGG G AGU U AGC Ser C AGA A AGG Arg G U GGU Asp Glu C GGC GGA GGG Gly C A G Third mRNA base (3 end) U Chapter 17: From Gene to Protein 1. 2. 3. 4. 5. 6. What is the “Central Dogma of Molecular Biology? How is this different in prokaryotes & eukaryotes? What are the stages of transcription? How is the mRNA altered (processed)? How is the mRNA read to go from NA language to aa language? In what organelle does translation take place? - Ribosome TRANSCRIPTION DNA mRNA Ribosome TRANSLATION Polypeptide Amino acids Polypeptide tRNA with amino acid Ribosome attached Gly tRNA A AA UGGU UU GG C Codons 5 mRNA Anticodon 3 Chapter 17: From Gene to Protein 1. 2. 3. 4. 5. 6. 7. What is the “Central Dogma of Molecular Biology? How is this different in prokaryotes & eukaryotes? What are the stages of transcription? How is the mRNA altered (processed)? How is the mRNA read to go from NA language to aa language? Where does translation take place? What does tRNA do? - Transfers amino acids to protein being made 3 A Amino acid C attachment site C A 5 C G G C C G U G U A A U A U U C * AG * C A C AG UA * CUC G * C * * G U G UC* C G A GA GG U G * * GAG C G C Hydrogen U A bonds * G A * A C * U A AG Anticodon 5 3 Amino acid attachment site Hydrogen bonds A A G 3 Anticodon Wobble – 3rd base doesn’t matter Anticodon 5 Ribosomes are made of rRNA & proteins in the nucleolus (Ch 6) P site (Peptidyl-tRNA binding site) A site (AminoacyltRNA binding site) E site (Exit site) Large subunit E mRNA binding site P A 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. Chapter 17: From Gene to Protein 1. 2. 3. 4. 5. 6. 7. 8. What is the “Central Dogma of Molecular Biology? How is this different in prokaryotes & eukaryotes? What are the stages of transcription? How is the mRNA altered (processed)? How is the mRNA read to go from NA language to aa language? Where does translation take place? What does tRNA do? What are the steps of translation? - Initiation - Elongation - Termination Figure 17.17 The initiation of translation Large ribosomal subunit P site 3 U A C 5 5 A U G 3 Initiator tRNA GTP GDP E A mRNA 5 Start codon mRNA binding site 1 Small subunit mRNA Initiator tRNA with Met 5 3 Small ribosomal subunit 3 Translation initiation complex 2 Energy from GTP allows large subunit to bind Figure 17.18 The elongation cycle of translation 1 Codon recognition. The anticodon TRANSCRIPTION Amino end of polypeptide DNA mRNA Ribosome 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. TRANSLATION Polypeptide E mRNA Ribosome ready for next aminoacyl tRNA 5 3 P A site site 2 GTP 2 GDP E E P P A 2 Peptide bond formation. An GDP 3 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. A GTP E P A 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. Figure 17.19 The termination of translation Release factor Free polypeptide 5 3 3 3 5 5 Stop codon (UAG, UAA, or UGA) 1 When a ribosome reaches a stop 2 The release factor hydrolyzes codon on mRNA, the A site of the ribosome accepts a protein called a release factor instead of tRNA. 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. 3 The two ribosomal subunits and the other components of the assembly dissociate. Chapter 5 The Structure and Function of Macromolecules 9. How are all amino acids similar? - Amino group (-NH2) - Carboxyl group (-COOH) - Variable “R” group - C in the center - H carbon R O H N C C OH H H Amino group Carboxyl group Figure 5.17 The 20 amino acids of proteins CH3 CH3 H H3N+ C CH3 O H3N+ C H Glycine (Gly) O– C H3N+ C H Alanine (Ala) O– CH CH3 CH3 O C CH2 CH2 O H3N+ C H Valine (Val) CH3 CH3 O– C O H3N+ C H Leucine (Leu) H3C O– CH C O C H Isoleucine (Ile) O– Nonpolar CH3 CH2 S NH CH2 CH2 H3N+ C H CH2 O H3N+ C O– Methionine (Met) C H CH2 O H3N+ C C O– Phenylalanine (Phe) H O H2C CH2 H2N C O C O– H C O– Tryptophan (Trp) Proline (Pro) Non-polar – equal sharing of electrons in these bonds of R-groups OH OH Polar CH2 H3N+ C CH O H3N+ C O– H Serine (Ser) C CH2 O H3N+ C O– H C CH2 O C H O– H3N+ C O H3N+ C O– H C Electrically charged CH2 H3N+ C O H H3N+ NH3+ O C C O C O– H Glutamine (Gln) C CH2 CH2 CH2 CH2 CH2 C O CH2 C O– H3N+ C H C O– Lysine (Lys) NH2+ H3N+ CH2 O CH2 H3N+ C H Glutamic acid (Glu) NH+ NH2 CH2 H Aspartic acid (Asp) C H3N+ – O C CH2 C O– CH2 Basic O– O O Asparagine (Asn) Acidic –O CH2 CH2 H Tyrosine (Tyr) Cysteine (Cys) Threonine (Thr) C NH2 O C SH CH3 OH NH2 O NH CH2 O C C O– H O C O– Arginine (Arg) Histidine (His) 10. Why are these polar? (Review in Ch 2 if needed) - Electronegative atoms – O & N – attract & hold electrons - Unequal sharing of electrons in R-groups where O & N are 11. Why acidic & basic? - Acids – donate H+ to environment (H+ = proton) - Bases – accept H+ from environment Chapter 5 The Structure and Function of Macromolecules 9. How are all amino acids similar? 10. Why are these polar? 11. Why acidic & basic? 12. How are amino acids connected? Dehydration (condensation) rxn Creates a peptide bond (C—N) Figure 5.18 Making a polypeptide chain Peptide bond OH CH2 SH CH2 H N H OH C C CH2 H H N C C OH H N C H O H O H (a) C OH O DESMOSOMES H2O OH DESMOSOMES DESMOSOMES OH CH2 H H N C C H O (b) Amino end (N-terminus) Side chains (R-groups) SH Peptide CH2 bond CH2 H H N C C H O N C C H O Carboxyl end (C-terminus) OH Backbone Chapter 5 The Structure and Function of Macromolecules 9. 10. 11. 12. 13. - How are all amino acids similar? Why are these polar? Why acidic & basic? How are amino acids connected? What are the 4 levels of protein structure? 1° (Primary) – aa sequence (determined by DNA sequence) 2° (Secondary) – based on H-bonds 3° (Tertiary) – overall globular shape – 3D structure 4° (Quaternary) – several 3° polypeptides (subunits) Figure 5.20 Primary structure of a protein • Based on amino acid sequence • Each protein has a unique sequence • Like the alphabet (letters = aa) – Order is important for proper function • great • grate • retag • arteg Gly Pro Thr Gly Thr +H N 3 Amino acid subunits Gly Amino end Leu Seu Pro Cys Lys Glu Met Val Lys Val Leu Asp Ala Val Arg Gly Ser Pro Ala Glu Lle Asp Thr Lys Ser Tyr Lys Trp Leu Ala Gly lle Ser Pro Phe His Glu His Ala Glu Ala Thr Phe Val Val Asn Asp Arg Ser Gly Pro Thr Tyr Thr lle Ala Ala Arg Leu Ser Tyr Ser Tyr Pro Leu Ser Thr Ala Val Val Thr Asn Pro Lys Glu c o o– Carboxyl end Figure 5.20 Secondary structure of a protein pleated sheet Amino acid subunits H O O H R C N H C C N C N C C R O H H O C C R H N O N O C H C H O R H C R H O C N O C C R N C H H R H C C N H C C N O H H R C H N HC N H C H C O R C H H C C N O R C C N O H H R O H H N H C N C H C O R C C C O R R O H H N H C N C H C O R H C N H C N H C O H H helix R H C R H O C N O R R C O N C H C C N H H C C N O H R R O H C R N C H H Based on H-bonds • α-helix • adjacent polar aa (every 4th aa is polar) - β-pleated sheet - after folding, polar aa become neighbors and form H-bonds Hydrogen Bonds • A hydrogen bond (Review in Ch 2 if needed) – Occurs between 2 molecules with polar covalent bonds – Forms when a hydrogen atom bonded to one electronegative atom is also attracted to another electronegative atom – + H Water (H2O) O H + – Ammonia (NH3) N H + Figure 2.15 H H + + A hydrogen bond results from the attraction between the partial positive charge on the hydrogen atom of water and the partial negative charge on the nitrogen atom of ammonia. Figure 5.20 Tertiary structure • overall globular shape – 3D structure • each protein has unique 3D shape • based on 1° structure (aa sequence) • Disulfide bridge--2 cysteine amino acids -(a type of covalent bond) • hydrophobic interactions • ionic bonds • occasional H-bonds CH CH2 Hydrogen bond H3C CH3 H3C CH3 CH O H O Hydrophobic interactions and van der Waals interactions OH C CH2 CH2 S S CH2 Disulfide bridge O CH2 NH3+ -O C CH2 Ionic bond Polypeptide backbone Figure 5.20 Quaternary structure • more than one 3° polypeptide (subunit) needed for biological activity • not all proteins have quaternary structure • # of subunits varies by protein Polypeptide chain Collagen Chains Iron Heme Chains Hemoglobin Chapter 5 The Structure and Function of Macromolecules 13. What are the 4 levels of protein structure? 14. How does structure (sequence) influence function? - Sickle cell anemia Figure 5.21 A single amino acid substitution in a protein causes sickle-cell disease Primary structure Normal hemoglobin Val His Leu Glu Glu Sickle-cell hemoglobin Val His Leu Molecules do not associate with one another; each carries oxygen Normal cells are full of individual hemoglobin molecules, each carrying oxygen Thr Pro Val Glu ... structure 1 2 3 4 5 6 7 Secondary subunit and tertiary structures Quaternary Hemoglobin A structure Red blood cell shape Pro 1 2 3 4 5 6 7 Secondary and tertiary structures Function Thr . . . Primary Quaternary structure subunit Function 10 m 10 m Red blood cell shape Hemoglobin S Molecules interact with one another to crystallize into a fiber, capacity to carry oxygen is greatly reduced Fibers of abnormal hemoglobin deform cell into sickle shape Chapter 5 The Structure and Function of Macromolecules 13. What are the 4 levels of protein structure? 14. How does structure (sequence) influence function? 15. What are polyribosomes? Figure 17.20 Polyribosomes Completed polypeptide Growing polypeptides Incoming ribosomal subunits Start of mRNA (5 end) 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 17.22 Coupled transcription and translation in bacteria RNA polymerase DNA mRNA Polyribosome RNA polymerase Direction of transcription 0.25 m DNA Polyribosome Polypeptide (amino end) Ribosome mRNA (5 end) Chapter 17: From Gene to Protein 13. What are the 4 levels of protein structure? 14. How does structure (sequence) influence function? 15. What are polyribosomes? 16. How is rough ER made? - SRP - signal recognition particle - Binds to signal peptide (1st couple amino acids) Figure 17.21 The signal mechanism for targeting proteins to the ER 1 Polypeptide synthesis begins on a free ribosome in the cytosol. 2 An SRP binds to the signal peptide, halting synthesis momentarily. 3 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. 4 The SRP leaves, and the polypeptide resumes growing, meanwhile translocating across the membrane. (The signal peptide stays attached to the membrane.) 5 The signalcleaving enzyme cuts off the signal peptide. 6 The rest of the completed polypeptide leaves the ribosome and folds into its final conformation. Ribosome mRNA Signal peptide Signalrecognition particle (SRP) SRP receptor CYTOSOL protein Signal peptide removed Translocation complex Which proteins are made in rough ER? - Secreted from cell or stay outside of cell - Embedded in plasma membrane ER membrane Protein Chapter 17: From Gene to Protein 13. What are the 4 levels of protein structure? 14. How does structure (sequence) influence function? 15. What are polyribosomes? 16. How is rough ER made? 17. How do mutations alter the genotype & phenotype of organisms? - Mutation – any change in the genetic material of a cell (heritable) - Point mutation – a change in 1 or only a few bases - Base-pair substitution - Insertions or deletions result in a frameshift Figure 17.24 Base-pair substitution Wild type mRNA A U G A A G U U U G G C U A A 5 Protein Met Lys 3 Phe Amino end Gly Stop Carboxyl end Base-pair substitution No effect on amino acid sequence U instead of C Silent mutation - No change in aa A U G A A G U U U G G U U A A Met Lys Missense Phe Gly Stop A instead of G A U G A A G U U U A G U U A A Met Lys Phe Ser Stop Nonsense U instead of A A U G U A G U U U G G C U A A Met Stop Missense – still translates but a mistake is made Nonsense – stop codon created NO complete protein Figure 17.25 Base-pair insertion or deletion Wild type mRNA 5 Protein A UG A A GU U U GG C U A A Met Lys Gly Phe 3 Stop Amino end Carboxyl end Base-pair insertion or deletion Frameshift causing immediate nonsense Extra U A U GU A A G U U U GG C U A Met Stop Frameshift causing extensive missense U Missing A U G A A G U U G G C U A A Met Lys Leu Ala Insertion or deletion of 3 nucleotides: no frameshift but extra or missing amino acid A A G Missing A U G U U UG G C U A A Met Phe Gly Stop Frameshift – reading frame is shifted creating new codons Figure 17.26 A summary of transcription and translation in a eukaryotic cell DNA TRANSCRIPTION 1 RNA is transcribed from a DNA template. 3 5 RNA transcript RNA polymerase RNA PROCESSING Exon 2 In eukaryotes, the RNA transcript (premRNA) is spliced and modified to produce mRNA, which moves from the nucleus to the cytoplasm. RNA transcript (pre-mRNA) Intron Aminoacyl-tRNA synthetase NUCLEUS Amino acid tRNA FORMATION OF INITIATION COMPLEX CYTOPLASM 3 After leaving the nucleus, mRNA attaches to the ribosome. mRNA AMINO ACID ACTIVATION 4 Each amino acid attaches to its proper tRNA with the help of a specific enzyme and ATP. Growing polypeptide Activated amino acid Ribosomal subunits 5 TRANSLATION A succession of tRNAs add their amino acids to the polypeptide chain Anticodon as the mRNA is moved through the ribosome one codon at a time. (When completed, the polypeptide is released from the ribosome.) 5 E A AAA UG GU U U A U G Codon Ribosome