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Chapter 17: From Gene to Protein

Chapter 17: From Gene to Protein. Chapter 16 answered the question “What is the genetic material?” Chapter 17 answers the question “How do genes give us our traits?”. Chapter 17: From Gene to Protein. What is the “Central Dogma of Molecular Biology? DNA RNA Protein

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Chapter 17: From Gene to Protein

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  1. Chapter 17: From Gene to Protein Chapter 16 answered the question “What is the genetic material?” Chapter 17 answers the question “How do genes give us our traits?”

  2. Chapter 17: From Gene to Protein • What is the “Central Dogma of Molecular Biology? • DNA RNA Protein • 2. How is this different in prokaryotes & eukaryotes? Transcription Translation

  3. 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

  4. Chapter 17: From Gene to Protein • What is the “Central Dogma of Molecular Biology? • How is this different in prokaryotes & eukaryotes? • What are the stages of transcription? • Initiation • Elongation • Termination

  5. Eukaryotic promoters 1 2 3 DNA TRANSCRIPTION RNA PROCESSING Pre-mRNA mRNA Ribosome TRANSLATION Polypeptide Promoter 5 3 A T A T A A A A T A T T T T 3 5 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 Chapter 17: From Gene to Protein • What is the “Central Dogma of Molecular Biology? • How is this different in prokaryotes & eukaryotes? • What are the stages of transcription? • Initiation • At promoter – promotes transcription • Transcription factors bind to TATA box • RNA polymerase makes RNA

  6. Non-template strand of DNA Elongation RNA nucleotides RNA polymerase T A C C A T A C T 3 U 3 end T G A U G G A C C G U A C A 5 A A T A G G T T Direction of transcription (“downstream) 5 Template strand of DNA Newly made RNA Chapter 17: From Gene to Protein • What is the “Central Dogma of Molecular Biology? • How is this different in prokaryotes & eukaryotes? • What are the stages of transcription? • Initiation • Elongation • Bases added to 3’ end • RNA polymerase – 60 nts/sec • opens DNA 10-20 bases at • a time

  7. 3 1 2 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 Chapter 17: From Gene to Protein • What is the “Central Dogma of Molecular Biology? • How is this different in prokaryotes & eukaryotes? • What are the stages of transcription? • Initiation • Elongation • Termination • AAUAAA – stop sequence • 10 – 35 bases later

  8. 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 Chapter 17: From Gene to Protein • 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 • 3’ poly-A tail

  9. Intron Exon 5 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 5 UTR Chapter 17: From Gene to Protein • 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

  10. Chapter 17: From Gene to Protein 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 • 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 • Codons do not overlap • Reading frame • The red dog ate the cat. • The red cat ate the dog. • There dc ata tet hed og. • “The Rosetta Stone of • Molecular Biology”

  11. 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 • Let’s translate: • UGG • ACC • GAA • There is redundancy • Only the 1st 2 bases matter • AUG – start • UAA, UAG, UGA – stop • The overview of translation • Trp • Thr • Glu Glu

  12. Chapter 17: From Gene to Protein 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 Codons 5 3 mRNA • 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?

  13. Chapter 17: From Gene to Protein 3 A Amino acid attachment site C Amino acid attachment site C 5 5 A 3 C G C G C G Hydrogen bonds 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 G A * A C 3 5 * U Anticodon A Anticodon G A Anticodon • 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 Wobble – 3rd base doesn’t matter

  14. 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. Ribosomes are made of rRNA & proteins in the nucleolus (Ch 6)

  15. Chapter 17: From Gene to Protein • 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

  16. 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 1 2 Figure 17.17 The initiation of translation Small subunit mRNA Initiator tRNA with Met Energy from GTP allows large subunit to bind

  17. 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

  18. 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

  19. Chapter 17: From Gene to Protein • 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? • What are polyribosomes?

  20. 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

  21. 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

  22. Upon further review…… • Test – Tuesday November 29 • Essay questions & Ch 16 & 17 ppts are available

  23. Chapter 17: From Gene to Protein • 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? • What are polyribosomes? • How are proteins targeted to the ER? • SRP • signal recognition particle • Binds to signal peptide (1st couple amino acids)

  24. 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.

  25. Chapter 17: From Gene to Protein • 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? • What are polyribosomes? • How are proteins targeted to the ER? • Let’s consider mutations…… • - Sickle cell disease

  26. 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

  27. Chapter 17: From Gene to Protein • 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? • What are polyribosomes? • How are proteins targeted to the ER? • Let’s consider mutations…… • 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

  28. 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

  29. 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

  30. 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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