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From Gene to Phenotype- part 3
TRANSCRIPTION RNA is transcribed from a DNA template. DNA RNA polymerase transcript RNA PROCESSING In eukaryotes, the RNA transcript (pre- mRNA) is spliced and modified to produce mRNA, which moves from the nucleus to the cytoplasm. Exon Poly-A RNA transcript (pre-mRNA) Intron NUCLEUS Cap FORMATION OF INITIATION COMPLEX After leaving the nucleus, mRNA attaches to the ribosome. CYTOPLASM mRNA Growing polypeptide Ribosomal subunits Aminoacyl-tRNA synthetase Amino acid tRNA AMINO ACID ACTIVATION Each amino acid attaches to its proper tRNA with the help of a specific enzyme and ATP. Activated amino acid TRANSLATION 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.) Anticodon A C U G E Ribosome 1 5 3 Codon DNA 2 mRNA 3 4 polypeptide 5
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Lecture Outline 11/9/05 Review translation:
Initiation, elongation, termination EPA model Post-translational modification of polypeptides Signal sequences Mutations (again) Exam 3 is next Monday. It will cover mitosis and meiosis, DNA synthesis, transcription, translation, genetics of viruses. (chapters 12, 13, 16, 17, part of 18 (to page 345))
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Translation: overview
TRANSCRIPTION TRANSLATION DNA mRNA Ribosome Polypeptide Amino acids tRNA with amino acid attached tRNA Anticodon Trp Phe Gly A G C U Codons 5 3 tRNA serves as an “adaptor” that brings the correct amino acid to each codon. The ribosome is the machine that builds the polypeptide
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The genetic code Second mRNA base Third mRNA base (3 end)
5 C G G C C G U G Second mRNA base U C A G UUU UUC UUA UUG CUU CUC CUA CUG AUU AUC AUA AUG GUU GUC GUA GUG Met or start Phe Leu lle Val UCU UCC UCA UCG CCU CCC CCA CCG ACU ACC ACA ACG GCU GCC GCA GCG Ser Pro Thr Ala UAU UAC UGU UGC Tyr Cys CAU CAC CAA CAG CGU CGC CGA CGG AAU AAC AAA AAG AGU AGC AGA AGG GAU GAC GAA GAG GGU GGC GGA GGG UGG UAA UAG Stop UGA Trp His Gln Asn Lys Asp Arg Gly First mRNA base (5 end) Third mRNA base (3 end) Glu U A A U U A U * C C A C A G U G A A C U C * G * * C G U G U * C G A G G * * U C * A G G * G A G C Hydrogen bonds G C U A * G * A A C * U A A G 5’-AUGCAAUUCGGAAAC Codon in the mRNA
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An aminoacyl-tRNA synthetase joins a specific amino acid to a tRNA
synthetase (enzyme) Active site binds the amino acid and ATP. 1 P P P Adenosine ATP Each tRNA has a slightly different shape Pyrophosphate P Adenosine P Pi Pi Pi tRNA Appropriate tRNA bonds to amino Acid, displacing AMP. 3 P Adenosine AMP Activated amino acid is released by the enzyme. 4
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How does the ribosome find AUG?
Prokaryotes have a special binding sequence upstream of the start codon. In Eukaryotes,the ribosome binds to the 5’ cap and “scans” the message for an AUG.
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See the Animation
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Inhibition of protein synthesis
NOTE: Prokaryotes (this generally includes protein synthesis in mitochondria and chloroplasts)
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Only the anticodon of tRNA determines which amino acid is added by a ribosome.
Experimental evidence: Convert cystein to alanine chemically, after it is attached to tRNA (remove SH group) Alanine shows up in Cystein sites
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The amino acid carried by a tRNA is independent of the anticodon sequence
Determined by the amino-acyl tRNA synthetase enzyme tRNA with mutations in the anticodon still have their normal amino acid at the 3’ end. Experiment:. mutate anticodon of tRNAthr (AGU-->AGG) Now binds to proline codon instead (CCU). Those tRNA still carry threonine, but now bind to proline sites. Threonine inserted into polypeptide where proline normally goes.
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Alananine tRNA synthetase
Aminoacyl tRNA synthetase enzyme is specific to a particular amino acid and a particular tRNA Alananine tRNA synthetase Glycine doesn’t fit . .
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Quality control Both cap and tail bind to initiation factors to start translation Ensures that mRNA is intact Small subunit can detect mis-paired tRNA and remove them Needs a short delay before peptide bond is formed (to give time for proofreading) Error rate: about 10-4
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Cost of protein biosynthesis
Synthesis of aminoacyl tRNAs 2 ATPs Formation of 1 peptide bond 2 GTPs 1 for codon recognition; 1 for translocation Proofreading 1 ATP/error Construction of a specific amino acid sequence is much more costly than formation of a random peptide bond!
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Transcription and translation can occur simultaneously
RNA polymerase DNA mRNA Polyribosome Direction of transcription 0.25 m RNA polymerase DNA Polypeptide Ribosome mRNA (5 end)
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Post translational modifications and sorting
Glycosylation Signal directs protein to the right compartment
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The signal mechanism for targeting proteins to the ER
Signal peptide removed Translation begins in the cytosol Attaches to translocation pore in ER membrane Polypeptide synthesized into the ER Folds to final shape 1 SRP binds to the signal peptide, 2 3 4 5 6 Ribosome mRNA Signal peptide ER membrane Signal peptide removed Signal- recognition particle (SRP) Protein SRP receptor protein CYTOSOL Translocation complex
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Destined for cytosol or other organelles Destined for ER
Signal peptide determines where it goes Destined for cytosol or other organelles Destined for ER Imported during translation Imported after translation Stays within the membrane system Brooker Figure 13.22
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Chaperones help fold proteins
Hsp 70 covers exposed hydrophobic patches until the protein can fold Hsp60 is like an isolation chamber
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Mis-folded proteins are marked for destruction with ubiquitin
Ubiquitin tail Proteosome acts as garbage disposal
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Mutations (again)
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The molecular basis of sickle-cell disease: a point mutation
In the DNA, the mutant template strand has an A where the wild-type template has a T. The mutant mRNA has a U instead of an A in one codon. The mutant (sickle-cell) hemoglobin has a valine (Val) instead of a glutamic acid (Glu). Mutant hemoglobin DNA Wild-type hemoglobin DNA mRNA Normal hemoglobin Sickle-cell hemoglobin Glu Val C T A G U 3 5
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Base-pair substitution
Wild type A U G C mRNA 5 Protein Met Lys Phe Gly Stop Carboxyl end Amino end 3 Base-pair substitution No effect on amino acid sequence U instead of C Ser Missense A instead of G Nonsense U instead of A
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Base-pair insertion or deletion
mRNA Protein Wild type A U G C 5 Met Lys Phe Gly Amino end Carboxyl end Stop Base-pair insertion or deletion Frameshift causing immediate nonsense Leu Ala Missing Extra U Frameshift causing extensive missense Insertion or deletion of 3 nucleotides: no frameshift but extra or missing amino acid 3
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Mutations in the 3rd position of a codon are often silent
Second mRNA base U C A G UUU UCU UAU UGU U For amino acids that have only two codons, the 3rd base will either both be purines or both be pyrimidines Phe Tyr Cys UUC UCC UAC UGC C U Ser UUA UCA UAA Stop UGA Stop A Leu UUG UCG UAG Stop UGG Trp G CUU CCU CAU CGU U His CUC CCC CAC CGC C C Leu Pro Arg CUA CCA CAA CGA A Gln CUG CCG CAG CGG Third mRNA base (3 end) G First mRNA base (5 end) AUU ACU AAU AGU U Asn Ser AUC lle ACC AAC AGC C A Thr AUA ACA AAA AGA A Lys Met or start Arg AUG ACG AAG AGG G GUU GCU GAU GGU U Asp GUC GCC GAC GGC C G Val Ala Gly GUA GCA GAA GGA A Glu GUG GCG GAG GGG G
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Wobble in 3rd position
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