Protein Metabolism (Chap 27) Translation: nucleic acid language amino acid language The Genetic Code is the basis for this translation: aminoacyl-trna synthetase xxx xxx xxx trna + amino acid xxx amino acyl xxx- trna trna anticodon is critical!!! Ribosomes = giant protein synthesis enzymes While early life forms may have relied on RNA molecules to perform most of the jobs necessary to maintain life, that clearly is not the current scheme for stayin alive (cue Bee Gees). 1
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I. The Genetic Code A. The Genetic Code was Cracked Using Artificial mrna Templates 1. Basic coding thoughts a) How many RNA letters per word are necessary to code for 20 amino acid words? 1codes for 2 codes for 3 codes for b) overlapping vs. non-overlapping coding system c) Crick s adaptor hypothesis (see fig. 27-2) & reading frame 2. Polynucleotide phosphorylase used to make RNAs a) simple for homopolymers b) not simple for others 3. Ribosome binding assays (Table 27-2) and cell free protein synthesis systems (Table 27-1) were developed to decode the polymers made in #2, above. (see next page). 3
4. The Genetic Code (Figure 27-7) a) initiation codon b) elongation(?) coding codons c) termination codons (orfs) d) the code is degenerate (Table 27-3) Aside thoughts on evolution of the code? e) is nearly invariant (see Box 27-1) (Consistent with common ancestry!) B. Wobble Allows Some trnas to Recognize More than One Codon st 1. The 1 two codon letters are the primary determinants of the read (See Fig. 27-8 a) 2. The 5' position of the anticodon sometimes wobbles 3. One way to manage the H Bonding aspect of this is with unusual bases like inosinate (see Fig 27-8 b) 4. Crick s Wobble Hypothesis: a) The first (from 5' end) bases (#1 & 2 in Fig 27-8) of the codon always form strong (traditional) W-C base pairs with the corresponding bases of the anticodon. b) Base #1 of the anticodon determines how many codons will be recognized by this trna. i) If anticodon base #1 is C or A, H Bonding is specific, and only one codon is recognized by that trna. ii) If anticodon base #1 is U or G, H Bonding is less specific, and two codons are recognized by that trna. 4
iii) If anticodon base #1 is I, H Bonding is even less specific, and three codons are recognized by that trna. This is the most possible. c) When an amino acid is heavily degenerate (has a large number of different codons), different trnas are required for codons that differ in the first to codon positions. d) This results in: i) 32 trnas are necessary to manage all 61 of the coding codons ii) 1 for initiation and 31 for elongation e) Summary in Table 27-4 C. Translational Frameshifting and RNA Editing Affect How the Code is Read 1. Translational frameshifting occurs in translation of the gag and pol genes of the Rous sarcoma virus. (see Chapt 26) a) Same mrna is used to make both the gag and pol proteins. b) pol reading frame is offset by -1 relative to gag read. 2. Guide RNAs manage specific editing of mrnas in some mitochondrial and chloroplast expressions 5
II. Protein Synthesis A. Summary: 5 stages 1. Activation of amino acids. Linkage of aa to correct trna. 2. Initiation. Formation of ternary complex. 3. Elongation. Successive addition of correct amino acids to nascent polypeptide chain. 4. Termination & ribosome recycling. 5. Protein folding and posttanslational modification. B. The Ribosome is a Complex Supramolecular machine 1. Components (prokaryotic) a) 50S subunit i) 23S and 5S rrnas ii) 33 different proteins (36 protein molecules) b) 30 S subunit i) 16S rrna ii) 21 different proteins (one molecule of each) 2. Combine w/ initiator trna, mrna, & factors (that bind and dissociate) to form functional unit (80S ribosome) Aside on Dr. M. Nomura 6
Aside on Box 27-2 RNA world to Protein World 1. Rxns catalyzed by extant ribozymes 2. Old thoughts on ribosomes function protein vs. rrna 3. New understanding of ribosome function 4. The transition??? C. Transfer RNAs have Characteristic Structural Features 1. 2-D structure (Figs. 27-16 to 17 2. 3-D structure of trnas D. Stage 1: trna Synthetases Attach the Correct Amino Acids to Their trnas 1. Basic rxn. x aa + trna + ATP aminoacyl-trna + AMP + PP x i 2. Two classes of trna sythetases (origin?) 3. Proofreading by aa-trna synthetases a) Two levels b) Edit requirement determined by? 7
4. Second genetic code: How do the aminoacyl trna synthetases recognize their trna substrates? (Fig. 27-21-3) E. Stage 2: Specific aa initiates protein synthesis (Shine-Delgarno) F. Stage 3: Peptide bond formation 1. Incoming aa-trna 2. Peptide bond formation 3. Translocation 4. Proofreading on the ribosome G. Stage 4: Termination 1. Factors (see Fig. 27-31 2. Energy cost > 4 XTP equiv. (122 kj/mol) 3. Polysomes H. Stage 5: Folding and processing 1. Terminal modification 2. Loss of signal sequences 3. Modification of aa side chains 4. Glycosylation 5. Attachment of lipid 6. Other prosthetic groups added 8
7. Proteolytic processing 8. Disulfide link oxidation I. Protein synthesis inhibitors as antibiotics Look at puromycin mechanism in Fig. 27-36 III. Protein targeting and degradation A. Significance of the signal sequence B. Posttranslational modification for manu eukaryotic proteins begins at the rough ER 9
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C. (Organelle specific) Glycosylation plays a role in targeting (re. vesicular sorting, etc.) 11
D. Golgi apparatus and sorting E. Sequences for nuclear transport are not cleaved F. Bacteria also do some targeting. What would they need to target? G. Review some signal sequences (Fig. 27-43) 12
H. Receptor mediated endocytosis (examples?) 13
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I. Degradation and ubiquitin 15
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3. Eukaryotic mrna processing summary (ovalbumin gene as example) 17
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G. rrnas and trnas also undergo processing (eukaryotes, prokaryotes, and archae) 1. rrnas (see Fig. 26-24 re. bacteria, Fig. 26-25 re. us, vertebrates) 19
III. Protein Targeting and Degradation 20