Fundamentals of Genetic Expression Translation and Genetic Code PDF

Translation & Genetic Code [1] TRANSLATION AND GENETIC CODE Paul J. McDermott, Ph.D. Office: (843) 792-3462 Email: [email protected] A. BASIC TRANSLATION MACHINERY 1. Ribosomes 2. mRNA 3. tRNA B. AMINOACYL-tRNA SYNTHETASES 1. Functions 2. Specificity 3. Proofreading 4. Aminoacyl-tRNA Synthetase: 2...

Translation & Genetic Code [1] TRANSLATION AND GENETIC CODE Paul J. McDermott, Ph.D. Office: (843) 792-3462 Email: [email protected] A. BASIC TRANSLATION MACHINERY 1. Ribosomes 2. mRNA 3. tRNA B. AMINOACYL-tRNA SYNTHETASES 1. Functions 2. Specificity 3. Proofreading 4. Aminoacyl-tRNA Synthetase: 2 step reaction C. GENETIC CODE 1. Features 2. Codon-Anticodon Base Pairing 3. Inosine Base Pairing D. STAGES OF TRANSLATION 1. Overview 2. Prokaryotic Translation 3. Initiation Stage in Prokaryotes 4. Initiation Stage in Eukaryotes 5. Differences in Translation Initiation between Prokaryotes and Eukaryotes 6. Elongation Stage 7. Characteristics of Polypeptide Chain Elongation 8. Protein Chaperones 9. Termination Stage E. EXAMPLES OF ANTIBIOTICS THAT TARGET TRANSLATION F. REGULATION OF GENE EXPRESSION BY miRNA 1. Components of miRNA Synthesis 2. Mechanism of Action G. PROCESSING BODIES (P-BODIES) Suggested Reading: Marks’ Basic Medical Biochemistry: A Clinical Approach, 5th Ed., Ch. 15 “Comparing the genetic code of E. coli to that of Xenopus and hamster, we found that the code is essentially universal. These results had a profound philosophical impact on me because they indicate that all forms of life on this planet use essentially the same language. Some dialects have been reported subsequently in some organisms, but all are modifications of the same genetic code” Marshall Nirenberg Translation & Genetic Code [2] OBJECTIVES 1. Specify the RNA and protein components of ribosome subunits. 2. Compare and contrast the main structural components of prokaryotic versus eukaryotic mRNAs. 3. Describe secondary and tertiary structure of the tRNA molecule and pinpoint the anticodon region and the amino acid attachment site. 4. Describe a "charged” tRNA molecule and how it is generated. 5. Describe the function of aminoacyl-tRNA synthetases in implementing the genetic code. 6. Explain why the genetic code is "degenerate". 7. Describe codon-anticodon base pairing and indicate the "wobble" bases. 8. Using the genetic code, translate a sequence of mRNA. Example: 5´-AUGCAAGCAAUCCAUUUCAUGUAA-3´ 9. Explain why inosine is a frequent base modification in the anticodon loop of tRNA. 10. Define the Shine-Dalgarno sequence and describe its role in translational initiation. 11. Compare the initiation step of translation in prokaryotes versus eukaryotes. Describe how the mechanisms for recognition of the AUG start codon differ. 12. Specify the roles of the following elongation factors: EF-Tu, EF-Ts, EF-G during the elongation phase of translation. Specify their counterparts in eukaryotes. 13. Specify two steps in the elongation phase of translation that require GTP hydrolysis. 14. Name the enzyme that catalyzes peptide bond formation. 15. Describe how proofreading occurs during the elongation step of elongation. 16. Explain why mRNA is read in the 5´ to 3´ direction and why proteins are synthesized from the amino terminus to the carboxy terminus. 17. Define polyribosomes and explain how they improve the efficiency of translation. 18. Describe the functions of protein chaperones. 19. Describe the mechanism by which translation is terminated. 20. Describe how miRNAs are generated and the mechanisms by which miRNAs regulate gene expression. Illustrations adapted from: • The Cell: A Molecular Approach © 2000 ASM Press and Sinauer Associates, Inc. • Marks Basic Medical Biochemistry: A Clinical Approach, © 2012 Lippincott Williams & Wilkins Translation & Genetic Code [3] A. BASIC TRANSLATION MACHINERY 1. Ribosomes a) Structure: Ribosomes are ribonucleoprotein complexes that are classified as non-membranous organelles. Ribosome Subunits 23S rRNA and 5S rRNA 34 r-Proteins 70S Prokaryotic Components 16S rRNA 21 r-Proteins 28S rRNA, 5.8S rRNA and 5S rRNA 50 r-Proteins 80S Eukaryotic 18S rRNA 33 r-Proteins b) Functions of Ribosomes • Ribosomes are the site of protein synthesis. • rRNA has enzyme activity (Peptidyl Transferase) to catalyze peptide bond formation. • r-Proteins form ribosome structure with rRNA (ribonucleoprotein complexes). 2. mRNA a) Prokaryotic Start Codon Fig Start Stop Codon Codon Stop Codon 5´- -3´ 5´-UTR Xter(5) Start Stop Codon Codon Protein 1 Protein 2 Protein 3 • 5´-Untranslated Region (5´-UTR) • Shine-Dalgarno sequences upstream of AUG start codons • Start Codon [AUG] • Protein Coding Sequences (polycistronic) • Stop Codon [UAA, UAG, or UGA] 3´-UTR Translation & Genetic Code [4] A. BASIC TRANSLATION MACHINERY 2. mRNA b) Eukaryotic Start Codon PolyA Sequence Stop Codon m7Gppp- -AAA(A)n Protein Coding Sequence 5´-UTR 3´-UTR • 5´ Cap [m7Gppp-] • 5´-Untranslated Region (5´-UTR) • Start Codon [AUG] in the Kozak consensus sequence: 5´ • Protein Coding Sequence (monocistronic) • Stop Codon [UAA, UAG, or UGA] • 3´-Untranslated Region (3´-UTR) • Polyadenylation Sequence [AAUAAA] • PolyA Tail 3. tRNA a) Secondary Structure 3´ -3 +1 +4 A G CCAUGG b) Tertiary Structure AA attachment site T Loop 5´ P T Stem Acceptor Stem 5´ Figs(2) CC A D Loop T Stem T Loop D Loop D Stem Anticodon Stem Anticodon Stem Anticodon Loop Anticodon Anticodon Anticodon Loop c) Processing of tRNA • Cleavage of tRNA precursor molecule by Ribonuclease (RNase) • Addition of CCA to the 3´end of tRNA to create AA attachment site in acceptor stem • Modifications of bases (7-15 bases in each tRNA) e.g. 2-Methylguanosine 5-Methylcytosine Dihydrouridine 3´ AA attachment site Acceptor Stem D Stem 3´ Inosine (deaminated Adenosine) Ribothymidine Pseudouridine Translation & Genetic Code [5] A. BASIC TRANSLATION MACHINERY 3. tRNA d) Functions of tRNA • Adapter molecule: binds to mRNA codon and carries the specified amino acid. • Anticodon Stem/Loop: translates genetic code of mRNA. • Attachment site (CCA-3´): terminal A of tRNA is ”charged” with AA via aminoacyl linkage. B. AMINOACYL-tRNA SYNTHETASES 1. Functions • Implementation of genetic code- attach each AA to its correct tRNA molecule. 3´end of tRNA • Catalyze formation of aminoacyl linkage on terminal 3´A of each tRNA with correct AA. 2. Specificity • At least one Aminoacyl-tRNA Synthetase is expressed for each of the 20 AA. • Enzyme must recognize both its cognate tRNA and the correct AA. 3. Proofreading: High fidelity (1 error/104 AA) • Recognition of correct AA occurs in the active site before an aminoacyl linkage with tRNA is catalyzed. Aminoacyl linkage • Incorrect aminoacyl linkages are removed by editing function. 4. Aminoacyl-tRNA Synthetase: 2 step reaction mechanism Step 1: AA activation by formation of an aminoacyl-AMP intermediate Step 1 Aminoacyl-AMP intermediate PPi Step 2: Charging of tRNA by transfer of AA from aminoacyl-AMP intermediate to terminal 3´A of tRNA to generate aminoacyl-tRNA -O- + Aminoacyl-AMP Step 2 AMP Aminoacyl-tRNA Translation & Genetic Code [6] C. GENETIC CODE 1. Features a) Number of Codons: 64 triplet Codons [43 = 64] b) Codon Utilization: All possible codons are used. • 20 Amino Acids • 3 Stop Codons [UAA, UGA, UAG] • 1 Start Codon [AUG] c) Degeneracy of Genetic Code: More than 1 codon can be used for the same AA. 5´-AUG UUU AAG CCA GUA UGA-3´ 5´-AUG UUC AAG CCA GUA UGA-3´ 5´-AUG UUU AGG CCA GUA UGA-3´ 5´-AUG UUU UAG CCA GUA UGA-3´ 2. Codon-Anticodon Base Pairing Complementary Watson-Crick base pairing occurs between the codon in mRNA and anticodon in tRNA. 3´ 5´ a) mRNA: Codons are read in 5´ to 3´ direction. Anticodon loop of Met-tRNA b) tRNA: Anticodons are read in 3´ to 5´ direction. UAC AUG mRNA 5´ 3´ c) “Wobble”: Third base of codon exhibits uncommon base pairings with the first base of the anticodon, allowing for degeneracy of genetic code. d) Allowed Wobble Pairings Anticodon 1st base Codon 3rd base C G A U U A or G G U or C I U, C or A 5´ 3´ Anticodon loop of Leu-tRNA GAU mRNA 5´ CUA G 1 Leu-tRNA can read 2 Leu codons. 3´ Translation & Genetic Code [7] C. GENETIC CODE 3. Inosine Base Pairing a) Deamination of Adenosine in 1st Base of tRNA Anticodon Deaminase Inosine Nota Bene: Inosine maximizes the number of codons that a tRNA molecule can read by forming non-standard base pairings. b) Cytidine : Inosine c) Uridine : Inosine D. STAGES OF TRANSLATION 1. Overview d) Adenosine : Inosine You don’t have to memorize these base pairings. • Translation is mRNA-directed polypeptide synthesis that occurs in 3 stages: initiation, elongation and termination. Each stage is regulated by a corresponding set of translation factors called Initiation Factors, Elongation Factors and Termination (Release) Factors. • Initiation stage of translation differs somewhat in prokaryotic versus eukaryotic organisms. • Elongation and termination stages are basically the same in prokaryotes and eukaryotes except that some of the elongation factors are named differently. 2. Prokaryotic Translation • In prokaryotes (bacteria), mRNA is neither processed or spliced. • Bacteria do not have a nuclear envelope to separate the nucleus from the cytoplasm. • Transcription and translation occur simultaneously. Bacterial chromosome Translation & Genetic Code [8] D. STAGES OF TRANSLATION 3. Initiation Stage in Prokaryotes a) Role of Initiation Factors (IF) in Prokaryotes Step 1: Binding of IF-1 and IF-3 to the 30S ribosomal subunit Step 2: Binding of IF-2•GTP fMet-tRNAf mRNA 30S 5´ 3´ (fMet = N´-formylmethionine) 5´ 3´ Step 3: Release of IF-1 and IF-3 50S Step 4: Binding of the 50S subunit Step 5: Hydrolysis of GTP Step 6: Release of IF-2•GDP 70S Initiation Complex 50S 5´ 3´ 30S b) Role of the Shine-Dalgarno Sequence • The Shine-Dalgarno sequence in prokaryotic mRNAs is located just upstream of the AUG start codon. It functions as a “marker” for recognition of the AUG start codon. • The 30S ribosome binds to mRNA in the vicinity of the AUG start codon by complementary base pairing between 16S rRNA and the Shine-Dalgarno sequence. • Multiple Shine-Dalgarno sequences in polycistronic mRNA enables initiation of translation at internal AUG start codons. Prokaryotic mRNA 5´- -3´ 30S Shine-Dalgarno Sequence 5´- Start Codon Prokaryotic mRNA -3´ AGGAGGUUUGACCUAUG UCCUCCA 3´- Complementary base pairing 16S rRNA (part of the 30S Ribosome) -5´ Translation & Genetic Code [9] D. STAGES OF TRANSLATION 4. Initiation Stage in Eukaryotes Met-tRNAi Step 1: Ternary Complex Formation of the ternary complex [Met-tRNAi-eIF-2•GTP] Step 2: 40S Binding of the ternary complex and other eukaryotic Initiation Factors [eIF-1, eIF-1A, eIF-3, eIF-5] to the 40S ribosomal subunit to form the pre-initiation complex Pre-initiation Complex eIF-5 Step 3: eIF-4F facilitates binding of pre-initiation complex to the m7Gppp cap on the 5´-end of mRNA Subunits of eIF-4F • 4E - Binds to m7Gppp cap • 4G -Binds to eIF-3 • 4A - RNA helicase 4A 4G Step 4: 4E 3 1A 1 ATP mRNA m7Gppp- AUG Note the AUG start codon in the Kozak sequence -3 A G +4 AUG G -AAAA... eIF-5 Recognition of the AUG initiation codon by linear scanning (5´ to 3´) of pre-initiation complex along the 5´-UTR of mRNA ATP 4A 4G m74E Gppp- 3 1A Step 5: 1 When the AUG start codon is recognized, eIF-5 triggers hydrolysis of eIF-2•GTP. This causes release of eIF-2•GDP and other eIFs, which then enables binding of the 60S ribosomal subunit to form the 80S initiation complex. eIF-2•GDP, other eIFs A AUG G G eIF-5 60S Met m7Gppp- -AAAA... 80S Initiation Complex -AAA... -AAA… Translation & Genetic Code [10] D. STAGES OF TRANSLATION 5. Differences in Translation Initiation Between Prokaryotes and Eukaryotes Prokaryotes Eukaryotes • Formation of ternary complex • Binding of IFs to 30S ribosome • Binding of ternary complex & Binding of mRNA • Shine-Dalgarno sequence other eIFs to 40S ribosome to and recognition of located upstream of AUG form the pre-initiation complex initiation codon binds by AUG start codon • Linear scanning of pre-initiation complementary base pairing complex along 5´-UTR of mRNA with sequence in 16S rRNA to AUG codon within Kozak sequence First AA-tRNA fMet-tRNAi fMet = formyl-Methionine Met-tRNAi Met = Methionine Initiation Factors IF-1, IF-2 and IF-3 eIF-1, eIF-1A, eIF-2, eIF-3, eIF-4F, eIF-5 and at least 6 more Ribosomes 70S (30S and 50S subunits) 80S (40S and 60S subunits) In contrast to prokaryotes, translation of eukaryotic mRNAs is separated in time and space. All mRNA is transcribed and processed in the nucleus prior to export into the cytoplasm. Translation occurs in the cytoplasm where components of the translation machinery such as ribosomes, tRNA and translation factors are located. mRNAs that are inactive with respect to translation can be stored in P-bodies. Translation of specific mRNAs can be repressed by miRNAs. Nuclear envelope Transcription 40S Met-tRNAi•eIF-2•GTP eIF1, eIF1a, eIF3 Pore Pre-Initiation complex mRNP Processing m7 G Export C eIF-4F An mRNP & Pre-miRNA Protein N eIF-5 AA ATP 60S tRNA P-body miRNA in RISC N AA-tRNA synthetase Initiation m7G An Termination RF N N AA-tRNA eEFs N Elongation m7G N An Polyribosomes Translation & Genetic Code [11] D. STAGES OF TRANSLATION 6. Elongation Stage In general, elongation in prokaryotes and eukaryotes is essentially the same. Elongation factors have different names in prokaryotes vs. eukaryotes, but their corresponding functions are similar. 60S a) tRNA Binding Sites in the Ribosome E P A A site = Aminoacyl site P site = Peptidyl site E site = Exit site mRNA 5´- -3´ AUG 40S b) Peptidyl Transferase • Peptidyl transferase catalyzes peptide bond formation. • Peptidyl transferase is a ribozyme. The catalytic activity is derived from 23S rRNA in prokaryotes and 28S rRNA in eukaryotes. • Peptidyl transferase shifts nascent peptide from P site to A site. A-site • Energy for peptide bond formation derived from ATP used in tRNA charging. NH3+ R1 P-site A-site CH O=C :NH3+ NH3+ R1 P-site R2 CH CH NH Peptidyl Transferase R2 CH O=C O=C O O O O tRNA tRNA tRNA tRNA O=C D. STAGES OF TRANSLATION 6. Elongation Stage c) Mechanism of Elongation in Prokaryotes Step 1: fMet-tRNAf positioned in the P site Step 2: The next AA-tRNA is recruited to the A site by [EF-Tu•GTP•AA-tRNA] Step 3: Complementary base pairing in the A site between codon in mRNA and anticodon in AA-tRNA. Hydrolysis of GTP by EF-Tu is dependent on correct codon:anticodon base pairing. Thus, eIF-Tu functions as a proofreading mechanism during elongation step of translation. Step 4: Peptide Bond formation and resulting transfer of AA to the A site to form peptidyl tRNA Step 5: EF-G stimulates GTP hydrolysis and translocation of peptidyl tRNA to the P site, the uncharged tRNA translocates to the E site Step 6: Binding of next AA-tRNA to the A site causes release of uncharged tRNA from the E site Translation & Genetic Code [12] Translation & Genetic Code [13] D. STAGES OF TRANSLATION 6. Elongation Stage d) Role of Elongation Factors in Prokaryotes versus Eukaryotes Prokaryotes Eukaryotes Role in Elongation EF-Tu eEF-1a Recruit AA-tRNA to A site; GTP hydrolysis; Proofreading of codon-anticodon base paring EF-Ts eEF-1bg Exchange GDP•Pi for GTP to activate EF-Tu or eEF-1a EF-G eEF-2 Translocation of peptidyl tRNA to P site; GTP hydrolysis Transport to A Site AA-tRNA Binding GTP Hydrolysis (proofreading step) Inactive Active GTP - GDP•Pi Exchange 7. Characteristics of Polypeptide Chain Elongation a) Reading of mRNA Template: Coding sequence of mRNA is read in the 5´ to 3´ direction. b) Direction of Polypeptide Synthesis: Polypeptide grows from amino end to carboxy end. c) Polyribosomes: Multiple ribosomes translating a single mRNA molecule to improve efficiency. d) Translational Efficiency = # Ribosomes mRNA Molecule NH3+ + HN 3 NH3+ mRNA NH3+ 5´ 3´ Direction of ribosome movement Translation & Genetic Code [14] D. STAGES OF TRANSLATION 8. Protein Chaperones • Chaperones facilitate proper protein folding by binding and stabilizing unfolded or partially folded polypeptides. Nota Bene: Chaperones function co-translationally • Stabilization by chaperones enables protein to fold into proper conformation. • Prevent incorrect protein folding and/or protein aggregation. • Correct 3-D conformation of protein is determined by its AA sequence. Examples of chaperones: Hsp70 & Hsp90 BiP & PDI (found in lumen of RER) NH3+ 9. Termination Stage of Translation The termination of translation is essentially the same in prokaryotes and eukaryotes. Step 1: Stop codon moves into the A site Step 2: Recognition of stop codon by Release Factors (RF) RF NH3+ Step 3: Binding of RFs to the A site stimulates hydrolysis of the bond between the C terminal AA of polypeptide chain and tRNA in the P site E. EXAMPLES OF ANTIBIOTICS THAT TARGET TRANSLATION Antibiotic Target Effect on Translation Streptomycin 30S subunit Inhibits formation of 70S initiation complexes Tetracycline 30S subunit Inhibits binding of aminoacyl-tRNAs to the A site Chloramphenicol 50S subunit Inhibits peptidyl transferase reaction to block formation of peptide bonds Erythromycin 50S subunit Inhibits translocation of peptidyl-tRNA from the A site to the P site Translation & Genetic Code [15] F. REGULATION OF GENE EXPRESSION BY miRNA 1. Components of miRNA Synthesis a) miRNA Genes: Recent studies indicate that the human genome contains thousands of miRNA genes of which nearly half are located within introns. Transcription by RNA polymerase II generates a primary miRNA transcript (pri-miRNA) that contains a stem-loop structure. b) Drosha: Nuclease that processes the stem-loop of pri-miRNA into a 70 nt pre-miRNA in the nucleus. Pre-miRNA is then transported through a nuclear pore into the cytoplasm. c) Dicer: Nuclease located in the cytoplasm that processes pre-miRNA into a miRNA duplex between 20-22 nt in length. Base pairings in the miRNA duplex are partly complementary. d) RISC: miRNA-Induced Silencing Complex that contains proteins involved in separation of the miRNA strands and complementary base pairing of single-stranded miRNA to the target sequence in mRNA. The target sequence is usually located in the 3´-UTR of the mRNA. Primary miRNA transcript (Pri-miRNA) 5´ Nuclease (Dicer) RISC Strand separation Exportin 3´ Nuclear envelope AUG UGA 3´ 5´ A(n) 7 miRNA genes Nuclease (Drosha) miRNA duplex (~20-22 nt) m G RNA Pol II Pre-miRNA (~70 nt) Complementary base pairing between miRNA and target sequence in 3´-UTR of mRNA 2. Mechanism of Action Sequence-specific binding between miRNA/RISC and the mRNA target sequence inhibits or silences gene expression by the following mechanisms: a) Repress translation of mRNA b) Promote degradation of mRNA Binding of miRNA to a target mRNA does not require 100% complementarity along the entire length of the sequence. In fact, only nucleotides 2-7 of a miRNA must be complementary. This is referred to as the “seed region”. Consequently, a single miRNA can bind to multiple target mRNAs and thereby coordinately control protein synthesis and mRNA levels. Additionally, a single mRNA can contain target sequences for multiple species of miRNAs. G. PROCESSING BODIES (P-BODIES) • P-bodies are nonmembranous cytosolic organelles that contain a large number of RNAs and proteins condensed into heterogeneous ribonucleoprotein granules. • The RNA and protein components are known to function in processes such as translation, mRNA decay and miRNA-induced silencing. • It was thought originally that P-bodies function mainly as part of mRNA decay mechanisms, but recent evidence indicates P-bodies function as a depot for storage of translationally-arrested mRNPs. Storage is reversible as mRNAs can be released from P-bodies and initiate translation in the cytoplasm in order to accommodate the needs of the cell (see schematic on P. 10).