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LECTURE 4 Translation of mRNA (Chapter 13) Slides 1-18; 28-60 On your own: Slides 19-27 1 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. INTRODUCTION The translation of the mRNA codons into amino acid sequences leads to the synthesis of proteins A variety of cellular components play important roles in translation These include proteins, RNAs and small molecules In this chapter we will discuss the current state of knowledge regarding the molecular features of mRNA translation Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 2 13.1 THE GENETIC BASIS FOR PROTEIN SYNTHESIS Proteins are the active participants in cell structure and function Genes that encode polypeptides are termed structural genes These are transcribed into messenger RNA (mRNA) The main function of the genetic material is to encode the production of cellular proteins In the correct cell, at the proper time, and in suitable amounts Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 3 Archibald Garrod First to propose (at the beginning of the 20th century) a relationship between genes and protein production Garrod studied patients who had defects in their ability to metabolize certain compounds Urine chemist He was particularly interested in alkaptonuria Patients bodies accumulate abnormal levels of homogentisic acid (alkapton) Disease characterized by Black urine and bluish black discoloration of cartilage and skin Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 4 5 Archibald Garrod He proposed that alkaptonuria was due to a missing enzyme, namely homogentisic acid oxidase Garrod also knew that alkaptonuria follows an autosomal recessive pattern of inheritance He proposed that a relationship exists between the inheritance of the trait and the inheritance of a defective enzyme Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 6 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Dietary protein H CH2 C COOH NH2 Phenylalanine Phenylketonuria Phenylalanine hydroxylase H HO CH2 C COOH NH2 Tyrosine Tyrosine aminotransferase p-hydroxyphenylpyruvic acid Tyrosinosis Hydroxyphenylpyruvate oxidase Homogentisic acid Alkaptonuria Homogentisic acid oxidase Maleylacetoacetic acid Figure 13.1 Metabolic pathway of phenylalanine metabolism and related genetic diseases 7 Beadle and Tatum’s Experiments In the early 1940s, George Beadle and Edward Tatum were also interested in the relationship between genes, enzymes and traits Experiments supported Garrod’s idea that each gene codes for one enzyme Their genetic model was Neurospora crassa (a common bread mold) Their studies involved the analysis of simple nutritional requirements Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 8 Beadle and Tatum’s Experiments They analyzed more than 2,000 strains that had been irradiated to produce mutations They analyzed enzyme pathways for synthesis of vitamins and amino acids Figure 13.2 shows an example of their findings on the synthesis of the amino acid methionine Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 9 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Neurospora growth WT 1 WT 1 24 4 3 Minimal WT 1 24 3 +O–acetylhomoserine WT 24 3 +Cystathionine WT 1 1 2 4 3 +Homocysteine 2 3 +Methionine (a) Growth of strains on minimal and supplemented growth media Homoserine O–acetylhomoserine Enzyme 1 Cystathionine Enzyme 2 Methionine Homocysteine Enzyme 3 Enzyme 4 (b) Simplified pathway for methionine biosynthesis Every mutant strain was blocked at one (and only one) particular step in the synthesis pathway, showing that each gene encoded one enzyme Figure 13.2 10 Beadle and Tatum’s Experiments In the normal strains, methionine was synthesized by cellular enzymes In the mutant strains, a genetic defect in one gene prevented the synthesis of one protein required in one step of the pathway to produce that amino acid Beadle and Tatum’s conclusion: A single gene controlled the synthesis of a single enzyme This was referred to as the one gene–one enzyme hypothesis Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 11 Beadle and Tatum’s Experiments In later decades, this theory had to be modified 1. Enzymes are only one category of proteins 2. Some proteins are composed of two or more different polypeptides The term polypeptide denotes structure The term protein denotes function So it is more accurate to say a structural gene encodes a polypeptide In eukaryotes, alternative splicing means that a structural gene can encode many different polypeptides 3. Many genes have been identified that do not encode polypeptides For instance, functional RNA molecules (tRNA, rRNA, etc.) Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 12 The Genetic Code Translation involves an interpretation of one language into another Translation relies on the genetic code In genetics, the nucleotide language of mRNA is translated into the amino acid language of proteins Refer to Table 13.1 The genetic information is coded within mRNA in groups of three nucleotides known as codons Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 13 Three codons do not encode an amino acid. These are read as STOP signals for translation Triplet codons correspond to a specific amino acid Multiple codons may encode the same amino acid. These are known as synonymous codons 14 Special codons: AUG (which specifies methionine) = start codon UAA, UAG and UGA = termination, or stop, codons The code is degenerate More than one codon can specify the same amino acid For example: GGU, GGC, GGA and GGG all code for glycine In most instances, the third base is the variable base This defines the reading frame for all following codons AUG specifies additional methionines within the coding sequence It is sometime referred to as the wobble base The code is nearly universal Only a few rare exceptions have been noted Refer to Table 13.3 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 15 Figure 13.3 provides an overview of gene expression Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Coding strand DNA Transcription 5′ 3′ A C T G C C C A T G A G C G A C C A C T T G G G G C T C G G G G A A T A AC C G T C G A G G T G A CG GG T A CT C G CT G G TG A A CC CC G A G CC CC T TA T TGGC AGC T C C 3′ 5′ Template strand 5′ mRNA A C UG C C C A UG A G C G AC C A CU UG G G G C U CG G G G A A UA A C C G UC G A G G 5′ − untranslated Start region codon Codons 3′ Stop 3′ − untranslated codon region Anticodons Translation UAC UCG CUG GUG A AC CCC GAG CCC CUU Polypeptide tRNA 5′ 3′ Figure 13.3 Met Ser Asp His Leu Gly Leu Gly Note that the start codon sets the reading frame for all remaining codons Glu 16 A Polypeptide Chain Has Directionality Polypeptide synthesis has a directionality that parallels the 5’ to 3’ orientation of mRNA During each cycle of elongation, a peptide bond is formed between the carboxyl group of the last amino acid in the polypeptide chain and the amino group in the amino acid being added The first amino acid has an exposed amino group The last amino acid has an exposed carboxyl group Said to be N-terminal or amino terminal end Said to be C-terminal or carboxy terminal end Refer to Figure 13.6 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 17 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. R1 O H3N+ C C H R2 O N C H H R1 O H3N+ C C H R3 O C C H H + H3N+ N C C H H O– R2 O N R4 O R3 O C N C H H C C C H O– R4 O N C C + H2O H H O– Last peptide bond formed in the growing chain of amino acids (a) Attachment of an amino acid to a peptide chain OH CH3 S CH2 OH CH2 CH2 H 3C H + H3N Amino terminal end C C H O Methionine N H C C H O Serine N SH CH3 CH CH2 H C C H O Valine N CH2 H C C H O Tyrosine N C C H O O– Carboxyl terminal end Cysteine Peptide bonds 5′ AUG AGC GU U UAC UGC 3′ Sequence in mRNA (b) Directionality in a polypeptide and mRNA Figure 13.6 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 18 There are 20 amino acids that may be found in polypeptides Each contains a different side chain, or R group Each R group has its own particular chemical properties Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. CH3 H H3N CH3 CH3 CH CH3 + COO– C + H3N H Glycine (Gly) G C COO– H Alanine (Ala) A + H3N C CH3 CH3 CH3 CH CH2 COO– H Valine (Val) V + H3N C CH2 CH3 COO– H Leucine (Leu) L S + H3N CH C SH CH2 CH2 CH2 COO– H Isoleucine (Ile) I + H2N C COO– H Proline (Pro) P CH2 CH2 CH2 + H3N C COO– + H3N C COO– H H Cysteine (Cys) C Methionine (Met) M (a) Nonpolar, aliphatic amino acids H OH N CH2 + H3N C CH2 COO– + H3N C CH2 COO– H H Phenylalanine (Phe) F Tyrosine (Tyr) Y + H3N C Nonpolar amino acids are hydrophobic COO– H Tryptophan (Trp) W They are often buried within the interior of a folded protein (b) Aromatic amino acids Figure 13.7 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 19 Polar and charged amino acids are hydrophilic They are more likely to be on the surface of a protein Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. O O OH HCOH CH2 + H3N COO– C H Serine (Ser) S + H3N CH2 CH2 COO– C NH2 C CH3 H Threonine (Thr) T + H3N C NH2 C CH2 COO– + H3N C COO– H H Asparagine (Asn) N Glutamine (Gln) Q (c) Polar, neutral amino acids NH2 O– O C O– O C H3N C + NH CH2 CH2 + HN CH2 COO– + H3N C CH2 COO– H H Aspartic acid (Asp) D Glutamic acid (Glu) E (d) Polar, acidic amino acids + H3N C NH3 C CH2 NH CH2 CH2 NH CH2 CH2 CH2 CH2 COO– H Histidine (His) H + H3N N + + C CH3 + H3N C C O CH2 CH2 COO– H Lysine (Lys) K (e) Polar, basic amino acids NH2 COO– H Arginine (Arg) R SeH CH2 CH2 + H3N C CH2 COO– + H3N C COO– H H Selenocysteine (Sec) Pyrrolysine (Pyl) (f) Nonstandard amino acids Figure 13.7 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 20 Levels of Structure in Proteins There are four levels of structure in proteins 1. 2. 3. 4. Primary Secondary Tertiary Quaternary A protein’s primary structure is its amino acid sequence Refer to Figure 13.8 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 21 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Arg Cys Glu Phe Gly Leu 1 Val Lys 10 Ala Ala Ala NH3+ Met Lys 20 Gly Arg His Tyr Asn Asp Leu Gly Tyr Ser Leu Arg Gly 30 Asn The amino acid sequence of the enzyme lysozyme Ala Ala Trp Val Cys Lys Phe Glu Ser Asn Phe Asn Arg Asn Thr Thr Ala Asp 40 Asn Within the cell, the protein will not be found in this linear state Rather, it will adopt a compact 3-D structure Gin Thr Gly 50 Ser 60 Thr Asp Tyr Gly lle Leu Asn lle Gln Ser Arg Trp Trp Cys Asn 70 Leu Asn Arg Ser Gly Pro Thr Cys Asp Gly Arg Indeed, this folding can begin during translation Asn lle 129 amino acids long Pro 80 Cys Leu Ser Ala Leu Ser Ser Asp lle Thr Ser Asp Gly Gly Met Asn Asp Ser Val lle Lys Lys Ala Cys Arg Asn Arg Cys Lys 129 Gly Leu Arg COO– Figure 13.8 Asn 100 Ala Trp Val Ala Trp 110 Cys Gly Arg lle 90 Ala Trp Ala Gln 120 Thr Val The progression from the primary structure to the 3-D structure is dictated by the amino acid sequence within the polypeptide Val Asp Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 22 Levels of Structures in Proteins The primary structure of a protein folds to form regular, repeating shapes known as secondary structures There are two types of secondary structures a helix b sheet Certain amino acids are good candidates for each structure These secondary structures are stabilized by the formation of hydrogen bonds between atoms located in the polypeptide backbone Refer to Figure 13.9 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 23 Levels of Structures in Proteins The short regions of secondary structure in a protein fold into a three-dimensional tertiary structure Refer to Figure 13.9 This is the final conformation of proteins that are composed of a single polypeptide Structure determined by hydrophobic and ionic interactions as well as hydrogen bonds and Van der Waals interactions Proteins made up of two or more polypeptides have a quaternary structure This is formed when the various polypeptides associate with one another to make a functional protein Refer to Figure 13.9 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 24 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Primary structure Tertiary structure Secondary structure Quaternary structure C Phe Glu C O H N O H H N C C C H Leu N Iso N C HO O N Tyr H C C C C O H C C H C N Two or more polypeptides may associate with each other. NH3+ COO– N C O C Regions of NH3+ secondary structure and irregularly shaped regions fold into a – three-dimensional COO conformation. NH3+ Val Depending on the amino acid sequence, some regions may fold into an α helix or β sheet. COO– Ala (c) C N Protein subunit O O α helix Ala (d) (a) β sheet (b) Figure 13.9 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 25 Functions of Proteins To a great extent, the characteristics of a cell depend on the types of proteins its makes Proteins can perform a variety of functions Refer to Table 13.5 A key category of proteins are enzymes Accelerate chemical reactions within a cell Can be divided into two main categories Anabolic enzymes Synthesize molecules and macromolecules Catabolic enzymes Break down large molecules into small ones Important in generating cellular energy Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 26 13-38 27 13-39 13.2 STRUCTURE AND FUNCTION OF tRNA In the 1950s, Francis Crick and Mahon Hoagland proposed the adaptor hypothesis tRNAs play a direct role in the recognition of codons in the mRNA In particular, the hypothesis proposed that tRNA has two functions 1. Recognizing a 3-base codon in mRNA 2. Carrying an amino acid that is specific for that codon Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 28 Recognition Between tRNA and mRNA During mRNA-tRNA recognition, the anticodon in tRNA binds to a complementary codon in mRNA Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Phenylalanine Proline tRNAs are named according to the amino acid they bear tRNAPhe tRNAPro A A G The anticodon is anti-parallel to the codon G GC Phenylalanine anticodon Proline anticodon U UC C C G 5′ 3′ mRNA Phenylalanine codon Figure 13.10 Proline codon Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 29 tRNAs Share Common Structural Features The secondary structure of tRNAs exhibits a cloverleaf pattern It contains Three stem-loop structures A few variable sites An acceptor stem with a 3’ single strand region The actual three-dimensional or tertiary structure involves additional folding In addition to the normal A, U, G and C nucleotides, tRNAs commonly contain modified nucleotides More than 80 of these can occur Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 30 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. NH3+ H C R C O 3′ A C C Found in all tRNAs 5′ OH O Covalent bond between tRNA and an amino acid A C C Acceptor stem PO4 70 Stem–loop 60 U UH2 G C A U U 10 A G T 50 G Not found in all tRNAs Other variable sites are shown in blue as well C P m 2G A G UH2 19 UH2 40 30 U P U mI I G C The modified bases are: I = inosine mI = methylinosine T = ribothymidine UH2 = dihydrouridine m2G = dimethylguanosine y = pseudouridine Anticodon Figure 13.12 Structure of tRNA 31 Charging of tRNAs The enzymes that attach amino acids to tRNAs are known as aminoacyl-tRNA synthetases There are 20 types One for each amino acid Aminoacyl-tRNA synthetases catalyze a two-step reaction involving three different molecules Amino acid, tRNA and ATP Refer to Figure 13.13 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 32 Charging of tRNAs The aminoacyl-tRNA synthetases are responsible for the “second genetic code” The selection of the correct amino acid must be highly accurate or the polypeptides may be nonfunctional Error rate is less than one in every 100,000 Sequences throughout the tRNA including but not limited to the anticodon are used as recognition sites Modified bases may affect translation rates recognition by aminoacyl-tRNA synthetases Codon-anticodon recognition Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 33 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Aminoacyl-tRNA synthetase Specific amino acid A P P P ATP An amino acid and ATP bind to the enzyme. AMP is covalently bound to the amino acid, and pyrophosphate is released. A P P P Pyrophosphate The correct tRNA binds to the enzyme. The amino acid becomes covalently attached to the 3′ end of the tRNA. AMP is released. tRNA 3′ 5′ 5′ 3′ A The amino acid is attached to the 3’ end of the tRNA by an ester bond P AMP The “charged” tRNA is released. 5′ Figure 13.13 3′ 34 tRNAs and the Wobble Rule As mentioned earlier, the genetic code is degenerate With the exception of serine, arginine and leucine, this degeneracy always occurs at the codon’s third position To explain this pattern of degeneracy, Francis Crick proposed in 1966 the wobble hypothesis In the codon-anticodon recognition process, the first two positions pair strictly according to the A – U /G – C rule However, the third position can actually “wobble” or move a bit Thus tolerating certain types of mismatches Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 35 Phenylalanine tRNAs that can recognize the same codon are termed isoacceptor tRNAs 5′ 3′ Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Nucleotide of of tRNA anticodon A A G U U U Wobble position 3′ 5′ G C A U inosine I 5-methyl-2-thiouridine xm5s2U 5-methyl-2’-O-methyluridine xm5Um 2’-O-methyluridine Um position 5-methyluridine xm5U Third nucleotide of mRNA codon C, U G U, C, G, (A) A, U, G, (C) U, C, A (a) Location of wobble You don’t need to memorize these rules 5-hydroxyuridine xo5U lysidine k2C A, (G) U, A, G A (b) Revised wobble rules Figure 13.14 Wobble position and base pairing rules Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Recognized very poorly by the tRNA 36 13.3 RIBOSOME STRUCTURE AND ASSEMBLY Translation occurs on the surface of a large macromolecular complex termed the ribosome Bacterial cells have one type of ribosome Found in their cytoplasm Eukaryotic cells have two types of ribosomes One type is found in the cytoplasm The other is found in organelles Mitochondria ; Chloroplasts Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 37 13.3 RIBOSOME STRUCTURE AND ASSEMBLY Unless otherwise noted the term eukaryotic ribosome refers to the ribosomes in the cytosol A ribosome is composed of structures called the large and small subunits Each subunit is formed from the assembly of Proteins rRNA Table 13.6 presents the composition of bacterial and eukaryotic ribosomes Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 38 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 39 Functional Sites of Ribosomes During bacterial translation, the mRNA lies on the surface of the 30S subunit Ribosomes contain three discrete sites As a polypeptide is being synthesized, it exits through a channel within the 50S subunit Peptidyl site (P site) Aminoacyl site (A site) Exit site (E site) Ribosomal structure is shown in Figure 13.15 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 40 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Polypeptide tRNA E P A 50S 30S mRNA 3 5 (c) Model for ribosome structure Figure 13.15 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 41 13.4 STAGES OF TRANSLATION Translation can be viewed as occurring in three stages Initiation Elongation Termination Refer to 13.16 for an overview of translation Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 42 Initiator tRNA Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. aa1 aa1 Initiator tRNA – tRNA with first amino acid Large E Ribosomal subunits UAC Anticodon A Initiation Small AUG Start codon mRNA UAG Stop codon 5′ P 3′ 5′ 3′ AUG Start codon Elongation (This step occurs many times.) aa1 aa2 aa3 aa4 Recycling of translational components Release factor Completed polypeptide E P aa5 E A P A Termination UAG Stop codon 5′ Figure 13.16 3′ 5′ Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 3′ 43 The Translation Initiation Stage The mRNA, initiator tRNA, and ribosomal subunits associate to form an initiation complex This process requires three Initiation Factors The initiator tRNA recognizes the start codon in mRNA In bacteria, this tRNA is designated tRNAfmet It carries a methionine that has been covalently modified to N-formylmethionine The start codon is AUG, but in some cases GUG or UUG In all three cases, the first amino acid is N-formylmethionine Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 44 The binding of mRNA to the 30S subunit is facilitated by a ribosomal-binding site or Shine-Dalgarno sequence This is complementary to a sequence in the 16S rRNA Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Component of the 30S subunit 3′ 5′ 16S rRNA Hydrogen bonding mRNA A UCU AGU A AGGAGGUUGU A UGGUU C AGCGC A CG Figure 13.18 A UUCC UC C A Shine-Dalgarno sequence CAG 3′ Start codon Figure 13.17 outlines the steps that occur during translational initiation in bacteria Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 45 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. IF1 and IF3 bind to the 30S subunit. IF3 IF1 The mRNA binds to the 30S subunit. The Shine-Dalgarno sequence is complementary to a portion of the 16S rRNA. Portion of 16S rRNA IF3 5′ 30S subunit IF1 Start Shinecodon Dalgarno sequence (actually 9 nucleotides long) 3′ IF2, which uses GTP, promotes the binding of the initiator tRNA to the start codon in the P site. Figure 13.17 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 46 tRNAfMet Initiator tRNA GTP IF2 IF1 IF3 3′ 5′ IF1 and IF3 are released. IF2 hydrolyzes its GTP and is released. The 50S subunit associates. tRNAfMet 70S initiation complex E Figure 13.17 5′ P A 70S initiation complex This marks the end of the initiation stage 3′ Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 47 The Translation Initiation Stage In eukaryotes, the assembly of the initiation complex is similar to that in bacteria However, additional factors are required Note that eukaryotic Initiation Factors are denoted eIF Refer to Table 13.7 The initiator tRNA is designated tRNAmet It carries a methionine rather than a formylmethionine Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 48 The start codon for eukaryotic translation is AUG Ribosome scans from the 5’ end of mRNA until it finds the AUG start codon (not all AUGs can act as a start) The consensus sequence for optimal start codon recognition is show here Most important positions for codon selection C C A U G G -2 -1 +1 +2 +3 +4 These rules are called Kozak’s rules G C C (A/G) -6 -5 -4 -3 Start codon After Marilyn Kozak who first proposed them With that in mind, the start codon for eukaryotic translation is usually the first AUG after the 5’ Cap! Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 49 Translational initiation in eukaryotes can be summarized as such: An initiation factor protein complex (eIF4) binds to the 5’ cap in mRNA These are joined by a complex consisting of the 40S subunit, tRNAmet, and other initiation factors The entire assembly moves along the mRNA scanning for the right start codon Once it finds this AUG, the 40S subunit binds to it The 60S subunit joins This forms the 80S initiation complex Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 50 The Translation Elongation Stage During this stage, amino acids are added to the polypeptide chain, one at a time The addition of each amino acid occurs via a series of steps outlined in Figure 13.19 This process, though complex, can occur at a remarkable rate In bacteria 15-20 amino acids per second In eukaryotes 2-6 amino acids per second Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 51 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. aa1 aa2 Ribosome aa3 E site A site P site 5′ Codon 4 Codon 3 aa1 aa2 aa3 3′ aa4 mRNA E A charged tRNA binds to the A site. EF-Tu facilitates tRNA binding and hydrolyzes GTP. The 23S rRNA (a component of the large subunit) is the actual peptidyl transferase Thus, the ribosome is a ribozyme! Figure 13.19 P A 3′ 5′ Peptidyltransferase, which is a component of the 50S subunit, catalyzes peptide bond formation between the polypeptide and the amino acid in the A site.The polypeptide is transferred to the A site. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 52 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. aa1 aa2 tRNAs at the P and A sites move into the E and P sites, respectively aa3 aa4 E P A aa1 aa2 aa3 3′ 5′ aa4 The ribosome translocates 1 codon to the right. This translocation is promoted by EF-G, which hydrolyzes GTP. aa3 aa4 aa2 E P A Codon 3 aa1 Codon 5 5′ E P Codon 4 3′ An uncharged tRNA is released from the E site. A Codon 3 5′ Codon 5 Codon 4 3′ This process is repeated, again and again, until a stop codon is reached. Figure 13.19 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 53 The Translation Termination Stage The final stage occurs when a stop codon is reached in the mRNA In most species there are three stop or nonsense codons UAG UAA UGA These codons are not recognized by tRNAs, but by proteins called release factors Indeed, the 3-D structure of release factors mimics that of tRNAs Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 54 The Translation Termination Stage Bacteria have three release factors RF1, which recognizes UAA and UAG RF2, which recognizes UAA and UGA RF3, which does not recognize any of the three codons It binds GTP and helps facilitate the termination process Eukaryotes only have one release factor eRF, which recognizes all three stop codons Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 55 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. tRNA in P site carries completed polypeptide E 5′ P A Stop codon in A site 3′ mRNA A release factor (RF) binds to the A site. E P A Release factor 3′ 5′ The polypeptide is cleaved from the tRNA in the P site. The tRNA is then released. 3′ 5′ The ribosomal subunits, mRNA, and release factor dissociate. + 50S subunit mRNA Figure 13.20 5′ 30S subunit 3′ 56 57 Bacterial Translation Can Begin Before Transcription Is Completed Bacteria lack a nucleus Therefore, both transcription and translation occur in the cytoplasm As soon an mRNA strand is long enough, a ribosome will attach to its 5’ end So translation begins before transcription ends This phenomenon is termed coupling Refer to Figure 13.21 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 58 Coupling between transcription and translation in bacteria Figure 13.21 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 59 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 60 Animations 61 Please note that due to differing operating systems, some animations will not appear until the presentation is viewed in Presentation Mode (Slide Show view). You may see blank slides in the “Normal” or “Slide Sorter” views. All animations will appear after viewing in Presentation Mode and playing each animation. Most animations will require the latest version of the Flash Player, which is available at http://get.adobe.com/flashplayer. 62 Please note that due to differing operating systems, some animations will not appear until the presentation is viewed in Presentation Mode (Slide Show view). You may see blank slides in the “Normal” or “Slide Sorter” views. All animations will appear after viewing in Presentation Mode and playing each animation. Most animations will require the latest version of the Flash Player, which is available at http://get.adobe.com/flashplayer. 63 Please note that due to differing operating systems, some animations will not appear until the presentation is viewed in Presentation Mode (Slide Show view). You may see blank slides in the “Normal” or “Slide Sorter” views. All animations will appear after viewing in Presentation Mode and playing each animation. Most animations will require the latest version of the Flash Player, which is available at http://get.adobe.com/flashplayer. 64 Please note that due to differing operating systems, some animations will not appear until the presentation is viewed in Presentation Mode (Slide Show view). You may see blank slides in the “Normal” or “Slide Sorter” views. All animations will appear after viewing in Presentation Mode and playing each animation. Most animations will require the latest version of the Flash Player, which is available at http://get.adobe.com/flashplayer. 65