1. © 2012 Pearson Education, Inc. Lectures by Kathleen Fitzpatrick Simon Fraser University Chapter 22 Gene Expression: II. Protein Synthesis and Sorting

2. © 2012 Pearson Education, Inc. Gene Expression: II. Protein Synthesis For some genes, the RNA is the final product But for most genes, the ultimate product is protein mRNAs encode instructions for translation, the assembling amino acids into a polypeptide

3. © 2012 Pearson Education, Inc. Translation: The Cast of Characters Ribosomes carry out the polypeptide synthesis tRNA molecules transport the amino acids Aminoacyl-tRNA synthetases attach amino acids to their appropriate tRNA molecules

4. © 2012 Pearson Education, Inc. The cast of characters mRNA molecules encode the amino acid sequence information Protein factors facilitate the steps of translation

5. © 2012 Pearson Education, Inc. The Ribosome Carries Out Polypeptide Synthesis Ribosomes: particles made of rRNA and protein In eukaryotes: found free in the cytoplasm, bound to ER and the outer nuclear envelope Ribosomes are built from dissociable subunits, the large and small subunits In prokaryotes, the ribosomes are smaller

6. © 2012 Pearson Education, Inc. Figure 22-1B

7. © 2012 Pearson Education, Inc. Table 22-1

8. © 2012 Pearson Education, Inc. Ribosomes, machines in polypeptide synthesis rRNA performs the key functions of ribosomes Ribosomes have four important sites: the mRNA binding site, the A (aminoacyl) site, the P (peptidyl) site, and an E (exit) site

9. © 2012 Pearson Education, Inc. Figure 22-2

10. © 2012 Pearson Education, Inc. Transfer RNA Molecules Bring Amino Acids to the Ribosome A tRNA molecule is an adaptor that binds both a specific amino acid and the mRNA sequences that specify the amino acid Each tRNA is linked to its amino acid by ester bond tRNAs are named for the amino acids attached to them, e.g., tRNA Ala for alanine

11. © 2012 Pearson Education, Inc. Figure 22-3A

12. © 2012 Pearson Education, Inc. Figure 22-3B

13. © 2012 Pearson Education, Inc. tRNAs tRNAs attached to an amino acid are said to be aminoacyl tRNAs—the tRNA is called charged, whereas the amino acid is called activated Each tRNA recognizes codons in mRNA due to their complementarity to the anticodon in the tRNA Some tRNAs recognize more than one codon

14. © 2012 Pearson Education, Inc. The wobble hypothesis mRNA and tRNA line up on the ribosome in a way that permits flexibility or wobble in the pairing between the third base of the codon and the corresponding base of the anticodon This is the wobble hypothesis, which allows for some unexpected base pairing

15. © 2012 Pearson Education, Inc. Figure 22-4

16. © 2012 Pearson Education, Inc. Inosine The nucleotide inosine is able to pair with U, C, or A and is often found in the wobble position of the anticodon This allows for several codons to specify one amino acid

17. © 2012 Pearson Education, Inc. Aminoacyl-tRNA Synthetases Link Amino Acids to the Correct Transfer RNAs Cells typically have 20 different aminoacyl-tRNA synthetases to attach each amino acid to the appropriate tRNA There is one aminoacyl-tRNA synthetase for each amino acid Cells with nontraditional amino acids have special tRNAs and aminoacyl-tRNA synthetases for these amino acids, too

18. © 2012 Pearson Education, Inc. Aminoacyl-tRNA synthesis Aminoacyl-tRNA synthetases catalyze the attachment of amino acids to the tRNAs via an ester bond, using ATP hydrolysis

19. © 2012 Pearson Education, Inc. Aminoacyl-tRNA synthesis Both the anticodon and the 3 end of the tRNA are needed to specify the correct amino acid After addition of an amino acid the synthetases proofread the final product to ensure the correct amino acid was added It is the tRNA that then recognizes the appropriate codon in mRNA

20. © 2012 Pearson Education, Inc. Figure 22-5, Steps 1, 2

21. © 2012 Pearson Education, Inc. Figure 22-5, Steps 3, 4

22. © 2012 Pearson Education, Inc. Messenger RNA Brings Polypeptide Coding Information to the Ribosome The sequence of codons in mRNA directs the order of amino acids in the polypeptide mRNA is exported to the cytoplasm via binding to proteins that contain nuclear export signals (NES) An untranslated sequence at the 5 end of the message precedes the start codon, the first to be translated (usually AUG)

23. © 2012 Pearson Education, Inc. Coding information There is an untranslated region at the 3 end of the mRNA that follows the stop codon, which signals the end of translation The stop codon may be UAG, UAA, or UGA 5 and 3 untranslated regions vary in length and are essential for mRNA function mRNAs also have a 5 cap and 3 poly(A) tail

24. © 2012 Pearson Education, Inc. Figure 22-6

25. © 2012 Pearson Education, Inc. Eukaryotic mRNAs are monocistronic Most mRNAs in eukaryotes are monocistronic, meaning they encode just one polypeptide In bacteria and archaea, some are polycistronic, encoding several polypeptides with related functions Polycistronic transcription units are called operons

26. © 2012 Pearson Education, Inc. Translation is an ordered, stepwise process that begins at the N-terminus of the polypeptide and adds amino acids to the growing chain until the C- terminus is reached The mRNA is read in the 5 to 3 direction Translation is divided into three stages: initiation (1), elongation (2), and termination (3) Protein Factors Are Required for the Initiation, Elongation, and Termination of Polypeptide Chains

27. © 2012 Pearson Education, Inc. Figure 22-7

28. © 2012 Pearson Education, Inc. The start codon in eukaryotes and archaea specifies methionine The initiation factors are called eIFs; there are about a dozen of these eIF2 (with GTP attached) binds to the initiator tRNA Met before the tRNA then binds the small ribosomal subunit The Initiation of Translation Requires Initiation Factors, Ribosomal Subunits, mRNA, and Initiator tRNA Eukaryotic Initiation

29. © 2012 Pearson Education, Inc. Eukaryotic initiation (continued) After binding the mRNA, the small ribosomal subunit (including the initiator tRNA) scans along the transcript and begins translation at the first AUG Nucleotides to either side of the start codon are involved in the recognition; e.g., a common start sequence is ACCAUGG, called a Kozak sequence After the initiator tRNA is base-paired with the start codon the large subunit joins the complex, facilitated by GTP hydrolysis

30. © 2012 Pearson Education, Inc. Chain Elongation Involves Sequential Cycles of Aminoacyl tRNA Binding, Peptide Bond Formation, and Translocation Once initiation has been completed a polypeptide chain is synthesized Amino acids are added in sequence to the growing chain (elongation) Elongation involves a repetitive cycle of three steps

31. © 2012 Pearson Education, Inc. Binding of Aminoacyl tRNA As elongation begins, the start codon is located at the P site and the next codon is at the A site Elongation begins as a tRNA with an anticodon complementary to the second codon binds the A site (1) This requires two elongation factors, EF-Tu and EF-Ts, and GTP hydrolysis

32. © 2012 Pearson Education, Inc. Figure 22-10

33. © 2012 Pearson Education, Inc. Binding of aminoacyl tRNA (continued) Elongation factors don’t recognize particular anticodons, so all types are brought to the A site Only those with an anticodon complementary to the codon stay at the A site long enough for GTP hydrolysis to take place The final error rate in translation is at most 1/10,000

34. © 2012 Pearson Education, Inc. Peptide Bond Formation Once the aminoacyl tRNA is bound to the A site, a peptide bond forms between the amino group of the amino acid at the A site and the carboxyl group of the amino acid at the P site The growing peptide chain is transferred to the tRNA at the A site (2) No ATP or GTP hydrolysis is required for this step

35. © 2012 Pearson Education, Inc. rRNA catalyzes peptide bond formation It was thought that the protein peptidyl transferase catalyzed peptide bond formation However, Noller and colleagues showed that rRNA contains the catalytic activity

36. © 2012 Pearson Education, Inc. Translocation After the peptide bond forms, the mRNA advances to bring the next codon into the proper position During this translocation, the peptidyl tRNA moves from the A to the P site, and the empty tRNA moves to the E site Hydrolysis of GTP bound to EF-G triggers a conformational change that completes these movements (3) Once the next mRNA codon reaches the A site, the ribosome is now set to receive the next aminoacyl tRNA

37. © 2012 Pearson Education, Inc. Termination of Polypeptide Synthesis Is Triggered by Release Factors That Recognize Stop Codons Codons are read on the mRNA one after the other, until a stop codon arrives at the A site Stop codons are recognized by protein release factors, rather than tRNAs Once release factors bind to the stop codons, translation is terminated through release of the completed polypeptide

38. © 2012 Pearson Education, Inc. Figure 22-11

39. © 2012 Pearson Education, Inc. Polypeptide Folding Is Facilitated by Molecular Chaperones Proteins must fold into their correct three- dimensional shapes before they can function Protein folding is usually facilitated by proteins called molecular chaperones; often several are required, acting in sequence Chaperones bind polypeptide chains during the early stages of folding

40. © 2012 Pearson Education, Inc. Molecular chaperones If folding goes awry, chaperones can sometimes rescue the proteins and fold them properly Alternatively, improperly folded proteins may be destroyed Some kinds of incorrectly folded proteins bind to each other and form insoluble aggregates within and between cells

41. © 2012 Pearson Education, Inc. Protein Synthesis Typically Utilizes a Substantial Fraction of a Cell’s Energy Budget Polypeptide elongation involves hydrolysis of at least four high-energy phosphoanhydride bonds Assuming each bond has a  G°of 7.3 kcal/mol, they represent a free energy input of 29.2 kcal/mol Additional GTPs are used during formation of the initiation complex, the binding of incorrect aminoacyl tRNAs, and termination

42. © 2012 Pearson Education, Inc. A Summary of Translation Translation converts information in mRNAs into a chain of amino acids linked by peptide bonds Most messages are read by many ribosomes simultaneously; a cluster of such ribosomes attached to the same mRNA is called a polyribosome RNA molecules play important roles in translation; mRNA, tRNA, rRNA

43. © 2012 Pearson Education, Inc. Mutations and Translation mRNAs may contain mutant codons that cause errors in the polypeptide chain synthesized Most codon mutations alter a single amino acid and some (in the third base of a codon) don’t alter the amino acid at all Mutations that add or remove stop codons or alter the reading frame can severely disrupt translation

44. © 2012 Pearson Education, Inc. Suppressor tRNA Overcomes the Effects of Some Mutations Mutations that convert amino acid-coding codons into stop codons, called nonsense mutations, typically lead to incomplete, nonfunctional polypeptides These mutations are often lethal, but can sometimes be overcome by an independent mutation affecting a tRNA gene This is called a suppressor tRNA

45. © 2012 Pearson Education, Inc. Figure 22-12A, B

46. © 2012 Pearson Education, Inc. Suppressor tRNAs Suppressor tRNAs recognize stop codons and insert amino acids, suppressing nonsense mutations Highly efficient suppressor tRNAs might lead to the production of many abnormal amino acids However, at the 3 ends of mRNAs, release factors bind stop codons more efficiently than suppressor tRNAs; this limits the effect of the suppressor to internal locations on the mRNA

47. © 2012 Pearson Education, Inc. Figure 22-12B, C

48. © 2012 Pearson Education, Inc. Nonsense-Mediated Decay and Nonstop Decay Promote the Destruction of Defective mRNAs Without a suppressor tRNA, a nonsense mutation will cause premature termination of translation and an incomplete polypeptide chain Eukaryotic cells use nonsense-mediated decay to destroy mRNAs containing premature stop codons In mammals, the exon junction complex (EJC) is used to detect premature stop codons

49. © 2012 Pearson Education, Inc. The EJC and nonsense mutations A multiprotein EJC is deposited wherever an intron is removed from pre-mRNA, so each spliced mRNA has at least one complex bound to it If an mRNA contains a stop codon prior to the final EJC, translation is terminated EJCs still associated with the tRNA target it for degradation

50. © 2012 Pearson Education, Inc. The fate of mRNAs with no stop codon In eukaryotes, translation is stalled when a ribosome reaches the end of a transcript that lacks a stop codon An RNA degrading enzyme binds the empty A site of the ribosome and degrades the defective mRNA via nonstop decay

51. © 2012 Pearson Education, Inc. Posttranslational Processing After polypeptide chains are synthesized, they often must undergo posttranslational modification before they can perform their functions In eukaryotes, the methionine at the N-terminus is released Sometimes whole blocks of amino acids are removed from the polypeptide, for instance certain enzymes synthesized as inactive precursors, these are activated by removal of sequences from one end of the protein

52. © 2012 Pearson Education, Inc. Posttranslational processing (continued) Other common processing events include chemical modification of amino acids—methylation, phosphorylation, acetylation Some proteins undergo a rare process called protein splicing Similar to RNA splicing, protein sequences called inteins are removed and the remaining sequences called exeins are spliced together