1. n Chapter 12~ From Gene to Protein

2. Protein Synthesis: overview n One gene-one enzyme hypothesis (Beadle and Tatum) n One gene-one polypeptide (protein) hypothesis n Transcription: synthesis of RNA under the direction of DNA (mRNA) n Translation: actual synthesis of a polypeptide under the direction of mRNA

3. Figure 17.3 The triplet code

4. The Central Dogma of Molecular Biology 4 codon 1codon 2 nontemplate strand template strand codon 3 polypeptide NN N CCC C CC R1R1 R2R2 R3R3 SerineAspartateProline OOO C G G CA G CC G C GTT AG CG C GCACCCAGC transcription in nucleus translation at ribosome mRNA 35 35 53 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. DNA

5. The Genetic Code of Life n RNA is a polymer of RNA nucleotides –RNA nucleotides contain the sugar ribose instead of deoxyribose –RNA nucleotides are of four types: uracil (U), adenine (A), cytosine (C), and guanine(G) –Uracil (U) replaces thymine (T) of DNA n Types of RNA Messenger (mRNA) - Takes a message from DNA in the nucleus to ribosomes in the cytoplasm Ribosomal (rRNA) - Makes up ribosomes, which read the message in mRNA Transfer (tRNA) - Transfers the appropriate amino acid to the ribosomes for protein synthesis 5

6. Structure of RNA 6 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. G U A C S S S S P P P P base is uracil instead of thymine one nucleotide ribose 3′ end 5′ end

7. RNA Structure Compared to DNA structure 7

8. The Genetic Code of Life n The unit of the genetic code consists of codons, each of which is a unique arrangement of symbols n Each of the 20 amino acids found in proteins is uniquely specified by one or more codons –The symbols used by the genetic code are the mRNA bases Function as “letters” of the genetic alphabet Genetic alphabet has only four “letters” (U, A, C, G) –Codons in the genetic code are all three bases (symbols) long Function as “words” of genetic information Permutations: –There are 64 possible arrangements of four symbols taken three at a time –Often referred to as triplets Genetic language only has 64 “words” 8

9. Messenger RNA Codons 9 UCAG U C A G U C A G U C A G U C A G U C A G Second Base Third Base First Base CGA arginine CGG arginine AGU serine AGC serine AGA arginine AGG arginine GGU glycine GGC glycine GGA glycine GGG glycine UGG tryptophan CGU arginine CGC arginine UGA stop AUG (start) methionine CAC histidine CAA glutamine CAG glutamine AAU asparagine AAC asparagine AAA lysine AAG lysine GAU aspartate GAC aspartate GAA glutamate GAG glutamate UUU phenylalanine CUU leucine CUC leucine CUA leucine CUG leucine AUU isoleucine AUC isoleucine AUA isoleucine GUU valine GUC valine GUA valine GUG valine UCA serine CCU proline CCC proline CCA proline CCG proline ACU threonine ACC threonine ACA threonine ACG threonine GCU alanine GCC alanine GCA alanine GCG alanine CAU histidine UAA stop UCU serine UAU tyrosine UGU cysteine UUC phenylalanine UCC serine UAC tyrosine UGU cysteine UUA leucine UCG serine UAG stop UUG leucine Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

10. The Genetic Code of Life n Properties of the genetic code: –Universal With few exceptions, all organisms use the code the same way Encode the same 20 amino acids with the same 64 triplets –Degenerate (redundant) There are 64 codons available for 20 amino acids Most amino acids encoded by two or more codons –Unambiguous (codons are exclusive) None of the codons code for two or more amino acids Each codon specifies only one of the 20 amino acids –Contains start and stop signals Punctuation codons Like the capital letter we use to signify the beginning of a sentence, and the period to signify the end 10

11. 12.4 First Step: Transcription n Transcription –A segment of DNA serves as a template for the production of an RNA molecule –The gene unzips and exposes unpaired bases –Serves as template for mRNA formation –Loose RNA nucleotides bind to exposed DNA bases using the C=G and A=U rule –When entire gene is transcribed into mRNA, the result is a pre-mRNA transcript of the gene –The base sequence in the pre-mRNA is complementary to the base sequence in DNA 11

12. First Step: Transcription n A single chromosome consists of one very long molecule encoding hundreds or thousands of genes n The genetic information in a gene describes the amino acid sequence of a protein –The information is in the base sequence of one side (the “sense” strand) of the DNA molecule –The gene is the functional equivalent of a “sentence” n The segment of DNA corresponding to a gene is unzipped to expose the bases of the sense strand –The genetic information in the gene is transcribed (rewritten) into an mRNA molecule –The exposed bases in the DNA determine the sequence in which the RNA bases will be connected together –RNA polymerase connects the loose RNA nucleotides together n The completed transcript contains the information from the gene, but in a mirror image, or complementary form 12

13. Transcription 13 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. T T G C A T T G C A terminator gene strand promoter template strand direction of polymerase movement RNA polymerase DNA template strand mRNA transcript to RNA processing 3′ 5′ 3′

14. RNA Polymerase 14 © Oscar L. Miller/Photo Researchers, Inc. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. a. 200m b. spliceosome DNA RNA polymerase RNA transcripts

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16. First Step: Transcription n Pre-mRNA is modified before leaving the eukaryotic nucleus. –Modifications to the ends of the primary transcript: Cap on the 5′ end –The cap is a modified guanine (G) nucleotide –Helps a ribosome determine where to attach when translation begins Poly-A tail of 150-200 adenines on the 3′ end –Facilitates the transport of mRNA out of the nucleus –Inhibits degradation of mRNA by hydrolytic enzymes. 16

17. First Step: Transcription n Pre-mRNA, is composed of exons and introns. –The exons will be expressed, –The introns, occur in between the exons. Allows a cell to pick and choose which exons will go into a particular mRNA –RNA splicing: Primary transcript consists of: –Some segments that will not be expressed (introns) –Segments that will be expressed (exons) Performed by spliceosome complexes in nucleoplasm –Introns are excised –Remaining exons are spliced back together n Result is a mature mRNA transcript 17

18. First Step: Transcription n In prokaryotes, introns are removed by “self-splicing”—that is, the intron itself has the capability of enzymatically splicing itself out of a pre-mRNA 18

19. Messenger RNA Processing in Eukaryotes 19 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. DNA

20. Messenger RNA Processing in Eukaryotes 20 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. exon intron exon DNA

21. Messenger RNA Processing in Eukaryotes 21 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. exon intron exon DNA transcription exon intron exon pre-mRNA 5 3

22. Messenger RNA Processing in Eukaryotes 22 exon intron exon transcription exon intron exon pre-mRNA 5 3 exon intron exon cap 3 5 DNA poly-A tail Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

23. Messenger RNA Processing in Eukaryotes 23 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. exon intron exon transcription exon intron exon pre-mRNA 5 3 exon intron exon cap poly-A tail spliceosome cap 3 3 5 5 DNA poly-A tail

24. Messenger RNA Processing in Eukaryotes 24 exon intron exon DNA transcription exon intron exon pre-mRNA 53 exon intron exon intron RNA exon pre-mRNA splicing exon cap poly-A tail spliceosome mRNA cappoly-A tail cappoly-A tail 3 3 3 5 5 5 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

25. Messenger RNA Processing in Eukaryotes 25 exon intron exon DNA transcription exon intron exon pre-mRNA 53 exon intron exon intron RNA exon pre-mRNA splicing exon cap poly-A tail spliceosome mRNA cytoplasm cap poly-A tail cappoly-A tail 3 3 3 5 5 5 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

26. Messenger RNA Processing in Eukaryotes 26 exon intron exon DNA transcription exon intron exon 53 intron exon intron RNA exon pre-mRNA splicing exon cappoly-A tail spliceosome nucleus mRNA cytoplasm nuclear pore in nuclear envelope cap poly-A tail cap poly-A tail 3 3 5 5 pre-mRNA 3 5 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

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28. First Step: Transcription n Functions of introns: –As organismal complexity increases; The number of protein-coding genes does not keep pace But the proportion of the genome that is introns increases –Possible functions of introns: Exons might combine in various combinations –Would allow different mRNAs to result from one segment of DNA Introns might regulate gene expression Introns may encourage crossing-over during meiosis –Exciting new picture of the genome is emerging 28

29. 12.5 Second Step: Translation n Translation –The sequence of codons in the mRNA at a ribosome directs the sequence of amino acids into a polypeptide –A nucleic acid sequence is translated into a protein sequence n tRNA molecules have two binding sites: –One associates with the mRNA transcript –The other associates with a specific amino acid –Each of the 20 amino acids in proteins associates with one or more of 64 types of tRNA n Translation –An mRNA transcript associates with the rRNA of a ribosome in the cytoplasm or a ribosome associated with the rough endoplasmic reticulum –The ribosome “reads” the information in the transcript –Ribosome directs various types of tRNA to bring in their specific amino acid “fares” –The tRNA specified is determined by the code being translated in the mRNA transcript 29

30. Second Step: Translation n tRNA molecules come in 64 different kinds n All are very similar except that –One end bears a specific triplet (of the 64 possible) called the anticodon –The other end binds with a specific amino acid type –tRNA synthetases attach the correct amino acid to the correct tRNA molecule n All tRNA molecules with a specific anticodon will always bind with the same amino acid 30

31. Messenger RNA Codons 31 UCAG U C A G U C A G U C A G U C A G U C A G Second Base Third Base First Base CGA arginine CGG arginine AGU serine AGC serine AGA arginine AGG arginine GGU glycine GGC glycine GGA glycine GGG glycine UGG tryptophan CGU arginine CGC arginine UGA stop AUG (start) methionine CAC histidine CAA glutamine CAG glutamine AAU asparagine AAC asparagine AAA lysine AAG lysine GAU aspartate GAC aspartate GAA glutamate GAG glutamate UUU phenylalanine CUU leucine CUC leucine CUA leucine CUG leucine AUU isoleucine AUC isoleucine AUA isoleucine GUU valine GUC valine GUA valine GUG valine UCA serine CCU proline CCC proline CCA proline CCG proline ACU threonine ACC threonine ACA threonine ACG threonine GCU alanine GCC alanine GCA alanine GCG alanine CAU histidine UAA stop UCU serine UAU tyrosine UGU cysteine UUC phenylalanine UCC serine UAC tyrosine UGU cysteine UUA leucine UCG serine UAG stop UUG leucine Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

32. 3’3’ 5’5’ hydrogen bonding amino acid leucine GAA Structure of a tRNA Molecule 32 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

33. Structure of a tRNA Molecule 33 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 3’3’ 5’5’ hydrogen bonding amino acid end amino acid leucine GAA

34. Structure of a tRNA Molecule 34 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. GAA 3’3’ 5’5’ hydrogen bonding amino acid end anticodon end amino acid leucine anticodon

35. Structure of a tRNA Molecule 35 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. G GA AA CCCCCCUUUU 3’3’ 5’5’ hydrogen bonding amino acid end anticodon end mRNA 5’5’3’3’ amino acid leucine anticodon

36. Structure of a tRNA Molecule 36 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. G GA AA CCCCCCUUUU 3’3’ 5’5’ hydrogen bonding amino acid end anticodon end mRNA 5’5’3’3’ amino acid leucine anticodon a.b. codon

37. Second Step: Translation n Ribosomes –Ribosomal RNA (rRNA): Produced from a DNA template in the nucleolus of a nucleus Combined with proteins into large and small ribosomal subunits –A completed ribosome has three binding sites to facilitate pairing between tRNA and mRNA The E (for exit) site The P (for peptide) site, and The A (for amino acid) site 37

38. Ribosome Structure and Function 38 tRNA binding sites outgoing tRNA 3 mRNA incoming tRNA polypeptide 5 small subunit mRNA large subunit a. Structure of a ribosome b. Binding sites of ribosome c. Function of ribosomes d. Polyribosome Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Courtesy Alexander Rich E P A

39. 39 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. Animation

40. Second Step: Translation n Initiation: –Components necessary for initiation are: Small ribosomal subunit mRNA transcript Initiator tRNA, and Large ribosomal subunit Initiation factors (special proteins that bring the above together) –Initiator tRNA: Always has the UAC anticodon Always carries the amino acid methionine Capable of binding to the P site 40

41. Second Step: Translation n Small ribosomal subunit attaches to mRNA transcript –Beginning of transcript always has the START codon (AUG) n Initiator tRNA (UAC) attaches to P site n Large ribosomal subunit joins the small subunit 41

42. Initiation 42 U A C A U G U AC A U G A small ribosomal subunit binds to mRNA; an initiator tRNA pairs with the mRNA start codon AUG. The large ribosomal subunit completes the ribosome. Initiator tRNA occupies the P site. The A site is ready for the next tRNA. Met amino acid methionine initiator tRNA mRNA 3 5 small ribosomal subunit large ribosomal subunit P site A site E site 3 5 Met Initiation start codon Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

43. Second Step: Elongation n “Elongation” refers to the growth in length of the polypeptide n RNA molecules bring their amino acid fares to the ribosome –Ribosome reads a codon in the mRNA Allows only one type of tRNA to bring its amino acid Must have the anticodon complementary to the mRNA codon being read The incoming tRNA joins the ribosome at its A site –Methionine of the initiator tRNA is connected to the amino acid of the 2 nd tRNA by a peptide bond 43

44. Second Step: Elongation n The second tRNA moves to P site (translocation) n The spent initiator tRNA moves to the E site and exits n The ribosome reads the next codon in the mRNA –Allows only one type of tRNA to bring its amino acid Must have the anticodon complementary to the mRNA codon being read Joins the ribosome at its A site –The dipeptide on the 2 nd amino acid is connected to the amino acid of the 3 rd tRNA by a peptide bond 44

45. Elongation 45 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. U A CA UG GAC 3 5 peptide bond Met Ala Trp Ser Val

46. Elongation 46 U A CA UG CUG GAC A tRNA–amino acid approaches the ribosome and binds at the A site. 3 5 anticodon tRNA peptide bond Met asp Ala Trp Ser Val 1 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

47. Elongation U A CA UG CUG GAC U A CA UGGAC CUG A tRNA–amino acid approaches the ribosome and binds at the A site. Two tRNAs can be at a ribosome at one time; the anticodons are paired to the codons. anticodon tRNA peptide bond Met asp Ala Trp Ser Val 3 5 3 5 Met Ala Trp Ser Val Asp 1 2 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 47

48. Elongation 48 U A CA UG CUG GAC U A CA UGGAC CUG A tRNA–amino acid approaches the ribosome and binds at the A site. Two tRNAs can be at a ribosome at one time; the anticodons are paired to the codons. anticodon tRNA peptide bond Met asp Ala Trp Ser Val Met Ala Trp Ser ValAsp 1 2 U A CA UGGAC CUG Peptide bond formation attaches the peptide chain to the newly arrived amino acid. 3 5 3 5 3 5 Met Val Asp Ala Trp Ser 3 peptide bond Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

49. Elongation 49 U A CA UG CUG GAC U A CA UGGAC CUG A tRNA–amino acid approaches the ribosome and binds at the A site. Two tRNAs can be at a ribosome at one time; the anticodons are paired to the codons. anticodon tRNApeptide bond asp 3 5 5 Asp 12 4 U A C A UG G A C CUG U C A GAC CUG AUG ACC Peptide bond formation attaches the peptide chain to the newly arrived amino acid. The ribosome moves forward; the “empty” tRNA exits from the E site; the next amino acid–tRNA complex is approaching the ribosome. 3 5 Met Val Asp Ala Trp Ser 33 5 Met Val Asp Ala Trp Ser 3 peptide bond Met Ala Trp Ser Val Met Ala Trp Ser Val Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

50. Elongation 50 U A CA UG CUG GAC U A CA UGGAC CUG A tRNA–amino acid approaches the ribosome and binds at the A site. Two tRNAs can be at a ribosome at one time; the anticodons are paired to the codons. anticodon tRNApeptide bond asp 3 5 5 Asp 12 4 U A C A UG G A C CUG U C A GAC CUG AUG U G G ACC Peptide bond formation attaches the peptide chain to the newly arrived amino acid. The ribosome moves forward; the “empty” tRNA exits from the E site; the next amino acid–tRNA complex is approaching the ribosome. 3 5 Met Val Asp Ala Trp Ser 33 5 Met Val Asp Ala Trp Ser Thr 3 peptide bond Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Met Ala Trp Ser Val Met Ala Trp Ser Val

51. Elongation 51 Elongation Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. U A CA UG CUG GAC U A CA UGGAC CUG A tRNA–amino acid approaches the ribosome and binds at the A site. Two tRNAs can be at a ribosome at one time; the anticodons are paired to the codons. anticodon tRNApeptide bond asp 3 5 5 Asp 12 4 U A C A UG G A C CUG U C A GAC CUG AUG U G G ACC Peptide bond formation attaches the peptide chain to the newly arrived amino acid. The ribosome moves forward; the “empty” tRNA exits from the E site; the next amino acid–tRNA complex is approaching the ribosome. 3 5 Met Val Asp Ala Trp Ser 33 5 Met Val Asp Ala Trp Ser Thr 3 peptide bond Met Ala Trp Ser Val Met Ala Trp Ser Val

52. Second Step: Translation n Termination: –Previous tRNA moves to the P site –Spent tRNA moves to the E site and exits –Ribosome reads the STOP codon at the end of the mRNA UAA, UAG, or UGA Does not code for an amino acid –A protein called a release factor binds to the stop codon and cleaves the polypeptide from the last tRNA –The ribosome releases the mRNA and dissociates into subunits –The same mRNA may be read by another ribosome 52

53. Termination 53 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. The ribosome comes to a stop codon on the mRNA. A release factor binds to the site. The release factor hydrolyzes the bond between the last tRNA at the P site and the polypeptide, releasing them. The ribosomal subunits dissociate. release factor Ala Asp Glu U A U A UGA A G A U G A U C U Termination stop codon Asp Ala Trp Val Glu 3′ 5′ 3′

54. Summary of Protein Synthesis in Eukaryotes 54 5 5 3 mRNA pre-mRNA DNA introns 6. During elongation, polypeptide synthesis takes place one amino acid at a time. 7. Ribosome attaches to rough ER. Polypeptide enters lumen, where it folds and is modified. 3. mRNA moves into cytoplasm and becomes associated with ribosomes. 4. tRNAs with anticodons carry amino acids to mRNA. TRANSLATION TRANSCRIPTION nuclear pore mRNA large and small ribosomal subunits amino acids anticodon tRNA peptide codon ribosome 5 3 3 CC GG G C G C G C C C C G U A C U A U A U A U U A U A A U C G A 1. DNA in nucleus serves as a template for mRNA. 2. mRNA is processed before leaving the nucleus. 5. During initiation, anticodon-codon complementary base pairing begins as the ribosomal subunits come together at a start codon. 8. During termination, a ribosome reaches a stop codon; mRNA and ribosomal subunits disband. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

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56. 12.6 Structure of the Eukaryotic Chromosome n Each chromosome contains a single linear DNA molecule, but is composed of more than 50% protein. n Some of these proteins are concerned with DNA and RNA synthesis n Histones play primarily a structural role –The most abundant proteins in chromosomes –Five primary types of histone molecules – Responsible for packaging the DNA The DNA double helix is wound at intervals around a core of eight histone molecules (called a nucleosome) Nucleosomes are joined by “linker” DNA. 56

57. Structure of Eukaryotic Chromosomes 57 a: © Ada L. Olins and Donald E. Olins/Biological Photo Service; b: Courtesy Dr. Jerome Rattner, Cell Biology and Anatomy, University of Calgary; c: Courtesy of Ulrich Laemmli and J.R. Paulson, Dept. of Molecular Biology, University of Geneva, Switzerland; d: © Peter Engelhardt/Department of Pathology and Virology, Haartman Institute/Centre of Excellence in Computational Complex Systems Research, Biomedical Engineering and Computational Science, Faculty of Information and Natural Sciences, Helsinki University of Technology, Helsinki, Finland Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 1. Wrapping of DNA around histone proteins. 2. Formation of a three-dimensional zigzag structure via histone H1 and other DNA-binding proteins. 3. Loose coiling into radial loops. 4. Tight compaction of radial loops to form heterochromatin. 5. Metaphase chromosome forms with the help of a protein scaffold. DNA double helix nucleosome euchromatin c. Radial loop domains d. Heterochromatin e. Metaphase chromosome 1,400 nm 700 nm 300 nm 30 nm histone H1 histones 11 nm 2nm a. Nucleosomes (“beads on a string”) b. 30-nm fiber

58. Mutations: genetic material changes in a cell n Point mutations: Changes in 1 or a few base pairs in a single gene n Base-pair substitutions: –silent mutations no effect on protein –missense ∆ to a different amino acid (different protein) –nonsense ∆ to a stop codon and a nonfunctional protein n Base-pair insertions or deletions: additions or losses of nucleotide pairs in a gene; alters the ‘reading frame’ of triplets~frameshift mutation n Mutagens: physical and chemical agents that change DNA

59. Figure 17.23 The molecular basis of sickle-cell disease: a point mutation

60. Figure 17.24 Categories and consequences of point mutations: Base-pair insertion or deletion

61. Figure 17.24 Categories and consequences of point mutations: Base-pair substitution