1. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display 8-1 Powerpoint to accompany Genetics: From Genes to Genomes Third Edition Hartwell ● Hood ● Goldberg ● Reynolds ● Silver ● Veres Chapter 8 Prepared by Malcolm Schug University of North Carolina Greensboro

2. 8-2 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Gene Expression The Flow of Genetic Information from DNA via RNA to Protein

3. 8-3 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Outline of Chapter 8 The genetic code The genetic code How triplets of the four nucleotides unambiguously specify 20 amino acids, making it possible to translate information from a nucleotide chain to a sequence of amino acids How triplets of the four nucleotides unambiguously specify 20 amino acids, making it possible to translate information from a nucleotide chain to a sequence of amino acids Transcription Transcription How RNA polymerase, guided by base pairing, synthesizes a single-stranded mRNA copy of a gene’s DNA template How RNA polymerase, guided by base pairing, synthesizes a single-stranded mRNA copy of a gene’s DNA template Translation Translation How base pairing between mRNA and tRNAs directs the assembly of a polypeptide on the ribosome How base pairing between mRNA and tRNAs directs the assembly of a polypeptide on the ribosome Significant differences in gene expression between prokaryotes and eukaryotes Significant differences in gene expression between prokaryotes and eukaryotes How mutations affect gene information and expression How mutations affect gene information and expression

4. 8-4 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display The triplet codon represents each amino acid. 20 amino acids encoded for by 4 nucleotides 20 amino acids encoded for by 4 nucleotides By deduction: By deduction: 1 nucleotide/amino acid = 4 1 = 4 triplet combinations. 1 nucleotide/amino acid = 4 1 = 4 triplet combinations. 2 nucleotides/amino acid = 4 2 = triplet combinations. 2 nucleotides/amino acid = 4 2 = triplet combinations. 3 nucleotides/amino acid = 4 3 = triplet combinations. 3 nucleotides/amino acid = 4 3 = triplet combinations. Must be at least triplet combinations that code for amino acids Must be at least triplet combinations that code for amino acids

5. 8-5 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display The Genetic Code: 61 triplet codons represent 20 amino acids; 3 triplet codons signify stop. Fig. 8.3

6. 8-6 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display A gene’s nucleotide sequence is colinear the amino acid sequence of the encoded polypeptide. Charles Yanofsky – E. coli genes for a subunit of tyrptophan synthetase compared mutations within a gene to particular amino acid substitutions. Charles Yanofsky – E. coli genes for a subunit of tyrptophan synthetase compared mutations within a gene to particular amino acid substitutions. Trp- mutants in trpA Trp- mutants in trpA Fine structure recombination map Fine structure recombination map Determined amino acid sequences of mutants Determined amino acid sequences of mutants

7. 8-7 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Fig. 8.4

8. 8-8 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display A codon is composed of more than one nucleotide. A codon is composed of more than one nucleotide. Different point mutations may affect same amino acid. Different point mutations may affect same amino acid. Codon contains more than one nucleotide. Codon contains more than one nucleotide. Each nucleotide is part of only a single codon. Each nucleotide is part of only a single codon. Each point mutation altered only one amino acid. Each point mutation altered only one amino acid.

9. 8-9 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display A codon is composed of three nucleotides and the starting point of each gene establishes a reading frame. studies of frameshift mutations in bacteriophage T4 rIIB gene Fig. 8.5

10. 8-10 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Most amino acids are specified by more than one codon. Most amino acids are specified by more than one codon. Phenotypic effect of frameshifts depends on if reading frame is restored. Phenotypic effect of frameshifts depends on if reading frame is restored. Fig. 8.6

11. 8-11 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Cracking the code: biochemical manipulations revealed which codons represent which amino acids. The discovery of messenger RNAs, molecules for transporting genetic information The discovery of messenger RNAs, molecules for transporting genetic information Protein synthesis takes place in cytoplasm deduced from radioactive tagging of amino acids. Protein synthesis takes place in cytoplasm deduced from radioactive tagging of amino acids. RNA, an intermediate molecule made in nucleus and transports DNA information to cytoplasm RNA, an intermediate molecule made in nucleus and transports DNA information to cytoplasm

12. 8-12 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Synthetic mRNAs and in vitro translation determines which codons designate which amino acids. 1961 – Marshall Nirenberg and Heinrich Mathaei created mRNAs and translated to polypeptides in vitro 1961 – Marshall Nirenberg and Heinrich Mathaei created mRNAs and translated to polypeptides in vitro Polymononucleotides Polymononucleotides Polydinucleotides Polydinucleotides Polytrinucleotides Polytrinucleotides Polytetranucleotides Polytetranucleotides Read amino acid sequence and deduced codons Read amino acid sequence and deduced codons Fig. 8.7

13. 8-13 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Ambiguities resolved by Nirenberg and Philip Leder using trinucleotide mRNAs of known sequence to tRNAs charged with radioactive amino acid with ribosomes Ambiguities resolved by Nirenberg and Philip Leder using trinucleotide mRNAs of known sequence to tRNAs charged with radioactive amino acid with ribosomes Fig. 8.8

14. 8-14 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display 5’ to 3’ direction of mRNA corresponds to N-terminal-to- C-terminal direction of polypeptide. 5’ to 3’ direction of mRNA corresponds to N-terminal-to- C-terminal direction of polypeptide. One strand of DNA is a template. One strand of DNA is a template. The other is an RNA-like strand. The other is an RNA-like strand. Nonsense codons cause termination of a polypeptide chain – UAA (ocher), UAG (amber), and UGA (opal). Nonsense codons cause termination of a polypeptide chain – UAA (ocher), UAG (amber), and UGA (opal). Fig. 8.9

15. 8-15 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Summary Codon consist of a triplet codon each of which specifies an amino acid. Codon consist of a triplet codon each of which specifies an amino acid. Code shows a 5’ to 3’ direction. Code shows a 5’ to 3’ direction. Codons are nonoverlapping. Codons are nonoverlapping. Code includes three stop codons, UAA, UAG, and UGA that terminate translation. Code includes three stop codons, UAA, UAG, and UGA that terminate translation. Code is degenerate. Code is degenerate. Fixed starting point establishes a reading frame. Fixed starting point establishes a reading frame. UAG in an initiation codon which specifies reading frame. UAG in an initiation codon which specifies reading frame. 5’- 3’ direction of mRNA corresponds with N-terminus to C- terminus of polypeptide. 5’- 3’ direction of mRNA corresponds with N-terminus to C- terminus of polypeptide. Mutaiton modify message encoded in sequence Mutaiton modify message encoded in sequence Frameshift mutaitons change reading frame. Frameshift mutaitons change reading frame. Missense mutations change codon of amino acid to another amino acid. Missense mutations change codon of amino acid to another amino acid. Nonsense mutations change a codon for an amino acid to a stop codon. Nonsense mutations change a codon for an amino acid to a stop codon.

16. 8-16 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Do living cells construct polypeptides according to same rules as in vitro experiments? Studies of how mutations affect amino-acid composition of polypeptides encoded by a gene Studies of how mutations affect amino-acid composition of polypeptides encoded by a gene Missense mutations induced by mutagens should be single nucleotide substitutions and conform to the code. Missense mutations induced by mutagens should be single nucleotide substitutions and conform to the code. Fig. 8.10 a

17. 8-17 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Proflavin treatment generates trp- mutants. Proflavin treatment generates trp- mutants. Further treatment generates trp+ revertants. Further treatment generates trp+ revertants. Single base insertion (trp-) and a deletion causes reversion (trp+). Single base insertion (trp-) and a deletion causes reversion (trp+). Fig. 8.10 b

18. 8-18 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Genetic code is almost universal but not quite. All living organisms use same basic genetic code. All living organisms use same basic genetic code. Translational systems can use mRNA from another organism to generate protein. Translational systems can use mRNA from another organism to generate protein. Comparisons of DNA and protein sequence reveal perfect correspondence between codons and amino acids among all organisms. Comparisons of DNA and protein sequence reveal perfect correspondence between codons and amino acids among all organisms.

19. 8-19 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Transcription RNA polymerase catalyzes transcription. RNA polymerase catalyzes transcription. Promoters signal RNA polymerase where to begin transcription. Promoters signal RNA polymerase where to begin transcription. RNA polymerase adds nucleotides in 5’ to 3’ direction. RNA polymerase adds nucleotides in 5’ to 3’ direction. Terminator sequences tell RNA when to stop transcription. Terminator sequences tell RNA when to stop transcription.

20. 8-20 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Initiation of transcription Fig. 8.11 a

21. 8-21 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Elongation Fig. 8.11 b

22. 8-22 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Termination Fig. 8.11 c

23. 8-23 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Information flow Fig. 8.11 d

24. 8-24 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Promoters of 10 different bacterial genes Fig. 8.12

25. 8-25 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display In eukaryotes, RNA is processed after transcription. A 5’ methylated cap and a 3’ Poly-A tail are added. Structure of the methylated cap Fig. 8.13

26. 8-26 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display How Poly-A tail is added to 3’ end of mRNA Fig. 8.14

27. 8-27 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display RNA splicing removes introns. Exons – sequences found in a gene’s DNA and mature mRNA (expressed regions) Exons – sequences found in a gene’s DNA and mature mRNA (expressed regions) Introns – sequences found in DNA but not in mRNA (intervening regions) Introns – sequences found in DNA but not in mRNA (intervening regions) Some eukaryotic genes have many introns. Some eukaryotic genes have many introns.

28. 8-28 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Dystrophin gene underlying Duchenne muscular dystrophy (DMD) is an extreme example of introns. Fig. 8.15

29. 8-29 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display How RNA processing splices out introns and adjoins adjacent exons Fig. 8.16

30. 8-30 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Splicing is catalyzed by spliceosomes. Splicing is catalyzed by spliceosomes. Ribozymes – RNA molecules that act as enzymes Ribozymes – RNA molecules that act as enzymes Ensures that all splicing reactions take place in concert Ensures that all splicing reactions take place in concert Fig. 8.17

31. 8-31 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Alternative splicing Alternative splicing Different mRNAs can be produced by same transcript. Different mRNAs can be produced by same transcript. Rare transplicing events combine exons from different genes. Rare transplicing events combine exons from different genes. Fig. 8.18

32. 8-32 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Translation Transfer RNAs (tRNAs) mediate translation of mRNA codons to amino acids. Transfer RNAs (tRNAs) mediate translation of mRNA codons to amino acids. tRNAs carry anticodon on one end. tRNAs carry anticodon on one end. Three nucleotides complementary to an mRNA codon Three nucleotides complementary to an mRNA codon Structure of tRNA Structure of tRNA Primary – nucleotide sequence Primary – nucleotide sequence Secondary – short complementary sequences pair and make clover leaf shape Secondary – short complementary sequences pair and make clover leaf shape Tertiary – folding into three dimensional space shape like an L Tertiary – folding into three dimensional space shape like an L Base pairing between an mRNA codon and a tRNA anticodon directs amino acid incorporation into a growing polypeptide. Base pairing between an mRNA codon and a tRNA anticodon directs amino acid incorporation into a growing polypeptide. Charged tRNA is covalently coupled to its amino acid. Charged tRNA is covalently coupled to its amino acid.

33. 8-33 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Many tRNAs contain modified bases. Fig. 8.19 a

34. 8-34 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Secondary and tertiary structure Fig. 8.19 b

35. 8-35 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Aminoacyl-tRNA syntetase catalyzes attachment of tRNAs to corresponding amino acid. Fig. 8.20

36. 8-36 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Base pairing between mRNA codon and tRNA anticodon determines where incorporation of amino acid occurs. Fig. 8.21

37. 8-37 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Wobble: Some tRNAs recognize more than one codon for amino acids they carry. Fig. 8.22

38. 8-38 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Rhibosomes are site of polypeptide synthesis. Ribosomes are complex structures composed of RNA and protein. Ribosomes are complex structures composed of RNA and protein. Fig. 8.23

39. 8-39 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Mechanism of translation Initiation sets stage for polypeptide synthesis. Initiation sets stage for polypeptide synthesis. AUG start codon at 5’ end of mRNA AUG start codon at 5’ end of mRNA Formalmethionine (fMet) on initiation tRNA Formalmethionine (fMet) on initiation tRNA First amino acid incorporated in bacteria First amino acid incorporated in bacteria Elongation during which amino acids are added to growing polypeptide Elongation during which amino acids are added to growing polypeptide Ribosomes move in 5’-3’ direction revealing codons. Ribosomes move in 5’-3’ direction revealing codons. Addition of amino acids to C terminus Addition of amino acids to C terminus 2-15 amino acids per second 2-15 amino acids per second Termination which halts polypeptide synthesis Termination which halts polypeptide synthesis Nonsense codon recognized at 3’ end of reading frame Nonsense codon recognized at 3’ end of reading frame Release factor proteins and halt polypeptide synthesis Release factor proteins and halt polypeptide synthesis

40. 8-40 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Initiation of translation Fig. 8.25 a

41. 8-41 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Elongation Fig. 8.25 b

42. 8-42 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Termination of translation Fig. 8.25 c

43. 8-43 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Posttranslational processing can modify polypeptide structure. Posttranslational processing can modify polypeptide structure. Fig. 8.26

44. 8-44 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Significant differences in gene expression between prokaryotes and eukaryotes Eukaryotes, nuclear membrane prevents coupling of transcription and translation. Eukaryotes, nuclear membrane prevents coupling of transcription and translation. Prokaryotic messages are polycistronic. Prokaryotic messages are polycistronic. Contain information for multiple genes Contain information for multiple genes Eukaryotes, small ribosomal subunit binds to 5’ methylated cap and migrates to AUG start codon. Eukaryotes, small ribosomal subunit binds to 5’ methylated cap and migrates to AUG start codon. 5’ untranslated leader sequence – between 5’ cap and AUG start 5’ untranslated leader sequence – between 5’ cap and AUG start Only a single polypeptide produced from each gene Only a single polypeptide produced from each gene Initiating tRNA in prokaryotes is fMet. Initiating tRNA in prokaryotes is fMet. Initiating tRNA in eukaryotes Met is unmodified. Initiating tRNA in eukaryotes Met is unmodified.

45. 8-45 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

46. 8-46 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display A computerized analysis of gene expression in C. elegans: A comprehensive example Computer programs search for possible exons by looking for strings of codons uninterrupted by nonsense codons. Computer programs search for possible exons by looking for strings of codons uninterrupted by nonsense codons. Look for splice donor and acceptor sites to identify introns. Look for splice donor and acceptor sites to identify introns. C. elegans genome contains roughly 19,000 genes. C. elegans genome contains roughly 19,000 genes. 15% encode worm’s genes or proteins. 15% encode worm’s genes or proteins.

47. 8-47 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Landmarks in a callogen gene of C. elegans and comparison of DNA and mRNA sequences Fig. 8.27

48. 8-48 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Mutations in a gene’s coding sequence can alter the gene product. Mutations in a gene’s coding sequence can alter the gene product. Missense mutations replace one amino acid with another. Missense mutations replace one amino acid with another. Nonsense mutations change an amino-acid-specifying codon to a stop codon. Nonsense mutations change an amino-acid-specifying codon to a stop codon. Frameshift mutations result from the insertion or deletion of nucleotides within the coding sequence. Frameshift mutations result from the insertion or deletion of nucleotides within the coding sequence. Silent mutations do not alter amino acid specified. Silent mutations do not alter amino acid specified. Fig. 8.28

49. 8-49 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Mutations outside of the coding sequence can also alter gene expression. Mutations outside of the coding sequence can also alter gene expression. Promoter sequences Promoter sequences Termination signals Termination signals Splice-acceptor and splice-donor sites Splice-acceptor and splice-donor sites Ribosome binding sites Ribosome binding sites Fig. 8.28 b

50. 8-50 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Loss-of-function alleles are usually recessive. Null or amorphic mutations are alleles that completely block the function of a protein. Null or amorphic mutations are alleles that completely block the function of a protein. Hypomorphic mutations produce much less of a protein or a protein with weak but detectable function. Hypomorphic mutations produce much less of a protein or a protein with weak but detectable function. Rocket immunoelectrophoresis reveals the amount of xanthine dehydrogenase produced in flies with different genotypes. Null allele 1 and hypomorphic allele 2 are recessive to wildtype. Fig. 8.29

51. 8-51 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Incomplete dominance arises when phenotype varies in proportion to the amount of protein.

52. 8-52 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Rarely, loss-of-function mutations are dominant. Haploinsufficiency – one wild-type allele does not provide enough of a gene product Haploinsufficiency – one wild-type allele does not provide enough of a gene product Heterozygotes for the null mutation of the T locus in mice have short tails because they have an insufficient amount of protein to produce a wild-type tail. Fig. 8.31 a

53. 8-53 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Dominant-negative mutations – alleles of a gene encoding subunits of multimers that block the activity of subunits produced by normal alleles Dominant-negative mutations – alleles of a gene encoding subunits of multimers that block the activity of subunits produced by normal alleles Rarely, loss-of-function mutations are dominant. Fig. 8.31 b

54. 8-54 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Kinky: A dominant-negative mutation in mice causing a kink in the tail Fig. 8.31 c

55. 8-55 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Gain-of-function mutations are almost always dominant. Rare mutations that enhance a protein function or even confer a new activity on a protein Rare mutations that enhance a protein function or even confer a new activity on a protein Antennapedia is a neomorphic mutation causing ectopic expression of a leg-determining gene in structures that normally produce antennae. Fig. 8.31 d

56. 8-56 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Mutations in genes encoding the molecules that implement expression may affect transcription, l mRNA splicing, or translation. Mutations in genes encoding the molecules that implement expression may affect transcription, l mRNA splicing, or translation. Usually lethal Usually lethal Mutations in tRNA genes can suppress mutations in protein-coding genes. Mutations in tRNA genes can suppress mutations in protein-coding genes. Nonsense suppressor tRNAs Nonsense suppressor tRNAs

57. 8-57 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

58. 8-58 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Nonsense suppression Nonsense suppression (a) Nonsense mutation that causes incomplete nonfunctional polypeptide (a) Nonsense mutation that causes incomplete nonfunctional polypeptide (b) Nonsense- suppressing mutation causes addition of amino acid at stop codon allowing production of full length polypeptide. (b) Nonsense- suppressing mutation causes addition of amino acid at stop codon allowing production of full length polypeptide. Fig. 8.32