1. The Molecular Biology of Genes and Gene Expression

2. Central Dogma First described by Francis Crick Information only flows from DNA → RNA → protein Transcription = DNA → RNA Translation = RNA → protein Retroviruses violate this order using reverse transcriptase to convert their RNA genome into DNA 2

3. DNA RNA Protein replication (mutation!) transcription translation (amino acids) (nucleotides: A,T,G,C) “software” ~ DNA, RNA “hardware” ~ proteins How Genes Work: A Primer genes (nucleotides: A,U,G,C)

4. The Nature of Genes Early ideas to explain how genes work came from studying human diseases Archibald Garrod – 1902 –Recognized that alkaptonuria is inherited via a recessive allele –Proposed that patients with the disease lacked a particular enzyme These ideas connected genes to enzymes 4

5. 5 Beadle and Tatum – 1941 Deliberately set out to create mutations in chromosomes and verify that they behaved in a Mendelian fashion in crosses Studied Neurospora crassa –Used X-rays to damage DNA –Looked for nutritional mutations Had to have minimal media supplemented to grow

6. 6 Beadle and Tatum looked for fungal cells lacking specific enzymes –The enzymes were required for the biochemical pathway producing the amino acid arginine –They identified mutants deficient in each enzyme of the pathway One-gene/one-enzyme hypothesis has been modified to one-gene/one-polypeptide hypothesis

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9. 9 Transcription –DNA-directed synthesis of RNA –Only template strand of DNA used –U (uracil) in DNA replaced by T (thymine) in RNA –mRNA used to direct synthesis of polypeptides Translation –Synthesis of polypeptides –Takes place at ribosome –Requires several kinds of RNA

10. RNA All synthesized from DNA template by transcription Messenger RNA (mRNA) Ribosomal RNA (rRNA) Transfer RNA (tRNA) Small nuclear RNA (snRNA) Signal recognition particle RNA Micro-RNA (miRNA) 10

11. Genetic Code Francis Crick and Sydney Brenner determined how the order of nucleotides in DNA encoded amino acid order Codon – block of 3 DNA nucleotides corresponding to an amino acid Introduced single nulcleotide insertions or deletions and looked for mutations –Frameshift mutations Indicates importance of reading frame 11

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13. 13 Marshall Nirenberg identified the codons that specify each amino acid Stop codons –3 codons (UUA, UGA, UAG) used to terminate translation Start codon –Codon (AUG) used to signify the start of translation Code is degenerate, meaning that some amino acids are specified by more than one codon

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15. Code practically universal Strongest evidence that all living things share common ancestry Advances in genetic engineering Mitochondria and chloroplasts have some differences in “stop” signals 15

16. 16 Prokaryotic transcription Single RNA polymerase Initiation of mRNA synthesis does not require a primer Requires –Promoter –Start siteTranscription unit –Termination site

17. 17 Promoter –Forms a recognition and binding site for the RNA polymerase –Found upstream of the start site –Not transcribed –Asymmetrical – indicate site of initiation and direction of transcription

18. 18 Upstream Downstream 5׳5׳ 3׳3׳ b. Coding strand Template strand 5׳5׳ 3׳3׳ Start site (+1) TATAAT– Promoter (–10 sequence) TTGACA–Promoter (–35 sequence) Holoenzyme    99  a. Prokaryotic RNA polymerase Core enzyme  binds to DNA  dissociates ATP Start site RNA synthesis begins Transcription bubble RNA polymerase bound to unwound DNA Helix opens at –10 sequence 5׳5׳3׳3׳ 5׳5׳ 3׳3׳ Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

19. Elongation –Grows in the 5′-to-3′ direction as ribonucleotides are added –Transcription bubble – contains RNA polymerase, DNA template, and growing RNA transcript –After the transcription bubble passes, the now-transcribed DNA is rewound as it leaves the bubble 19

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21. 21 Termination –Marked by sequence that signals “stop” to polymerase Causes the formation of phosphodiester bonds to cease RNA–DNA hybrid within the transcription bubble dissociates RNA polymerase releases the DNA DNA rewinds –Hairpin

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23. Prokaryotic transcription is coupled to translation –mRNA begins to be translated before transcription is finished –Operon Grouping of functionally related genes Multiple enzymes for a pathway Can be regulated together 23

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25. 25 Eukaryotic Transcription 3 different RNA polymerases –RNA polymerase I transcribes rRNA –RNA polymerase II transcribes mRNA and some snRNA –RNA polymerase III transcribes tRNA and some other small RNAs Each RNA polymerase recognizes its own promoter

26. 26 Initiation of transcription –Requires a series of transcription factors Necessary to get the RNA polymerase II enzyme to a promoter and to initiate gene expression Interact with RNA polymerase to form initiation complex at promoter Termination –Termination sites not as well defined

27. 27 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Other transcription factors RNA polymerase II TATA box Transcription factor Eukaryotic DNA Initiation complex

28. Enhancers determine the temporal and spatial transcription patterns of genes (drawing modified from Tijan, R., Molecular Machines That Control Genes, Scientific American 272, Feb, 1995) Activators = + Auxiliary Transcription Factors Enhancers Silencer Repressor = - auxiliary transcription factor Co-activators TATA BOX REPRESSOR CORE PROMOTER transcription Basal Transcription Factors RNA Polymerase H E F B A

29. 29 In eukaryotes, the primary transcript must be modified to become mature mRNA –Addition of a 5′ cap Protects from degradation; involved in translation initiation –Addition of a 3′ poly-A tail Created by poly-A polymerase; protection from degradation –Removal of non-coding sequences (introns) Pre-mRNA splicing done by spliceosome mRNA modifications

30. 30 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. A A A A A A A Methyl group 3´ poly-A tail 5´ cap CH 2 HOOH P P P G mRNA P P P + N+N+ CH 3 5´ 3´ CH 3

31. Eukaryotic pre-mRNA splicing Introns – non-coding sequences Exons – sequences that will be translated Small ribonucleoprotein particles (snRNPs) recognize the intron–exon boundaries snRNPs cluster with other proteins to form spliceosome –Responsible for removing introns 31

32. 32 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. E1I1E2I2E3I3E4I4 Transcription Introns are removed a. Exons Introns Mature mRNA 33׳ poly-A tail5׳ cap 3׳ poly-A tail Primary RNA transcript DNA template

33. 33 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. E1I1E2I2E3I3E4I4 Transcription Introns are removed Intron Exon DNA 1 2 3 4 5 6 7 a. b.c. Exons Introns mRNA Mature mRNA 3׳ poly-A tail 5׳ cap 3׳ poly-A tail Primary RNA transcript DNA template b: Courtesy of Dr. Bert O’Malley, Baylor College of Medicine

34. 34 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. snRNPs Exon 2Exon 1Intron Branch point A snRNA 5´ 3´ 1. snRNA forms base-pairs with 5´ end of intron, and at branch site. A

35. 35 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. snRNPs Exon 2Exon 1 Intron Branch point A snRNA Spliceosome 5´ 3´ 2. snRNPs associate with other factors to form spliceosome. 1. snRNA forms base-pairs with 5´ end of intron, and at branch site. A A

36. 36 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. snRNPs Exon 2 Exon 1 Intron Branch point A snRNA Spliceosome Lariat 5´ 3´ 5´3´ 2. snRNPs associate with other factors to form spliceosome. 1. snRNA forms base-pairs with 5´ end of intron, and at branch site. 3. 5´ end of intron is removed and forms bond at branch site, forming a lariat. The 3´ end of the intron is then cut. A A A

37. 37 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. snRNPs Exon 2Exon 1Intron Branch point A snRNA Exon 1Exon 2 Lariat 5´ 3´ 5´ 3´ 2. snRNPs associate with other factors to form spliceosome. 4. Exons are joined; spliceosome disassembles. 1. snRNA forms base-pairs with 5´ end of intron, and at branch site. 3. 5´ end of intron is removed and forms bond at branch site, forming a lariat. The 3´ end of the intron is then cut. Mature mRNA Excised intron Spliceosome A A A

38. Alternative splicing Single primary transcript can be spliced into different mRNAs by the inclusion of different sets of exons 15% of known human genetic disorders are due to altered splicing 35 to 59% of human genes exhibit some form of alternative splicing Explains how 25,000 genes of the human genome can encode the more than 80,000 different mRNAs 38

39. DNA hnRNA mRNA 1 mRNA 2 protein 1 protein 2 translation RNA splicing Differential RNA splicing can lead to different protein products Differential RNA splicing can lead to different protein products 5’ ut 3’ ut exon 1 exon 2 exon 3 intron 1 intron 2

40. tRNA and Ribosomes tRNA molecules carry amino acids to the ribosome for incorporation into a polypeptide –Aminoacyl-tRNA synthetases add amino acids to the acceptor stem of tRNA –Anticodon loop contains 3 nucleotides complementary to mRNA codons 40

41. 41 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. c: Created by John Beaver using ProteinWorkshop, a product of the RCSB PDB, and built using the Molecular Biology Toolkit developed by John Moreland and Apostol Gramada (mbt.sdsc.edu). The MBT is fi nanced by grant GM63208 2D “Cloverleaf” Model Acceptor end Anticodon loop 3׳3׳ 5׳5׳ 3D Ribbon-like Model Acceptor end Anticodon loop 3D Space-filled Model Anticodon loop Acceptor end Icon Anticodon end Acceptor end

42. tRNA charging reaction Each aminoacyl-tRNA synthetase recognizes only 1 amino acid but several tRNAs Charged tRNA – has an amino acid added using the energy from ATP –Can undergo peptide bond formation without additional energy Ribosomes do not verify amino acid attached to tRNA 42

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44. 44 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. PiPi PiPi Amino group Carboxyl group Trp ATP Amino acid site Aminoacyl-tRNA synthetase tRNA site NH 3 + C O–O– O

45. 45 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. tRNA PiPi PiPi NH 3 + O–O– C O C O C O OH O AMP O OH AMP Amino group Carboxyl group Trp NH 3 + Trp ATP Amino acid site Accepting site Anticodon specific to tryptophan Aminoacyl-tRNA synthetase tRNA site

46. 46 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. tRNA PiPi PiPi NH 3 + O–O– C O C O C O C O C O OH O AMP O OH AMP O O Charged tRNA travels to ribosome Amino group Carboxyl group Trp NH 3 + Trp ATP Amino acid site Accepting site Anticodon specific to tryptophan Aminoacyl-tRNA synthetase tRNA site Charged tRNA dissociates Trp

47. 47 The ribosome has multiple tRNA binding sites –P site – binds the tRNA attached to the growing peptide chain –A site – binds the tRNA carrying the next amino acid –E site – binds the tRNA that carried the last amino acid

48. 48 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. mRNA 90 ° 0 ° 3´ 5´5´ Large subunit Small subunit Large subunit Small subunit Large subunit Small subunit

49. 49 The ribosome has two primary functions –Decode the mRNA –Form peptide bonds Peptidyl transferase –Enzymatic component of the ribosome –Forms peptide bonds between amino acids

50. 50 Translation In prokaryotes, i nitiation complex includes –Initiator tRNA charged with N-formylmethionine –Small ribosomal subunit –mRNA strand Ribosome binding sequence (RBS) of mRNA positions small subunit correctly Large subunit now added Initiator tRNA bound to P site with A site empty

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52. Initiations in eukaryotes similar except –Initiating amino acid is methionine –More complicated initiation complex –Lack of an RBS – small subunit binds to 5′ cap of mRNA 52

53. 53 Elongation adds amino acids –2 nd charged tRNA can bind to empty A site –Requires elongation factor called EF-Tu to bind to tRNA and GTP –Peptide bond can then form –Addition of successive amino acids occurs as a cycle

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56. 56 There are fewer tRNAs than codons Wobble pairing allows less stringent pairing between the 3′ base of the codon and the 5′ base of the anticodon This allows fewer tRNAs to accommodate all codons

57. 57 Termination –Elongation continues until the ribosome encounters a stop codon –Stop codons are recognized by release factors which release the polypeptide from the ribosome

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59. 59 Protein targeting In eukaryotes, translation may occur in the cytoplasm or the rough endoplasmic reticulum (RER) Signal sequences at the beginning of the polypeptide sequence bind to the signal recognition particle (SRP) The signal sequence and SRP are recognized by RER receptor proteins Docking holds ribosome to RER Beginning of the protein-trafficking pathway

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61. 61 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 1. RNA polymerase II in the nucleus copies one strand of the DNA to produce the primary transcript. Primary RNA transcript RNA polymerase II Primary RNA transcript 5´ 3´ 5´ 3´

62. 62 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Primary RNA transcript Poly-A tail Mature mRNA Cut intron 5´ cap 1. RNA polymerase II in the nucleus copies one strand of the DNA to produce the primary transcript. 2. The primary transcript is processed by addition of a 5´ methyl-G cap, cleavage and polyadenylation of the 3´ end, and removal of introns. The mature mRNA is then exported through nuclear pores to the cytoplasm. Primary RNA transcript RNA polymerase II Primary RNA transcript Poly-A tail Mature mRNA Cut intron 5´ cap RNA polymerase II Primary RNA transcript 5´ 3´ 5´ 3´

63. 63 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Primary RNA transcript Poly-A tail Mature mRNA Cut intron 5´ cap 1. RNA polymerase II in the nucleus copies one strand of the DNA to produce the primary transcript. 2. The primary transcript is processed by addition of a 5´ methyl-G cap, cleavage and polyadenylation of the 3´ end, and removal of introns. The mature mRNA is then exported through nuclear pores to the cytoplasm. 3. The 5´ cap of the mRNA associates with the small subunit of the ribosome. The initiator tRNA and large subunit are added to form an initiation complex. Primary RNA transcript RNA polymerase II Primary RNA transcript Poly-A tail Mature mRNA Cut intron 5´ cap Large subunit mRNA 5´ cap Small subunit Cytoplasm RNA polymerase II Primary RNA transcript 5´ 3´ 5´ 3´

64. 64 Primary RNA transcript Cut intron 5´ cap Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 1. RNA polymerase II in the nucleus copies one strand of the DNA to produce the primary transcript. 2. The primary transcript is processed by addition of a 5´ methyl-G cap, cleavage and polyadenylation of the 3´ end, and removal of introns. The mature mRNA is then exported through nuclear pores to the cytoplasm. 3. The 5´ cap of the mRNA associates with the small subunit of the ribosome. The initiator tRNA and large subunit are added to form an initiation complex. 4. The ribosome cycle begins with the growing peptide attached to the tRNA in the P site. The next charged tRNA binds to the A site with its anticodon complementary to the codon in the mRNA in this site. Primary RNA transcript RNA polymerase II Primary RNA transcript Cut intron 5´ cap Large subunit mRNA 5´ cap Small subunit Cytoplasm tRNA arrivesin A siteAmino acids mRNA A site P site E site Cytoplasm RNA polymerase II Primary RNA transcript 5´ 3´ 5´ 3´ Poly-A tail Mature mRNA Poly-A tail Mature mRNA

65. 65 Primary RNA transcript Poly-A tail Mature mRNA Cut intron 5´ cap Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 1. RNA polymerase II in the nucleus copies one strand of the DNA to produce the primary transcript. 2. The primary transcript is processed by addition of a 5´ methyl-G cap, cleavage and polyadenylation of the 3´ end, and removal of introns. The mature mRNA is then exported through nuclear pores to the cytoplasm. 3. The 5´ cap of the mRNA associates with the small subunit of the ribosome. The initiator tRNA and large subunit are added to form an initiation complex. 4. The ribosome cycle begins with the growing peptide attached to the tRNA in the P site. The next charged tRNA binds to the A site with its anticodon complementary to the codon in the mRNA in this site. 5. Peptide bonds form between the amino terminus of the next amino acid and the carboxyl terminus of the growing peptide. This transfers the growing peptide to the tRNA in the A site, leaving the tRNA in the P site empty. Primary RNA transcript RNA polymerase II Primary RNA transcript Poly-A tail Mature mRNA Cut intron 5´ cap Large subunit mRNA Small subunit Cytoplasm tRNA arrivesin A siteAmino acids mRNA A site P site E site Lengthening polypeptide chain Emptyt RNA Cytoplasm RNA polymerase II Primary RNA transcript 5´ 3´ 5´ 3´ 5´ 3´ 5´ cap

66. 66 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 1. RNA polymerase II in the nucleus copies one strand of the DNA to produce the primary transcript. 2. The primary transcript is processed by addition of a 5´ methyl-G cap, cleavage and polyadenylation of the 3´ end, and removal of introns. The mature mRNA is then exported through nuclear pores to the cytoplasm. 3. The 5´ cap of the mRNA associates with the small subunit of the ribosome. The initiator tRNA and large subunit are added to form an initiation complex. 4. The ribosome cycle begins with the growing peptide attached to the tRNA in the P site. The next charged tRNA binds to the A site with its anticodon complementary to the codon in the mRNA in this site. 5. Peptide bonds form between the amino terminus of the next amino acid and the carboxyl terminus of the growing peptide. This transfers the growing peptide to the tRNA in the A site, leaving the tRNA in the P site empty. 6. Ribosome translocation moves the ribosome relative to the mRNA and its bound tRNAs. This moves the growing chain into the P site, leaving the empty tRNA in the E site and the A site ready to bind the next charged tRNA. Primary RNA transcript RNA polymerase II mRNA Small subunit Cytoplasm tRNA arrivesin A siteAmino acids mRNA A site P site E site Lengthening polypeptide chain Emptyt RNA Empty tRNA moves into E site and is ejected Cytoplasm RNA polymerase II Primary RNA transcript 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ Primary RNA transcript Poly-A tail Mature mRNA Cut intron 5´ cap Primary RNA transcript Poly-A tail Mature mRNA Cut intron 5´ cap Large subunit 5´ cap

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69. Mutation: Altered Genes Point mutations alter a single base Base substitution – substitute one base for another –Silent mutation – same amino acid inserted –Missense mutation – changes amino acid inserted Transitions Transversions –Nonsense mutations – changed to stop codon 69

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72. 72 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Polar Normal HBB Sequence Abormal HBB Sequence Nonpolar (hydrophobic) Amino acids Nucleotides Amino acids Nucleotides Leu CCCCGTTTAGGAGAA ThrProGlu CTTGAA LysSer Leu CCCCGTTTAGGGAA ThrPro val Glu CTTTGAA LysSer 11 11 Normal Deoxygenated Tetramer Abnormal Deoxygenated Tetramer Tetramers form long chains when deoxygenated. This distorts the normal red blood cell shape into a sickle shape. Hemoglobin tetramer "Sticky" non- polar sites 22 22 11 11 22 22

73. 73 Frameshift mutations –Addition or deletion of a single base –Much more profound consequences –Alter reading frame downstream –Triplet repeat expansion mutation Huntington disease Repeat unit is expanded in the disease allele relative to the normal

74. 74 Chromosomal mutations Change the structure of a chromosome –Deletions – part of chromosome is lost –Duplication – part of chromosome is copied –Inversion – part of chromosome in reverse order –Translocation – part of chromosome is moved to a new location

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77. 77 Mutations are the starting point for evolution Too much change, however, is harmful to the individual with a greatly altered genome Balance must exist between amount of new variation and health of species