Gene Expression: From Gene to Protein 14 Gene Expression: From Gene to Protein

Figure 14.1 Figure 14.1 How does a single faulty gene result in the dramatic appearance of an albino deer? 2

DNA TRANSCRIPTION mRNA Ribosome TRANSLATION Polypeptide Figure 14.4a-2 DNA TRANSCRIPTION mRNA Ribosome TRANSLATION Polypeptide Figure 14.4a-2 Overview: the roles of transcription and translation in the flow of genetic information (part 1, step 2) (a) Bacterial cell 3

Nuclear envelope DNA TRANSCRIPTION Pre-mRNA (b) Eukaryotic cell Figure 14.4b-1 Nuclear envelope DNA TRANSCRIPTION Pre-mRNA Figure 14.4b-1 Overview: the roles of transcription and translation in the flow of genetic information (part 2, step 1) (b) Eukaryotic cell 4

Nuclear envelope DNA TRANSCRIPTION Pre-mRNA RNA PROCESSING mRNA Figure 14.4b-2 Nuclear envelope DNA TRANSCRIPTION Pre-mRNA RNA PROCESSING mRNA Figure 14.4b-2 Overview: the roles of transcription and translation in the flow of genetic information (part 2, step 2) (b) Eukaryotic cell 5

Nuclear envelope DNA TRANSCRIPTION Pre-mRNA RNA PROCESSING mRNA Figure 14.4b-3 Nuclear envelope DNA TRANSCRIPTION Pre-mRNA RNA PROCESSING mRNA Figure 14.4b-3 Overview: the roles of transcription and translation in the flow of genetic information (part 2, step 3) TRANSLATION Ribosome Polypeptide (b) Eukaryotic cell 6

DNA template strand 3 5 A C C A A A C C G A G T T G G T T T G G C T Figure 14.5 DNA template strand 3 5 A C C A A A C C G A G T T G G T T T G G C T C A 5 3 TRANSCRIPTION U G G U U U G G C U C A mRNA 5 3 Codon Figure 14.5 The triplet code TRANSLATION Protein Trp Phe Gly Ser Amino acid 7

First mRNA base (5 end of codon) Third mRNA base (3 end of codon) Figure 14.6 Second mRNA base U C A G UUU UCU UAU UGU U Phe Tyr Cys UUC UCC UAC UGC C U Ser UUA UCA UAA Stop UGA Stop A Leu UUG UCG UAG Stop UGG Trp G CUU CCU CAU CGU U His CUC CCC CAC CGC C C Leu Pro Arg CUA CCA CAA CGA A Gln CUG CCG CAG CGG G First mRNA base (5 end of codon) Third mRNA base (3 end of codon) AUU ACU AAU AGU U Asn Ser AUC IIe ACC AAC AGC C A Thr Figure 14.6 The codon table for mRNA AUA ACA AAA AGA A Lys Arg AUG Met or start ACG AAG AGG G GUU GCU GAU GGU U Asp GUC GCC GAC GGC C G Val Ala Gly GUA GCA GAA GGA A Glu GUG GCG GAG GGG G 8

(a) Tobacco plant expressing a firefly gene Figure 14.7a Figure 14.7a Expression of genes from different species (part 1: tobacco plant) (a) Tobacco plant expressing a firefly gene 9

(b) Pig expressing a jellyfish gene Figure 14.7b Figure 14.7b Expression of genes from different species (part 2: pig) (b) Pig expressing a jellyfish gene 10

Synthesis of an RNA Transcript The three stages of transcription Initiation Elongation Termination 11 11

RNA polymerase attaches to the promoter DNA sequence 12 12

Molecular Components of Transcription RNA polymerases assemble polynucleotides in the 5 to 3 direction Animation: Transcription Introduction 13 13

Promoter Transcription unit Start point RNA polymerase Initiation Figure 14.8-1 Promoter Transcription unit 5 3 3 5 Start point RNA polymerase 1 Initiation 5 3 3 5 Unwound DNA RNA transcript Template strand of DNA Figure 14.8-1 The stages of transcription: initiation, elongation, and termination (step 1) 14

Promoter Transcription unit Start point RNA polymerase Initiation Figure 14.8-2 Promoter Transcription unit 5 3 3 5 Start point RNA polymerase 1 Initiation 5 3 3 5 Unwound DNA RNA transcript Template strand of DNA 2 Elongation Rewound DNA 5 3 3 3 5 5 Figure 14.8-2 The stages of transcription: initiation, elongation, and termination (step 2) Direction of transcription (“downstream”) RNA transcript 15

Completed RNA transcript Figure 14.8-3 Promoter Transcription unit 5 3 3 5 Start point RNA polymerase 1 Initiation 5 3 3 5 Unwound DNA RNA transcript Template strand of DNA 2 Elongation Rewound DNA 5 3 3 3 5 5 Figure 14.8-3 The stages of transcription: initiation, elongation, and termination (step 3) Direction of transcription (“downstream”) RNA transcript 3 Termination 5 3 3 5 5 3 Completed RNA transcript 16

DNA TRANSCRIPTION Pre-mRNA RNA PROCESSING mRNA Ribosome TRANSLATION Figure 14.UN02 DNA TRANSCRIPTION Pre-mRNA RNA PROCESSING mRNA Figure 14.UN02 In-text figure, transcription, p. 275 Ribosome TRANSLATION Polypeptide 17

Several transcription factors bind to DNA. 3 5 Figure 14.9 Promoter Nontemplate strand DNA 5 T A T A A A A 3 3 A T A T T T T 5 1 A eukaryotic promoter TATA box Start point Template strand Transcription factors 5 3 2 Several transcription factors bind to DNA. 3 5 RNA polymerase II Transcription factors Figure 14.9 The initiation of transcription at a eukaryotic promoter 3 Transcription initiation complex forms. 5 3 3 3 5 5 RNA transcript Transcription initiation complex 18

Elongation of the RNA Strand As RNA polymerase moves along DNA, untwists double helix, 10 to 20 bases at a time gene can be transcribed simultaneously by several RNA polymerases 19 19

Direction of transcription Template strand of DNA Figure 14.10 Nontemplate strand of DNA RNA nucleotides RNA polymerase T C C A A A 3 T 5 U C T 3 end T G U A G A C C A U C C A C A 5 A 3 T A G G T T Figure 14.10 Transcription elongation 5 Direction of transcription Template strand of DNA Newly made RNA 20

DNA TRANSCRIPTION Pre-mRNA RNA PROCESSING mRNA Ribosome TRANSLATION Figure 14.UN03 DNA TRANSCRIPTION Pre-mRNA RNA PROCESSING mRNA Figure 14.UN03 In-text figure, RNA processing, p. 277 Ribosome TRANSLATION Polypeptide 21

Concept 14.3: Eukaryotic cells modify RNA after transcription Enzymes in eukaryotes modify pre-mRNA nucleotide 5 cap and Poly A tail are added Introns are edited out Exons are sometimes used 22 22

Transcript Modification unit of transcription in a DNA strand exon intron exon intron exon cap poly-A tail snipped out snipped out mature mRNA transcript Fig. 14-4, p.221

Polyadenylation signal Figure 14.11 50–250 adenine nucleotides added to the 3 end A modified guanine nucleotide added to the 5 end Polyadenylation signal Protein-coding segment 5 3 G P P P AAUAAA AAA … AAA Start codon Stop codon 5 Cap 5 UTR 3 UTR Poly-A tail Figure 14.11 RNA processing: addition of the 5' cap and poly-A tail 25

Spliceosome Small RNAs 5 Pre-mRNA Exon 1 Exon 2 Intron Spliceosome Figure 14.13 Spliceosome Small RNAs 5 Pre-mRNA Exon 1 Exon 2 Intron Figure 14.13 A spliceosome splicing a pre-mRNA Spliceosome components mRNA Cut-out intron 5 Exon 1 Exon 2 26

Ribozymes Ribozymes - RNA molecules that function as enzymes RNA splicing can occur without proteins, or even additional RNA molecules The introns can catalyze their own splicing 27 27

DNA TRANSCRIPTION mRNA Ribosome TRANSLATION Polypeptide Figure 14.UN04 Figure 14.UN04 In-text figure, translation, p. 279 Polypeptide 28

Amino acids Polypeptide tRNA with amino acid attached Ribosome tRNA Figure 14.14 Amino acids Polypeptide tRNA with amino acid attached Ribosome Trp Phe Gly Figure 14.14 Translation: the basic concept tRNA C C C C Anticodon A G A A A U G G U U U G G C 5 Codons 3 mRNA 29

(b) Three-dimensional structure (c) Symbol used in this book Figure 14.15 3 Amino acid attachment site A C C A 5 Amino acid attachment site C G G C 5 C G U G 3 U A A U U A U * C C A G G A C U A * A C G * C U * G U G U Hydrogen bonds C * C G A G G * * C A G U * G * G A G C Hydrogen bonds G C U A Figure 14.15 The structure of transfer RNA (tRNA) * G * A A C A A G * U 3 5 A G A Anticodon Anticodon Anticodon (b) Three-dimensional structure (c) Symbol used in this book (a) Two-dimensional structure 30

tRNA Structure codon in mRNA anticodon amino-acid attachment site OH Figure 14.7 Page 223

tRNA Structure codon in mRNA anticodon in tRNA amino acid Fig. 14-7, p.223

small ribosomal subunit large ribosomal subunit Ribosomes funnel small ribosomal subunit + large ribosomal subunit intact ribosome Fig. 14-8, p.223

Three Stages of Translation Initiation Elongation Termination

Initiation Initiator tRNA binds to small ribosomal subunit Small subunit/tRNA complex attaches to mRNA and moves an AUG “start” codon Large ribosomal subunit joins complex

Binding Sites on Large Subunit binding site for mRNA P site (Peptidyl) first binding site for 1st tRNA A site (Aminoacyl) (1st binding site for all other tRNA’s)

(a) Computer model of functioning ribosome Figure 14.17a Growing polypeptide Exit tunnel tRNA molecules Large subunit E P A Small subunit Figure 14.17a The anatomy of a functioning ribosome (part 1: computer model) 5 3 mRNA (a) Computer model of functioning ribosome 37

(b) Schematic model showing binding sites Figure 14.17b P site (Peptidyl-tRNA binding site) Exit tunnel A site (Aminoacyl- tRNA binding site) E site (Exit site) E P A Large subunit Figure 14.17b The anatomy of a functioning ribosome (part 2: binding sites) mRNA binding site Small subunit (b) Schematic model showing binding sites 38

(c) Schematic model with mRNA and tRNA Figure 14.17c Growing polypeptide Amino end Next amino acid to be added to polypeptide chain E tRNA mRNA 3 Figure 14.17c The anatomy of a functioning ribosome (part 3: mRNA and tRNA) Codons 5 (c) Schematic model with mRNA and tRNA 39

Elongation mRNA passes through ribosomal subunits tRNAs deliver amino acids to ribosomal binding site Peptide bonds form

Elongation

Termination Ribosome reaches “stop codon” No tRNA with anticodon Release factors bind to the ribosome mRNA and polypeptide released mRNA new polypeptide chain

Overview Transcription Translation mRNA rRNA tRNA Mature mRNA transcripts ribosomal subunits mature tRNA Translation

First mRNA base (5 end of codon) Third mRNA base (3 end of codon) Figure 14.6 Second mRNA base U C A G UUU UCU UAU UGU U Phe Tyr Cys UUC UCC UAC UGC C U Ser UUA UCA UAA Stop UGA Stop A Leu UUG UCG UAG Stop UGG Trp G CUU CCU CAU CGU U His CUC CCC CAC CGC C C Leu Pro Arg CUA CCA CAA CGA A Gln CUG CCG CAG CGG G First mRNA base (5 end of codon) Third mRNA base (3 end of codon) AUU ACU AAU AGU U Asn Ser AUC IIe ACC AAC AGC C A Thr Figure 14.6 The codon table for mRNA AUA ACA AAA AGA A Lys Arg AUG Met or start ACG AAG AGG G GUU GCU GAU GGU U Asp GUC GCC GAC GGC C G Val Ala Gly GUA GCA GAA GGA A Glu GUG GCG GAG GGG G 44

Amino end of polypeptide Codon recognition 1 E E P A 3 mRNA 5 GTP Figure 14.19-1 Amino end of polypeptide 1 Codon recognition E 3 mRNA P site A site 5 GTP GDP  P i E P A Figure 14.19-1 The elongation cycle of translation (step 1) 45

Amino end of polypeptide Codon recognition Peptide bond formation 1 E Figure 14.19-2 Amino end of polypeptide 1 Codon recognition E 3 mRNA P site A site 5 GTP GDP  P i E P A Figure 14.19-2 The elongation cycle of translation (step 2) 2 Peptide bond formation E P A 46

Amino end of polypeptide Codon recognition Ribosome ready for Figure 14.19-3 Amino end of polypeptide 1 Codon recognition E 3 mRNA Ribosome ready for next aminoacyl tRNA P site A site 5 GTP GDP  P i E E P A P A Figure 14.19-3 The elongation cycle of translation (step 3) GDP  P i 2 Peptide bond formation 3 Translocation GTP E P A 47

Completing and Targeting the Functional Protein Polypeptide chains are modified after translation 48 48

Making Multiple Polypeptides in Bacteria and Eukaryotes In bacteria and eukaryotes multiple ribosomes translate an mRNA at the same time 49 49

(a) Several ribosomes simultaneously translating one mRNA molecule Figure 14.22 Growing polypeptides Completed polypeptide Incoming ribosomal subunits Polyribosome Start of mRNA (5 end) End of mRNA (3 end) (a) Several ribosomes simultaneously translating one mRNA molecule Ribosomes Figure 14.22 Polyribosomes mRNA (b) A large polyribosome in a bacterial cell (TEM) 0.1 m 50

RNA polymerase RNA transcript RNA PROCESSING Figure 14.24 DNA TRANSCRIPTION 3 Poly-A RNA polymerase 5 RNA transcript Exon RNA PROCESSING RNA transcript (pre-mRNA) Intron Aminoacyl-tRNA synthetase Poly-A NUCLEUS Amino acid AMINO ACID ACTIVATION CYTOPLASM tRNA mRNA 5 Cap 3 A Aminoacyl (charged) tRNA P Poly-A E Ribosomal subunits Figure 14.24 A summary of transcription and translation in a eukaryotic cell 5 Cap TRANSLATION A C C U A E A C Anticodon A A A U G G U U U A U G Codon Ribosome 51

Types of Small-Scale Mutations 2 categories of Point mutations substitutions insertions or deletions 52 52

Sickle-cell hemoglobin Figure 14.25 Wild-type hemoglobin Sickle-cell hemoglobin Wild-type hemoglobin DNA Mutant hemoglobin DNA 3 C T C 5 3 C A C 5 5 G A G 3 5 G T G 3 mRNA mRNA 5 G A G 3 5 G U G 3 Figure 14.25 The molecular basis of sickle-cell disease: a point mutation Normal hemoglobin Sickle-cell hemoglobin Glu Val 53

Nucleotide-pair substitution: silent Figure 14.26a Wild type DNA template strand 3 T A C T T C A A A C C G A T T 5 5 A T G A A G T T T G G C T A A 3 mRNA 5 A U G A A G U U U G G C U A A 3 Protein Met Lys Phe Gly Stop Amino end Carboxyl end Nucleotide-pair substitution: silent A instead of G 3 T A C T T C A A A C C A A T T 5 Figure 14.26a Types of small-scale mutations that affect mRNA sequence (part 1: silent) 5 A T G A A G T T T G G T T A A 3 U instead of C 5 A U G A A G U U U G G U U A A 3 Met Lys Phe Gly Stop 54

Insertions and Deletions Insertion or deletions that alter the reading frame produce a frameshift mutation 55 55

Nucleotide-pair insertion: frameshift causing immediate nonsense Figure 14.26d Wild type DNA template strand 3 T A C T T C A A A C C G A T T 5 5 A T G A A G T T T G G C T A A 3 mRNA 5 A U G A A G U U U G G C U A A 3 Protein Met Lys Phe Gly Stop Amino end Carboxyl end Nucleotide-pair insertion: frameshift causing immediate nonsense Extra A 3 T A C A T T C A A A C C G A T T 5 Figure 14.26d Types of small-scale mutations that affect mRNA sequence (part 4: frameshift, nonsense) 5 A T G T A A G T T T G G C T A A 3 Extra U 5 A U G U A A G U U U G G C U A A 3 Met Stop 56

Nucleotide-pair deletion: frameshift causing extensive missense Figure 14.26e Wild type DNA template strand 3 T A C T T C A A A C C G A T T 5 5 A T G A A G T T T G G C T A A 3 mRNA 5 A U G A A G U U U G G C U A A 3 Protein Met Lys Phe Gly Stop Amino end Carboxyl end Nucleotide-pair deletion: frameshift causing extensive missense A missing 3 T A C T T C A A C C G A T T 5 Figure 14.26e Types of small-scale mutations that affect mRNA sequence (part 5: frameshift, missense) 5 A T G A A G T T G G C T A A 3 U missing 5 A U G A A G U U G G C U A A 3 Met Lys Leu Ala 57

3 nucleotide-pair deletion: no frameshift, but one amino acid missing Figure 14.26f Wild type DNA template strand 3 T A C T T C A A A C C G A T T 5 5 A T G A A G T T T G G C T A A 3 mRNA 5 A U G A A G U U U G G C U A A 3 Protein Met Lys Phe Gly Stop Amino end Carboxyl end 3 nucleotide-pair deletion: no frameshift, but one amino acid missing T T C missing 3 T A C A A A C C G A T T 5 Figure 14.26f Types of small-scale mutations that affect mRNA sequence (part 6: missing amino acid) 5 A T G T T T G G C T A A 3 A A G missing 5 A U G U U U G G C U A A 3 Met Phe Gly Stop 58