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

Central Dogma

DNA to RNA to Polypeptide (Protein) Transcription: the synthesis of RNA using information in the DNA DNA Serves as a template for making a complementary sequence of mRNA

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 5

mRNA leaves the nucleus and goes to the ribosomes Translation: the synthesis of a polypeptide using the information in the mRNA

Remember: RNA: Contains uracil instead of thymine Contains ribose instead of deoxyribose Is usually single stranded

The Genetic Code Triplets of nucleotide bases code for all of the amino acids (20) In other words, the genetic instructions for a polypeptide chain are written in the DNA as a series of non-overlaping, three nucleotide bases

The complementary RNA nucleotides are also called codons They are read in the 5’ – 3’ direction along the mRNA

Codon Table for mRNA

Evolutionary Significance Genetic code is nearly universal Genes can be transcribed and translated after being transplanted from species to another

(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 15

(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 16

Synthesis of an RNA Transcript The three stages of transcription Initiation Elongation Termination 17 17

RNA polymerase attaches to the promoter DNA sequence 18 18

Molecular Components of Transcription RNA polymerases assemble polynucleotides in the 5 to 3 direction https://usclip.net/video/WgvnFYyJGZQ/dna-transcription-animation-by-interact-medical.html Animation: Transcription Introduction 19 19

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) 20

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 21

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 22

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 23

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 3 Transcription initiation complex forms. Figure 14.9 The initiation of transcription at a eukaryotic promoter 5 3 3 3 5 5 RNA transcript Transcription initiation complex 24

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 25 25

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 26

Termination of Transcription The mechanisms of termination are different in bacteria and eukaryotes Prokaryotic cells have a sequence of nucleotide bases called the terminator which when reached releases the pre-mRNA In eukaryotic cells, RNA polymerase II transcribes a different sequence called the polyadenylation signal 27 27

Polyadenylation Site

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 29

Concept 14.3: Eukaryotic cells modify RNA after transcription Enzymes in eukaryotes modify pre-mRNA: RNA processing G nucleotide 5 cap and Poly A tail are added 30 30

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 31

Functions of Caps: 1. Facilitate the export of the mature mRNA from nucleus 2. Protect mRNA from hydrolytic enzymes 3. Help ribosomes attach to the 5’ end of the mRNA

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

Non-coding segments of the RNA are spliced out: introns Exons: sequences of RNA that are not spliced out Code for eukaryotic polypeptides Includes the UTRs: are not translated into protein, but assist with binding to the ribosome

How does splicing occur? Spliceosome: protein and RNA complex that removes the introns Also joins the remaining exons RNA in the spliceosome catalyze this process (not proteins/enzymes)

Ribozymes Ribozymes - RNA molecules that function as enzymes RNA splicing can occur without proteins (enzymes), or even additional RNA molecules The introns can catalyze their own splicing 36 36

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 37

DNA TRANSCRIPTION mRNA Ribosome TRANSLATION Polypeptide Figure 14.UN04 Figure 14.UN04 In-text figure, translation, p. 279 Polypeptide 38

Translation Mature mRNA moves out of the nucleus to the ribosome Ribosomes made of rRNA (ribosomal) and proteins Ribosomes attaches each amino acid brought to it be tRNA

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 40

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

tRNA carries a specific amino acid at one end and a nucleotide triplet that can base-pair with the complementary codon on the mRNA Anticodon: nucleotide triplet that base-pairs to a specific mRNA codon

(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 * G Figure 14.15 The structure of transfer RNA (tRNA) * 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 43

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

Three Stages of Translation Initiation Elongation Termination

Initiation Initiator tRNA first binds to the smaller ribosomal subunit Small subunit/tRNA complex attaches to mRNA and reads an AUG “start” codon Large ribosomal subunit joins complex

(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 mRNA binding site Figure 14.17b The anatomy of a functioning ribosome (part 2: binding sites) Small subunit (b) Schematic model showing binding sites 48

(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 49

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

Elongation

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

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) 53

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 54

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 55

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 57

Completing and Targeting the Functional Protein Polypeptide chains are modified after translation 58 58

Making Multiple Polypeptides in Bacteria and Eukaryotes In bacteria and eukaryotes multiple ribosomes translate an mRNA at the same time 59 59

(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 60

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 61

Mutations: changes to the genetic information

Small scale mutations: changes in one or just a few nucleotide pairs Point mutations: changes in a single nucleotide pair of a gene If occurs in the gametes can be passed on to offspring

Types of Small-Scale Mutations Substitutions: replacement of a single nucleotide and its partner with another pair of nucleotides (point mutation) Insertions or deletions: additions or losses of a nucleotide base pair in a gene (may be a point mutation) 64 64

Substitution Substitution may have no observable effect: silent mutation May code for the same amino acid

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 66

Substitution May cause a different amino acid to form: missense mutation Ex. Sickle cell anemia

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 68

Sickle Cell Anemia

Substitution The change may code for a stop codon: nonsense mutation

Insertions and Deletions Insertion or deletions: alter the reading frame produce a frameshift mutation 71 71

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 72

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 73

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 74