Fig. 17-1

Ch. 17 From Gene to Protein Now we begin to understand the story beyond DNA . . . Thanks to the many “shoulders”.

Let’s start at the end of this chapter Page 347 Gene = a region of DNA that can be expressed to produce a final functional product, either a Polypeptide (or set of related polypeptides via alternative splicing) RNA molecule Gene Expression is by Transcription into RNA Translation into a polypeptide that forms a specific protein Proteomics = proteins interact to bring about a phenotype Cell Differentiation = a given type of cell expresses only a subset of its genes. Gene expression is precisely regulated –Ch.18

Search for Definition of Gene 1940’s Beadle & Tatum mutated mold genes with x-rays & notice they then were unable to produce a specific nutrient in order to grow. When they identified the missing nutrient they knew the mutated gene could not produce the enzyme (protein) needed for that reaction Conclusion: support for the one gene – one enzyme hypothesis

Beadle & Tatum EXPERIMENT RESULTS CONCLUSION Fig. 17-2 Growth: Wild-type cells growing and dividing No growth: Mutant cells cannot grow and divide Minimal medium RESULTS Classes of Neurospora crassa Wild type Class I mutants Class II mutants Class III mutants Minimal medium (MM) (control) MM + ornithine Condition MM + citrulline MM + arginine (control) CONCLUSION Class I mutants (mutation in gene A) Class II mutants (mutation in gene B) Class III mutants (mutation in gene C) Wild type Precursor Precursor Precursor Precursor Gene A Enzyme A Enzyme A Enzyme A Enzyme A Ornithine Ornithine Ornithine Ornithine Gene B Enzyme B Enzyme B Enzyme B Enzyme B Citrulline Citrulline Citrulline Citrulline Gene C Enzyme C Enzyme C Enzyme C Enzyme C Arginine Arginine Arginine Arginine

EXPERIMENT Growth: Wild-type cells growing and dividing No growth: Fig. 17-2a EXPERIMENT Growth: Wild-type cells growing and dividing No growth: Mutant cells cannot grow and divide Minimal medium

RESULTS Classes of Neurospora crassa Wild type Minimal medium (MM) Fig. 17-2b RESULTS Classes of Neurospora crassa Wild type Class I mutants Class II mutants Class III mutants Minimal medium (MM) (control) MM + ornithine Condition MM + citrulline MM + arginine (control)

CONCLUSION Wild type Precursor Precursor Precursor Precursor Gene A Fig. 17-2c CONCLUSION Class I mutants (mutation in gene A) Class II mutants (mutation in gene B) Class III mutants (mutation in gene C) Wild type Precursor Precursor Precursor Precursor Gene A Enzyme A Enzyme A Enzyme A Enzyme A Ornithine Ornithine Ornithine Ornithine Gene B Enzyme B Enzyme B Enzyme B Enzyme B Citrulline Citrulline Citrulline Citrulline Gene C Enzyme C Enzyme C Enzyme C Enzyme C Arginine Arginine Arginine Arginine

DNA (compared to) RNA Double helix Sugar is Deoxyribose Bases are A, T, C, G Contains heredity is segments called genes Remains in the nucleus Single strand Sugar is Ribose Base U replaces T Can serve many functions depending upon type of RNA May leave the nucleus

Fig. 17-25 DNA TRANSCRIPTION 3 RNA polymerase 5 RNA transcript Poly-A RNA polymerase 5 RNA transcript RNA PROCESSING Exon RNA transcript (pre-mRNA) Intron Aminoacyl-tRNA synthetase Poly-A NUCLEUS Amino acid AMINO ACID ACTIVATION CYTOPLASM tRNA mRNA Growing polypeptide Cap 3 A Activated amino acid Poly-A P Ribosomal subunits E Cap 5 TRANSLATION E A Anticodon Codon Ribosome

In Eukaryotic cells, RNA is processed from pre-mRNA to mRNA in nucleus Fig. 17-3 DNA TRANSCRIPTION mRNA Ribosome TRANSLATION Polypeptide (a) Bacterial cell In Eukaryotic cells, RNA is processed from pre-mRNA to mRNA in nucleus Nuclear envelope DNA TRANSCRIPTION Pre-mRNA RNA PROCESSING mRNA TRANSLATION Ribosome Polypeptide (b) Eukaryotic cell

After processing mRNA leaves nucleus and moves to ribosomes Fig. 17-3b-3 Nuclear envelope DNA TRANSCRIPTION Pre-mRNA RNA PROCESSING mRNA After processing mRNA leaves nucleus and moves to ribosomes TRANSLATION Ribosome Polypeptide (b) Eukaryotic cell

Gene 2 Gene 1 Gene 3 DNA template strand mRNA Codon TRANSLATION Fig. 17-4 Gene 2 DNA molecule Gene 1 Gene 3 DNA template strand TRANSCRIPTION mRNA Codon TRANSLATION Protein Amino acid

Completed RNA transcript Fig. 17-7 Promoter Transcription unit 5 3 3 5 DNA Start point RNA polymerase 1 Initiation Elongation Nontemplate strand of DNA RNA nucleotides 5 3 RNA polymerase 3 5 RNA transcript Template strand of DNA Unwound DNA 3 2 Elongation 3 end Rewound DNA 5 5 3 3 3 5 5 5 Direction of transcription (“downstream”) RNA transcript Template strand of DNA 3 Termination Newly made RNA 5 3 3 5 5 3 Completed RNA transcript

Transcription: Definition & Stages = DNA copies its genetic code onto RNA 1. Initiation RNA polymerase binds to promoter,DNA unwinds 2. Elongation RNA nucleotides constructed on template DNA Multiple copies of a section can be made Different kinds of RNA result 3. Termination RNA is released Animation

Cracking the Code How can DNA with only 4 different bases carry a code for 20 different amino acids that will be put together to form specific proteins? 4=4 42=16 43=64 triplet codons 1960’s Tie Club discloses the amino acid translations of each of the 64 RNA codons

First mRNA base (5 end of codon) Third mRNA base (3 end of codon) Fig. 17-5 Second mRNA base First mRNA base (5 end of codon) Third mRNA base (3 end of codon)

Theme: Unity & Diversity Unity - Triplet codon translation into specific amino acid is nearly universal i.e. RNA codon CCG is translated into amino acid proline in all organisms whose genetic code has been examined Therefore, genes can be transplanted from one species to another leading to Gene Therapy Diversity - Order of amino acids affects final end product, the protein, which interacts with other proteins to cause the unique phenotype of the organism

(a) Tobacco plant expressing a firefly gene (b) Pig expressing a Fig. 17-6 (a) Tobacco plant expressing a firefly gene (b) Pig expressing a jellyfish gene

Completed RNA transcript Fig. 17-7a-4 Promoter Transcription unit 5 3 3 5 DNA Start point RNA polymerase 1 Initiation 5 3 3 5 RNA transcript Template strand of DNA Unwound DNA 2 Elongation Rewound DNA 5 3 3 3 5 5 RNA transcript 3 Termination 5 3 3 5 5 3 Completed RNA transcript

Nontemplate Elongation strand of DNA RNA nucleotides RNA polymerase 3 Fig. 17-7b Elongation Nontemplate strand of DNA RNA nucleotides RNA polymerase 3 3 end 5 5 Direction of transcription (“downstream”) Template strand of DNA Newly made RNA

Pre-mRNA processing Occurs in Eukaryotic cells Both ends are altered with a cap on one end and a poly-A-tail on the other end to Protect the mRN and facilitate its export from nucleus and its binding to ribosomes Splicing out of noncoding segments called introns leaving coding segments, exons, that exit the nucleus on mRNA In humans, about 98.5% of DNA is noncoding!

Protein-coding segment Polyadenylation signal 5 3 Fig. 17-9 Protein-coding segment Polyadenylation signal 5 3 G P P P AAUAAA AAA … AAA 5 Cap 5 UTR Start codon Stop codon 3 UTR Poly-A tail

exons spliced together Coding segment Fig. 17-10 5 Exon Intron Exon Intron Exon 3 Pre-mRNA 5 Cap Poly-A tail 1 30 31 104 105 146 Introns cut out and exons spliced together Coding segment mRNA 5 Cap Poly-A tail 1 146 5 UTR 3 UTR

RNA transcript (pre-mRNA) 5 Exon 1 Intron Exon 2 Fig. 17-11-3 RNA transcript (pre-mRNA) 5 Exon 1 Intron Exon 2 Protein Other proteins snRNA snRNPs Spliceosome 5 Spliceosome components Cut-out intron mRNA 5 Exon 1 Exon 2

Translation: Definition & Stages = triple codons on mRNA are interpreted to bring a specific amino acid to ribosome(rRNA) where the polypeptide is synthesized The interpreters are tRNA molecules with a anticodon on one end and the corresponding amino acid on the other 1. Initiation=a start codon begins the process 2. Elongation=amino acids are covalently bonded by the ribosome via peptide bonds to form a polypeptide 3. Termination=1 of 3 stop codons releases the polypeptide

Gene DNA Exon 1 Intron Exon 2 Intron Exon 3 Transcription Fig. 17-12 Gene DNA Exon 1 Intron Exon 2 Intron Exon 3 Transcription RNA processing Translation Domain 3 Domain 2 Domain 1 Polypeptide

Amino acids tRNA with amino acid attached Ribosome tRNA Anticodon 5 Fig. 17-13 Amino acids Polypeptide tRNA with amino acid attached Ribosome Trp Phe Gly tRNA Anticodon 5 Codons 3 animation mRNA

Fig. 17-14 3 Amino acid attachment site 5 Hydrogen bonds Anticodon (a) Two-dimensional structure 5 Amino acid attachment site 3 Hydrogen bonds 3 5 Anticodon Anticodon (c) Symbol used in this book (b) Three-dimensional structure

(a) Computer model of functioning ribosome Fig. 17-16a Growing polypeptide Exit tunnel tRNA molecules Large subunit E P A Small subunit 5 3 mRNA (a) Computer model of functioning ribosome

(b) Schematic model showing binding sites Fig. 17-16b P site (Peptidyl-tRNA binding site) A site (Aminoacyl- tRNA binding site) E site (Exit site) E P A Large subunit mRNA binding site Small subunit (b) Schematic model showing binding sites Growing polypeptide Amino end Next amino acid to be added to polypeptide chain E tRNA mRNA 3 Codons 5 (c) Schematic model with mRNA and tRNA

GDP GDP Amino end of polypeptide E 3 mRNA Ribosome ready for Fig. 17-18-4 Amino end of polypeptide E 3 mRNA Ribosome ready for next aminoacyl tRNA P site A site 5 GTP GDP E E P A P A GDP GTP E P A

Protein synthesis video Fig. 17-19-3 Release factor Free polypeptide 5 3 3 3 2 5 5 GTP Stop codon (UAG, UAA, or UGA) 2 GDP Protein synthesis video

Protein Processing 1. Free ribosomes deliver polypeptides that are destine for the cytosol 2. Bound ribosomes move polypeptides from ER to Golgi Apparatus for modification and may secreted out of cell via vesicles i.e. Insulin is a protein that is secreted

Ribosome mRNA Signal peptide ER membrane Signal peptide removed Fig. 17-21 Ribosome mRNA Signal peptide ER membrane Signal peptide removed Signal- recognition particle (SRP) Protein CYTOSOL Translocation complex ER LUMEN SRP receptor protein

Point Mutations in Proteins Substitutions i.e. CCG on template mutated to GCG Could be “Silent”, good thing last base does not always determine the amino acid i.e. CCG mutates to CCA Insertions or Deletions Frame shift mutation occurs The dog ate the cat T hed oga tet hec at

Fig. 17-25 DNA TRANSCRIPTION 3 RNA polymerase 5 RNA transcript Poly-A RNA polymerase 5 RNA transcript RNA PROCESSING Exon RNA transcript (pre-mRNA) Intron Aminoacyl-tRNA synthetase Poly-A NUCLEUS Amino acid AMINO ACID ACTIVATION CYTOPLASM tRNA mRNA Growing polypeptide Cap 3 A Activated amino acid Poly-A P Ribosomal subunits E Cap 5 TRANSLATION E A Anticodon Codon Ribosome

Fig. 17-UN4

Practice Coding DNA template to RNA to amino acids Fig. 17-UN6 Practice Coding DNA template to RNA to amino acids Next slide shows result without then with a mutation

Fig. 17-UN7

List all types of RNA & their Functions Much current research on small RNA

Fig. 17-UN8