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Gene Expression: From Gene to Protein
17 Gene Expression: From Gene to Protein Lecture Presentation by Nicole Tunbridge and Kathleen Fitzpatrick
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The Flow of Genetic Information
The information content of genes is in the specific sequences of nucleotides The DNA inherited by an organism leads to specific traits by dictating the synthesis of proteins Proteins are the links between genotype and phenotype Gene expression, the process by which DNA directs protein synthesis, includes two stages: transcription and translation
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Figure 17.1 Figure 17.1 How does a single faulty gene result in the dramatic appearance of an albino deer?
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Figure 17.1a An albino racoon
Figure 17.1a How does a single faulty gene result in the dramatic appearance of an albino deer? (part 1: albino raccoon) An albino racoon
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Proteins Proteins have many structures, resulting in a wide range of functions Proteins do most of the work in cells and act as enzymes Proteins are made of monomers called amino acids
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An overview of protein functions
Table 5.1
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Enzymes Are a type of protein that acts as a catalyst, speeding up chemical reactions Substrate (sucrose) Enzyme (sucrase) Glucose OH H O H2O Fructose 3 Substrate is converted to products. 1 Active site is available for a molecule of substrate, the reactant on which the enzyme acts. Substrate binds to enzyme. 2 4 Products are released. Figure 5.16
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Amino acids Are organic molecules possessing both carboxyl and amino groups Differ in their properties due to differing side chains, called R groups
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Twenty Amino Acids 20 different amino acids make up proteins
H H3N+ C CH3 CH CH2 NH H2C H2N Nonpolar Glycine (Gly) Alanine (Ala) Valine (Val) Leucine (Leu) Isoleucine (Ile) Methionine (Met) Phenylalanine (Phe) Tryptophan (Trp) Proline (Pro) H3C Figure 5.17 S 20 different amino acids make up proteins
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Polar Electrically charged
OH CH2 C H H3N+ O CH3 CH SH NH2 Polar Electrically charged –O NH3+ NH2+ NH+ NH Serine (Ser) Threonine (Thr) Cysteine (Cys) Tyrosine (Tyr) Asparagine (Asn) Glutamine (Gln) Acidic Basic Aspartic acid (Asp) Glutamic acid (Glu) Lysine (Lys) Arginine (Arg) Histidine (His)
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Amino Acid Polymers Amino acids Are linked by peptide bonds
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Polypeptides Polypeptides A protein
Are polymers (chains) of amino acids A protein Consists of one or more polypeptides
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Protein Conformation and Function
A protein’s specific conformation (shape) determines how it functions
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Four Levels of Protein Structure
Primary structure Is the unique sequence of amino acids in a polypeptide Figure 5.20 – Amino acid subunits +H3N Amino end o Carboxyl end c Gly Pro Thr Glu Seu Lys Cys Leu Met Val Asp Ala Arg Ser lle Phe His Asn Tyr Trp Lle
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Secondary structure Is the folding or coiling of the polypeptide into a repeating configuration Includes the helix and the pleated sheet O C helix pleated sheet Amino acid subunits N H R H Figure 5.20
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Animation: Secondary Protein Structure
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Tertiary structure Is the overall three-dimensional shape of a polypeptide Results from interactions between amino acids and R groups CH2 CH O H O C HO NH3+ -O S CH3 H3C Hydrophobic interactions and van der Waals interactions Polypeptide backbone Hydrogen bond Ionic bond Disulfide bridge
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Animation: Tertiary Protein Structure
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Quaternary structure Is the overall protein structure that results from the aggregation of two or more polypeptide subunits Polypeptide chain Collagen Chains Chains Hemoglobin Iron Heme
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Animation: Quaternary Protein Structure
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Review of Protein Structure
+H3N Amino end Amino acid subunits helix
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Sickle-Cell Disease: A Simple Change in Primary Structure
Results from a single amino acid substitution in the protein hemoglobin
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Sickle-cell hemoglobin
Primary structure Secondary and tertiary structures Quaternary structure Function Red blood cell shape Hemoglobin A Molecules do not associate with one another, each carries oxygen. Normal cells are full of individual hemoglobin molecules, each carrying oxygen 10 m Hemoglobin S Molecules interact with one another to crystallize into a fiber, capacity to carry oxygen is greatly reduced. subunit 1 2 3 4 5 6 7 Normal hemoglobin Sickle-cell hemoglobin . . . Figure 5.21 Exposed hydrophobic region Val Thr His Leu Pro Glul Glu Fibers of abnormal hemoglobin deform cell into sickle shape.
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What Determines Protein Conformation?
Protein conformation Depends on the physical and chemical conditions of the protein’s environment Temperature, pH, etc. affect protein structure
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Denaturation is when a protein unravels and loses its native conformation (shape)
Renaturation Denatured protein Normal protein Figure 5.22
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Denaturation is when a protein unravels and loses its native conformation (shape)
Renaturation Denatured protein Normal protein Figure 5.22
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The Protein-Folding Problem
Most proteins Probably go through several intermediate states on their way to a stable conformation Denaturated proteins no longer work in their unfolded condition Proteins may be denaturated by extreme changes in pH, temperature, salinity or heavy metals
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Chaperonins Are protein molecules that assist in the proper folding of other proteins Hollow cylinder Cap Chaperonin (fully assembled) Steps of Chaperonin Action: An unfolded poly- peptide enters the cylinder from one end. The cap attaches, causing the cylinder to change shape in such a way that it creates a hydrophilic environment for the folding of the polypeptide. The cap comes off, and the properly folded protein is released. Correctly folded protein Polypeptide 2 1 3 Figure 5.23
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What happens if a protein isn’t folded correctly?
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Prions can be formed – misfolded versions of normal proteins
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Prions Prions are slow-acting, virtually indestructible infectious proteins that cause brain diseases in mammals Prions propagate by converting normal proteins into the prion version Scrapie in sheep, mad cow disease, and Creutzfeldt- Jakob disease in humans are all caused by prions
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Overview: The Flow of Genetic Information
The information content of DNA Is in the form of specific sequences of nucleotides along the DNA strands
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Intro to Protein Synthesis
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The DNA inherited by an organism
Leads to specific traits by dictating the synthesis of proteins The process by which DNA directs protein synthesis, gene expression Includes two stages, called transcription and translation
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The ribosome Is part of the cellular machinery for translation, polypeptide synthesis Figure 17.1
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Concept 17.1: Genes specify proteins via transcription and translation
How was the fundamental relationship between genes and proteins discovered?
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Evidence from the Study of Metabolic Defects
In 1902, British physician Archibald Garrod first suggested that genes dictate phenotypes through enzymes that catalyze specific chemical reactions He thought symptoms of an inherited disease reflect an inability to synthesize a certain enzyme Cells synthesize and degrade molecules in a series of steps, a metabolic pathway
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Nutritional Mutants in Neurospora: Scientific Inquiry
George Beadle and Edward Tatum exposed bread mold to X-rays, creating mutants that were unable to survive on minimal media Using crosses, they and their coworkers identified three classes of arginine-deficient mutants, each lacking a different enzyme necessary for synthesizing arginine They developed a one gene–one enzyme hypothesis, which states that each gene dictates production of a specific enzyme
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Figure 17.2 Precursor Results Table Classes of Neurospora crassa Enzyme A Wild type Class I mutants Class II mutants Class III mutants Ornithine Minimal medium (MM) (control) Enzyme B Citrulline Enzyme C MM + ornithine Arginine Condition MM + citrulline MM + arginine (control) Growth: Wild-type cells growing and dividing No growth: Mutant cells cannot grow and divide Can grow with or without any supplements Can grow on ornithine, citrulline, or arginine Can grow only on citrulline or arginine Summary of results Require arginine to grow Control: Minimal medium Gene (codes for enzyme) Class I mutants (mutation in gene A) Class II mutants (mutation in gene B) Class III mutants (mutation in gene C) Figure 17.2 Inquiry: Do individual genes specify the enzymes that function in a biochemical pathway? 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
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Figure 17.2a Precursor Enzyme A Ornithine Enzyme B Citrulline Enzyme C
Figure 17.2a Inquiry: Do individual genes specify the enzymes that function in a biochemical pathway? (part 1: metabolic pathway) Arginine
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Figure 17.2b Growth: No growth: Wild-type Mutant cells cells growing
and dividing No growth: Mutant cells cannot grow and divide Control: Minimal medium Figure 17.2b Inquiry: Do individual genes specify the enzymes that function in a biochemical pathway? (part 2: control)
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Figure 17.2c Results Table Classes of Neurospora crassa Wild type
Class I mutants Class II mutants Class III mutants Minimal medium (MM) (control) MM + ornithine Condition MM + citrulline Figure 17.2c Inquiry: Do individual genes specify the enzymes that function in a biochemical pathway? (part 3: results table) MM + arginine (control) Can grow on ornithine, citrulline, or arginine Can grow with or without any supplements Can grow only on citrulline or arginine Summary of results Require arginine to grow
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Figure 17.2d Gene (codes for enzyme) 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 Enzyme A Enzyme A Enzyme A Enzyme A Gene A Ornithine Ornithine Ornithine Ornithine Enzyme B Enzyme B Enzyme B Enzyme B Gene B Citrulline Citrulline Citrulline Citrulline Enzyme C Enzyme C Enzyme C Enzyme C Gene C Figure 17.2d Inquiry: Do individual genes specify the enzymes that function in a biochemical pathway? (part 4: conclusion) Arginine Arginine Arginine Arginine
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The Products of Gene Expression: A Developing Story
Some proteins aren’t enzymes, so researchers later revised the hypothesis: one gene–one protein Many proteins are composed of several polypeptides, each of which has its own gene Therefore, Beadle and Tatum’s hypothesis is now restated as the one gene–one polypeptide hypothesis It is common to refer to gene products as proteins rather than polypeptides
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Basic Principles of Transcription and Translation
RNA is the bridge between genes and the proteins for which they code Transcription is the synthesis of RNA using information in DNA Transcription produces messenger RNA (mRNA) Translation is the synthesis of a polypeptide, using information in the mRNA Ribosomes are the sites of translation
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In prokaryotes, translation of mRNA can begin before transcription has finished
In a eukaryotic cell, the nuclear envelope separates transcription from translation Eukaryotic RNA transcripts are modified through RNA processing to yield the finished mRNA
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Figure 17.3 Nuclear envelope DNA TRANSCRIPTION Pre-mRNA RNA PROCESSING
NUCLEUS mRNA DNA TRANSCRIPTION CYTOPLASM CYTOPLASM mRNA TRANSLATION Ribosome Figure 17.3 Overview: the roles of transcription and translation in the flow of genetic information Ribosome TRANSLATION Polypeptide Polypeptide (a) Bacterial cell (b) Eukaryotic cell
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Figure 17.3a-1 DNA TRANSCRIPTION CYTOPLASM mRNA (a) Bacterial cell
Figure 17.3a-1 Overview: the roles of transcription and translation in the flow of genetic information (part 1, step 1) (a) Bacterial cell
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Figure 17.3a-2 DNA TRANSCRIPTION CYTOPLASM mRNA Ribosome TRANSLATION
Polypeptide Figure 17.3a-2 Overview: the roles of transcription and translation in the flow of genetic information (part 1, step 2) (a) Bacterial cell
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Figure 17.3b-1 Nuclear envelope DNA TRANSCRIPTION Pre-mRNA NUCLEUS
CYTOPLASM Figure 17.3b-1 Overview: the roles of transcription and translation in the flow of genetic information (part 2, step 1) (b) Eukaryotic cell
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Figure 17.3b-2 Nuclear envelope DNA TRANSCRIPTION Pre-mRNA
RNA PROCESSING NUCLEUS mRNA CYTOPLASM Figure 17.3b-2 Overview: the roles of transcription and translation in the flow of genetic information (part 2, step 2) (b) Eukaryotic cell
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Figure 17.3b-3 Nuclear envelope DNA TRANSCRIPTION Pre-mRNA
RNA PROCESSING NUCLEUS mRNA CYTOPLASM Figure 17.3b-3 Overview: the roles of transcription and translation in the flow of genetic information (part 2, step 3) TRANSLATION Ribosome Polypeptide (b) Eukaryotic cell
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A primary transcript is the initial RNA transcript from any gene prior to processing
The central dogma is the concept that cells are governed by a cellular chain of command: DNA → RNA → protein
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Figure 17.UN01 DNA RNA Protein
Figure 17.UN01 In-text figure, central dogma, p. 337
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The Genetic Code How are the instructions for assembling amino acids into proteins encoded into DNA? There are 20 amino acids, but there are only four nucleotide bases in DNA How many nucleotides correspond to an amino acid?
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Codons: Triplets of Nucleotides
The flow of information from gene to protein is based on a triplet code: a series of nonoverlapping, three-nucleotide words The words of a gene are transcribed into complementary nonoverlapping three-nucleotide words of mRNA These words are then translated into a chain of amino acids, forming a polypeptide
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Figure 17.4 DNA template strand 5′ 3′ A C C A A A C C G A G T T G G T
TRANSCRIPTION U G G U U U G G C U C A mRNA 5′ 3′ Codon Figure 17.4 The triplet code TRANSLATION Protein Trp Phe Gly Ser Amino acid
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During transcription, one of the two DNA strands, called the template strand, provides a template for ordering the sequence of complementary nucleotides in an RNA transcript The template strand is always the same strand for a given gene
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During translation, the mRNA base triplets, called codons, are read in the 5′ → 3′ direction
Each codon specifies the amino acid (one of 20) to be placed at the corresponding position along a polypeptide
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Cracking the Code All 64 codons were deciphered by the mid-1960s
Of the 64 triplets, 61 code for amino acids; 3 triplets are “stop” signals to end translation The genetic code is redundant (more than one codon may specify a particular amino acid) but not ambiguous; no codon specifies more than one amino acid Codons must be read in the correct reading frame (correct groupings) in order for the specified polypeptide to be produced
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Figure 17.5 Second mRNA base First mRNA base (5′ end of codon)
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 First mRNA base (5′ end of codon) CUG CCG CAG CGG G Third mRNA base (3′ end of codon) AUU ACU AAU AGU U Asn Ser AUC Ile ACC AAC AGC C A Thr Figure 17.5 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
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Evolution of the Genetic Code
The genetic code is nearly universal, shared by the simplest bacteria to the most complex animals Genes can be transcribed and translated after being transplanted from one species to another
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Figure 17.6 (a) Tobacco plant expressing a firefly gene (b)
Figure 17.6 Expression of genes from different species (a) Tobacco plant expressing a firefly gene (b) Pig expressing a jellyfish gene
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Figure 17.6a (a) Tobacco plant expressing a firefly gene
Figure 17.6a Expression of genes from different species (part 1: tobacco plant) (a) Tobacco plant expressing a firefly gene
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Figure 17.6b (b) Pig expressing a jellyfish gene
Figure 17.6b Expression of genes from different species (part 2: pig) (b) Pig expressing a jellyfish gene
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Concept 17.2: Transcription is the DNA-directed synthesis of RNA: A closer look
Transcription is the first stage of gene expression
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Molecular Components of Transcription
RNA synthesis is catalyzed by RNA polymerase, which pries the DNA strands apart and joins together the RNA nucleotides The RNA is complementary to the DNA template strand RNA polymerase does not need any primer RNA synthesis follows the same base-pairing rules as DNA, except that uracil substitutes for thymine
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Figure 17.7-1 Promoter Transcription unit Start point RNA polymerase 1
5′ 3′ 3′ 5′ Start point RNA polymerase 1 Initiation 5′ 3′ 3′ 5′ Template strand of DNA Unwound DNA RNA transcript Figure The stages of transcription: initiation, elongation, and termination (step 1)
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Figure 17.7-2 Promoter Transcription unit Start point RNA polymerase 1
5′ 3′ 3′ 5′ Start point RNA polymerase 1 Initiation 5′ 3′ 3′ 5′ Template strand of DNA Unwound DNA RNA transcript 2 Elongation Rewound DNA 5′ 3′ 3′ 3′ 5′ 5′ Direction of transcription (“downstream”) Figure The stages of transcription: initiation, elongation, and termination (step 2) RNA transcript
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Figure 17.7-3 Promoter Transcription unit Start point RNA polymerase 1
5′ 3′ 3′ 5′ Start point RNA polymerase 1 Initiation 5′ 3′ 3′ 5′ Template strand of DNA Unwound DNA RNA transcript 2 Elongation Rewound DNA 5′ 3′ 3′ 3′ 5′ 5′ Direction of transcription (“downstream”) Figure The stages of transcription: initiation, elongation, and termination (step 3) RNA transcript 3 Termination 5′ 3′ 3′ 5′ 5′ 3′ Completed RNA transcript
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Animation: Transcription
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The DNA sequence where RNA polymerase attaches is called the promoter; in bacteria, the sequence signaling the end of transcription is called the terminator The stretch of DNA that is transcribed is called a transcription unit
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Synthesis of an RNA Transcript
The three stages of transcription Initiation Elongation Termination
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RNA Polymerase Binding and Initiation of Transcription
Promoters signal the transcriptional start point and usually extend several dozen nucleotide pairs upstream of the start point Transcription factors mediate the binding of RNA polymerase and the initiation of transcription The completed assembly of transcription factors and RNA polymerase II bound to a promoter is called a transcription initiation complex A promoter called a TATA box is crucial in forming the initiation complex in eukaryotes
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Figure 17.8 Promoter Nontemplate strand DNA 5′ 3′ 1 A eukaryotic
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 17.8 The initiation of transcription at a eukaryotic promoter 5′ 3′ 3′ 3 Transcription initiation complex forms. 3′ 5′ 5′ RNA transcript Transcription initiation complex
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Elongation of the RNA Strand
As RNA polymerase moves along the DNA, it untwists the double helix, 10 to 20 bases at a time Transcription progresses at a rate of 40 nucleotides per second in eukaryotes A gene can be transcribed simultaneously by several RNA polymerases Nucleotides are added to the 3′ end of the growing RNA molecule
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Figure 17.9 Nontemplate strand of DNA RNA nucleotides RNA polymerase
3′ C U 5′ T T 3′ end G U A A G U C C A C C A C A 5′ 3′ A T A G G T T Figure 17.9 Transcription elongation 5′ Direction of transcription Template strand of DNA Newly made RNA
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Termination of Transcription
The mechanisms of termination are different in bacteria and eukaryotes In bacteria, the polymerase stops transcription at the end of the terminator and the mRNA can be translated without further modification In eukaryotes, RNA polymerase II transcribes the polyadenylation signal sequence; the RNA transcript is released 10–35 nucleotides past this polyadenylation sequence
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