From Gene to Protein Lecture Notes Biol 100 K'Marr

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From Gene to Protein Lecture NotesBiol 100
K.Marr

• Topics for the next few lectures
• Transcription From DNA to RNA
• Translation From RNA to Protein
• Understanding Cystic Fibrosis
• Chapter 10 in Essential Biology by Campbell et al
  
• 2. Lab 7. Modeling DNA Structure, DNA Replication
  and Protein Synthesis Read the introduction
  carefully
• Part 1 (through page 9)modeling DNA Structure
  and Replication
• Part 2modeling transcription and translation

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The Flow of Genetic Information DNA to RNA to
Protein ? Phenotype
Cytoplasm

• Transcription DNA copied into mRNA molecule
• Translation ribosomes translate mRNA into
  proteina chain of amino acids
• Proteins control phenotype. How?

Nucleus
DNA
Transcription
mRNA
Translation
Protein
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The one geneone protein hypothesisThe function
of a gene is to dictate the production of a
specific protein. Why are proteins so important?

• A few of the many roles played by proteins
• Enzymes catalysts for nearly all chemical
  reactions in cells Determine what cells can make
  and digest
• Structural components muscles (actin and
  myosin), connective tissue (collagen, elastin)
• Receptors on cell surface for growth factors,
  hormones, etc.
• Hormones e.g. insulin, growth hormone, prolactin
• Transport e.g. hemoglobin, spindle fibers
• Immune system antibodies

4

• CF phenotype
• Genes determine which proteins a cell can make
• Proteins control phenotype
• e.g. CFTR Gene codes for CFTR protein

5
CFTR Protein The cystic fibrosis transmembrane
regulator protein
Carbohydrate

• CFTR Protein
• Pumps chloride ions (salt) into cells lining
  ducts or the lungs
• What are the consequences when CFTR doesnt work?
• How does a gene control the production of a
  protein?

Cytoplasm of cell lining duct or lungs
Chloride ions
CFTR Protein
Water
Inside of duct or Air sac in lungs
Cell membrane
Water
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The order of Bases in a gene determines the
order of amino acids in the protein it codes for
Is the order of amino acids in a protein
important?
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Transcription copying DNA into RNA

• View animation of transcription
• Questions to answer
• 1. What do we start with and end with?
• 2. Where does transcription occur? When?
• 3. What is needed for transcription to occur?
• 4. What is the sequence of events?

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An RNA Nucleotide
Phosphate
Base (Uracil, U)
Sugar ribose
This oxygen is absent in deoxyribose
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Transcription of a gene by RNA
polymerase
RNA nucleotides
RNA polymerase
Newly made RNA
Direction of transcription
Template strand of DNA
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Transcription copying DNA into RNA ( 1 of 2)
(a) Parent DNA
(b) Transcription begins
RNA polymerase
Complementary base pairing
Strand separation
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Transcription copying DNA into RNA ( 2 of 2)
(d) Products of transcription
(c) Transcription continues
Non-coding strand
Coding strand
New RNA strand (actually several hundred
base pairs long)
Nucleotide joining
Parent DNA totally conserved
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Comparing DNA and RNA
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Transcription in Eukaryotic Cells Differential
RNA splicing can result in one gene producing
more than one protein
(a) Gene
Intron 1
Intron 2
Intron 3
Intron 4
Intron 5
Exon 1
Exon 2
Exon 3
Exon 4
Exon 5
Exon 6
Transcription
(b) Primary transcript
RNA splicing Differential splicing can result
in different mRNA molecules and, therefore,
different proteins
(c) Spliced RNA
RNA Processing
(d) Mature RNA
Translation
Fig. 7.07
(d) protein
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• Processing of Eukaryotic RNA

Intron
Exon

• RNA Processing includes
• Adding a cap and tail
• Removing introns
• Splicing exons together
• Differential splicing produces different mRNA
  molecules

Exon
Intron
Exon
Gene (DNA)
Transcription the Addition of cap and tail
Cap
RNA transcript with cap and tail
Tail
Introns removed
Exons spliced together
mRNA
Coding sequence
Nucleus
Cytoplasm
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Translation Ribosomes reading mRNA to
produce a polypeptide

• View animation of translation
• Questions to answer
• What do we start with and end with?
• 2. Where does translation occur?
• 3. What is needed for translation to occur?
• What is the sequence of events?
• What are the roles of mRNA, ribosomes, start
  codon, tRNA, anticodons, stop codon?

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Transfer RNA tRNA
Amino acid attachment site

• tRNA
• Acts as a molecular interpreter
• Carries amino acids
• Matches amino acids with codons in mRNA using
  anticodons

Hydrogen bond
RNA polynucleotide chain
Anticodon
Anticodon
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A portion of an mRNA molecule attached to a tRNA
Codon on mRNA
mRNA
Each Codon codes Specifies a specific
tRNAamino acid complex
Amino acid
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A ribosome translating mRNA into protein
Small subunit

• Ribosomes
• Organelle that makes protein
• Reads mRNA 5 ? 3
• Made of rRNA and protein
• Consist of 2 subunits

mRNA
Large subunit
Protein under construction
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1. Initiation of Translation
Codon
mRNA
Anticodon
Ribosome
tRNA
Amino acid
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2. Elongation
Peptide bond forms
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2. Elongation continues Translocation of
Ribosome
Ribosome moves
tRNA ejected
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3. Termination of Translation
Termination factor binds
Ribosome moves
tRNA ejected
Peptide bond forms
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3. Termination continued Disassembly of
Ribosome
tRNA
Polypeptide chain
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Transcription Translation of the CRTR Gene in
Healthy People

• Part of a normal CFTR gene
• 5...ATCATCTTTGGTGTT...3 non-coding strand
• 3...TAGTAGAAACCACAA...5 coding strand
• Transcribe this portion of the gene.
• The whole gene codes for 1480 amino acids in CFTR
  protein!
• What is the order of bases in the resulting mRNA
  molecule?
• Translate this portion of the gene.
• What is the order of amino acids in the resulting
  protein?

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Table of Codons found on mRNA

• Each codon specifies a specific amino acid
• The same genetic code is used by nearly all
  organisms!!

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Transcription Translation of the CRTR Gene in
Healthy People

• Part of a normal CFTR gene
• 5...ATCATCTTTGGTGTT...3 non-coding strand
• 3...TAGTAGAAACCACAA...5 coding strand

Transcription
5...AUCAUCUUUGGUGUU...3
Translation
.....Ile-Ile-Phe-Gly-Val (only 5 of the
1480 amino acids in protein!!)
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Transcription Translation of the CRTR Gene in
People with CF

• Part of CFTR gene associated with Cystic
  Fibrosis
• 5...ATCATTGGTGTT...3 non-coding strand
• 3...TAGTAACCACAA...5 coding strand
• Transcribe this portion of the gene.
• What is the order of bases in the resulting mRNA
  molecule?
• Translate this portion of the gene.
• What is the order of amino acids in the resulting
  protein?
• What is different about the gene and the protein
  in people with cystic fibrosis?

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Transcription Translation of the CRTR Gene in
People with CF

• Part of CFTR gene associated with Cystic
  Fibrosis
• 5...ATCATTGGTGTT...3 non-coding strand
• 3...TAGTAACCACAA...5 coding strand

Transcription
5...AUCAUUGGUGUU...3
Translation
.....Ile-Ile-Gly-Val.. Phenylalanine
(Phe) is missing
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Explaining the symptoms of CF

• Why does CF only affect certain parts of the
  body?
• What do the characteristics of CF have in common?
• Mucus build-up in the lungs
• Lung infections (e.g. pneumonia)
• Male sterility (blocked vas deferens)
• Salty sweat
• Trouble digesting food (blocked pancreatic duct)

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Explaining the symptoms of CF

• In CF, the faulty CFTR protein never makes it to
  cell membrane
• What builds up outside of cells? Why?
• Why salty sweat?
• Why does mucus collect in lungs?
• Why respiratory infections?
• Why problems with digestion?
• Why male sterility?

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Understanding Cystic Fibrosis at the Cellular
Level

• How does CFTR protein get from where its
  produced to its home in the cell membrane?
• Where is the CFTR protein produced?
• CFTR is a glycoproteinwhere does it go for
  modification?
• How does it get there?
• How does the modified CFTR protein get to the
  plasma membrane?
• The defective CFTR protein is recognized at the
  ER as defective
• Where is the defective CFTR protein sent?

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CF symptoms may be mild or severe
CFTR Gene
Several hundred different mutations are
associated with CF
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Whats a Mutation?

• Any change in the nucleotide sequence of DNA
• Types of Mutations
• Substitution, insertion or deletion
• Occur during DNA replication
• Mutations may Result from
• Errors in DNA replication
• Mutagens
• physical or chemical agents that may cause errors
  during DNA replication
• chemicals in cigarette smoke
• Radiation (e.g. U.V. light, X-rays)

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DF508 deletion the most common cause of cystic
fibrosis

• Why does isoleucine (Ile) at amino acid position
  507 remain unchanged?

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Mutations responsible for Sickle Cell Anemia

• Only one amino acid in 146 is incorrect in
  sickle-cell hemoglobin!

Normal hemoglobin DNA
Mutant hemoglobin DNA
mRNA
mRNA
Normal hemoglobin
Sickle-cell hemoglobin
Glu
Val
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Types of Mutations Base Substitutions,
Insertions or deletions

• Base substitutions
• May result in changes in the amino acid sequence
  in a protein, or
• May be silent (have no effect)

mRNA
Protein
Met
Lys
Phe
Gly
Ala
(a) Base substitution
Met
Lys
Phe
Ser
Ala
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• Types of Mutations Base Insertions and
  deletions

• Can have disastrous effects
• Change the reading frame of the genetic message

mRNA
Protein
Met
Lys
Phe
Gly
Ala
(b) Nucleotide deletion
Met
Lys
Leu
Ala
His
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• Although mutations are often harmful

• They are the source of the rich diversity of
  genes in the living world
• They contribute to the process of evolution by
  natural selection

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SUMMARY OF KEY CONCEPTS

• DNA and RNA Polymers of Nucleotides

Nitrogenous base
Phosphate group
Sugar
DNA
Nucleotide
Polynucleotide
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Review DNA? RNA? Protein
RNA Polymerase
1
1. Transcription
Nucleus
RNA transcript
DNA
2. RNA processing
2
Intron
Amino acid
CAP
Tail
mRNA
Intron
Enzyme
tRNA
3. Amino acid attachment
Ribosomal subunits
4
4. Initiation of translation
Stop codon
Anticodon
Codon
6. Termination
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5. Elongation