Transcript From Gene to Protein

From Gene to Protein Lecture Notes
Biol 100 – K.Marr
1.
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 2—modeling transcription and translation
The Flow of Genetic Information: DNA to RNA to Protein  Phenotype
Cytoplasm
• Transcription: DNA
copied into mRNA
molecule
• Translation:
ribosomes translate
mRNA into protein—
a chain of amino
acids
• Proteins control
phenotype. How?
Nucleus
DNA
Transcription
mRNA
Translation
Protein
The one gene–one protein hypothesis:
The 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:
1.
Enzymes: catalysts for nearly all chemical reactions in cells;
Determine what cells can make and digest
2.
Structural components: muscles (actin and myosin), connective
tissue (collagen, elastin)
3.
Receptors on cell surface for growth factors, hormones, etc.
4.
Hormones: e.g. insulin, growth hormone, prolactin
5.
Transport: e.g. hemoglobin, spindle fibers
6.
Immune system: antibodies
CF phenotype
• Genes
determine
which
proteins a cell
can make
• Proteins
control
phenotype
• e.g. CFTR
Gene codes
for CFTR
protein
CFTR Protein: The cystic fibrosis transmembrane regulator protein
Carbohydrate
Chloride ions
Cytoplasm of cell
lining duct or lungs
CFTR Protein
•
Pumps
chloride ions
(salt) into cells
lining ducts or
the lungs
•
What are the
consequences
when CFTR
doesn’t work?
•
How does a
gene control
the production
of a protein?
CFTR Protein
Water
Inside of duct
or
Air sac in lungs
Cell
membrane
Water
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?
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?
An RNA Nucleotide
Phosphate
Base
(Uracil, U)
Sugar:
ribose
This oxygen is
absent in
deoxyribose
Transcription of a gene by RNA polymerase
RNA nucleotides
RNA
polymerase
Newly made
RNA
Direction of
transcription
Template
strand of DNA
Transcription: copying DNA into RNA ( 1 of 2)
(a) Parent DNA
(b) Transcription begins
RNA
polymerase
Complementary
base pairing
Strand
separation
Transcription: copying DNA into RNA ( 2 of 2)
(d) Products of transcription
(c) Transcription continues
Noncoding
strand
Coding
strand
Nucleotide
joining
New RNA strand
(actually several
hundred base
pairs long)
Parent DNA
totally
conserved
Comparing DNA and RNA
DNA
Number of
Strands
Sugars
Bases
RNA
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:
(c) Spliced RNA
RNA Processing
(d) Mature RNA
Differential splicing
can result in
different mRNA
molecules and,
therefore, different
proteins
Translation
(d) protein
Fig. 7.07
Processing of Eukaryotic RNA
Intron
RNA Processing includes
• Adding a cap and tail
• Removing introns
• Splicing exons together
– Differential splicing
produces different
mRNA molecules
Exon
Gene
(DNA)
Cap
RNA
transcript
with cap
and tail
Intron Exon
Exon
Transcription + the
Addition of cap and tail
Tail
Introns removed
Exons spliced together
mRNA
Coding sequence
Nucleus
Cytoplasm
Translation:
Ribosomes reading mRNA to produce a polypeptide
•
View animation of translation
Questions to answer
1.
2.
3.
4.
5.
What do we start with and end with?
Where does translation occur?
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?
Transfer RNA: tRNA
tRNA
1. Acts as a
molecular
interpreter
2. Carries amino
acids
3. Matches amino
acids with codons
in mRNA using
anticodons
Amino acid attachment site
Hydrogen bond
RNA
polynucleotide
chain
Anticodon
Anticodon
A portion of an mRNA molecule attached to a tRNA
Codon on mRNA
mRNA
Each Codon codes
Specifies a specific
tRNA—amino acid
complex
Amino acid
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
1. Initiation of Translation
Codon
mRNA
Anticodon
tRNA
Amino
acid
Ribosome
2. Elongation
Peptide bond forms
2. Elongation continues: Translocation of Ribosome
Ribosome
moves
tRNA ejected
3. Termination of Translation
Termination
factor binds
Ribosome
moves
tRNA ejected
Peptide bond forms
3. Termination continued:
Disassembly of Ribosome
tRNA
Polypeptide chain
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
1.
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?
2.
Translate this portion of the gene.
–
What is the order of amino acids in the resulting protein?
Table of
Codons
found on
mRNA
•
•
Each codon
specifies a
specific
amino acid
The same
genetic code
is used by
nearly all
organisms!!
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!!)
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
1. Transcribe this portion of the gene.

2.
Translate this portion of the gene.
–
3.
What is the order of bases in the resulting mRNA molecule?
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?
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
Explaining the symptoms of CF
•
Why does CF only affect certain parts of the body?
•
What do the characteristics of CF have in common?
1. Mucus build-up in the lungs
• Lung infections (e.g. pneumonia)
2. Male sterility (blocked vas deferens)
3. Salty sweat
4. Trouble digesting food (blocked pancreatic duct)
Explaining the symptoms of CF
•
In CF, the faulty CFTR protein
never makes it to cell membrane
1.
What builds up outside of cells?
Why?
2.
Why salty sweat?
3.
Why does mucus collect in
lungs?
4.
Why respiratory infections?
5.
Why problems with digestion?
6.
Why male sterility?
CFTR Protein: Pumps
Chloride ions into cell
Chloride ions
outside of cell
Chloride ions
in cell
Understanding Cystic Fibrosis at the Cellular Level
How does CFTR protein get from where it’s produced to its home
in the cell membrane?
1.
Where is the CFTR protein produced?
2.
CFTR is a glycoprotein—where does it go for modification?
 How does it get there?
3.
How does the modified CFTR protein get to the plasma
membrane?
4.
The defective CFTR protein is recognized at the ER as defective
 Where is the defective CFTR protein sent?
CF symptoms may be mild or severe
CFTR
Gene
Several hundred different mutations are associated with CF
What’s 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)
DF508 deletion: the most common cause of cystic fibrosis
• Why does
isoleucine
(Ile) at
amino acid
position
507 remain
unchanged?
Mutations responsible for Sickle Cell Anemia
• Only one amino acid in 146 is incorrect in sickle-cell
hemoglobin!
Normal hemoglobin DNA
mRNA
Normal hemoglobin
Glu
Mutant hemoglobin DNA
mRNA
Sickle-cell hemoglobin
Val
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
Phe
Ser
Ala
(a) Base substitution
Met
Lys
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
• 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
SUMMARY OF KEY CONCEPTS
DNA and RNA: Polymers of Nucleotides
Nitrogenous
base
Phosphate
group
Sugar
DNA
Nucleotide
Polynucleotide
Review:
DNA RNA Protein
RNA Polymerase
1 Transcription
1.
Nucleus
RNA
transcript
DNA
22. RNA processing
Intron
Amino acid
CAP
Tail
mRNA
Intron
Enzyme
3. Amino acid attachment
Ribosomal
subunits
44. Initiation of translation
Stop codon
Anticodon
Codon
6. Termination
55. Elongation