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From Gene to Protein. Chapter 17. I. Connection of Genes to Protein. A. Early Evidence - from the study of metabolic defects 1. Gerrod’s Hypothesis 1909 Archibald Gerrod - first to suggest that genes dictate phenotype through enzymes. - PowerPoint PPT Presentation
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From Gene to Protein
Chapter 17
I. Connection of Genes to Protein
A. Early Evidence - from the study of metabolic defects
1. Gerrod’s Hypothesis 1909
Archibald Gerrod - first to suggest that genes dictate phenotype through
enzymes.
A. Early Evidence - from the study of metabolic defects
1. Gerrod’s Hypothesis 1909
Studied Alkaptonuria - absence of an enzyme that breaks down
Alkapton
Symptoms of disease due to inability to make an enzyme
Enzymes needed for metabolic pathways
A. Early Evidence - from the study of metabolic defects
2. Beadle and Ephrussi, 1930s
Mutations affecting drosophila eye color
Blocks pigment by absence of a
pathway enzyme.
Mere hypothesis.
A. Early Evidence - from the study of metabolic defects
3. Beadle and Tatum
Finally established the link between genes and enzymes
Studied nutrition of bread mold
Neurospora crassa
(Study text for details)
A. Early Evidence - from the study of metabolic defects
4. One Gene - One Polypeptide Hypothesis
Refined from Beadle and Tatum
Not all proteins are enzymes
Specific proteins require specific genes
Used to be
One gene - one protein
Why the change?
B. The Process of Protein Synthesis - General Overview
1. Genes = instructions for a protein
2. RNA = bridge between DNA and protein
RNA distinctives
Single stranded
Ribose sugar
Uracil instead of Thymine
B. The Process of Protein Synthesis - General Overview
3. DNA nucleotide sequence
translates to Amino Acid sequence
B. The Process of Protein Synthesis - General Overview
4. Two Stages in Protein Synthesis
Transcription
DNA code to RNA copy
Messenger RNA (mRNA)
Translation
mRNA code
to Amino Acid sequence
B. The Process of Protein Synthesis - General Overview
5. Eukaryotes and Prokaryotes
Prokaryotes - no nucleus
Transcription and translation are
closely coupled
Eukaryotes
Transcription - in nucleus
RNA processing - in nucleus
Translation - cytoplasmic ribosome
B. The Process of Protein Synthesis - General Overview
6. Pathway summary
DNA - RNA - Protein
C. Structure of the Genetic Code
1. Triplets of nucleotides specify amino acids
3 letters = dictate one amino acid
With 4 letters = 64 triplet combinations
2. Each Triplet called a Codon
C. Structure of the Genetic Code
3. Matching codons to their amino acids began in the 1960s
First - Marshall Nirenberg
Mid 1960s - completed
61 code for amino acids
AUG = start of a gene
3 codons = termination of a gene
C. Structure of the Genetic Code
4. Redundant, but not ambiguous
Several codons can code for 1 amino acid
(redundant)
One codon can mean only 1 amino acid
(not ambiguous)
II. Transcription and RNA
A. DNA in the nucleus
but Proteins built in cytoplasm
Need an RNA copy of the gene to take
code to the cytoplasm.
Transcription - making the RNA copy
II. Transcription and RNA
B. RNA Polymerase - opens up the specific gene and adds RNA nucleotides according to the DNA sequence.
Prokaryotes - 1 kind RNA polymerase
Eukaryotes - RNA Polymerases I, II, III
C. Gene is read 3’ to 5’
RNA built 5’ to 3’
II. Transcription and RNA
D. Beginning and end of gene
Promotor sequence begins
Terminator signals the end
II. Transcription and RNA
E. Three stages in Transcription
Initiation
Elongation
Termination
II. Transcription and RNA F. Initiation
Promotor sequence
start of a gene
determines which strand is template
provides a binding site for
RNA Polymerase
II. Transcription and RNA F. Initiation
In Eukaryotes
Transcription factors find promotor
(Often a TATA sequence)
Bind to promotor
RNA Polymerase binds onto Trans Fact
makes Transcription Initiation Complex
II. Transcription and RNA G. Elongation
RNA Polymerase moves down DNA
Untwists and unzips DNA (10-20 bases)
Adds in RNA nucleotides to 3’ end of
growing RNA chain
Behind this - DNA connects back and twists
RNA copy peels away.
II. Transcription and RNA G. Elongation
Many RNA Polymerases can be making a mRNA copy of the same gene at the same time.
= More mRNAs
increases the speed of protein production
II. Transcription and RNA H. Termination
At end, RNA Polymerase copies a
terminator
In prokaryotes
Transcription ends at end of terminator
In Eukaryotes
Transcription continues far beyond it
This longer “pre-RNA” is then released
I. Modification of mRNA after transcription
Enzymes modify the “pre-RNA” before its sent out to make protein
1. At 5’ end, a modified Guanine is added
the 5’ Cap
Function?
Prevents erosion
“Attach here” signal for
ribosomes
I. Modification of mRNA after transcription
Enzymes modify the “pre-RNA” before its sent out to make protein
2. At 3’ end - Poly A tail - 50 -250 letters
Protects against hydrolysis damage
Facilitates ribosome attachment
Facilitates export from nucleus
I. Modification of mRNA after transcription
3. RNA Splicing
mRNA has long stretches of non-coding nucleotides in between codes
Called Introns
Coding regions called Exons
Splicing removes many intron portions
I. Modification of mRNA after transcription
3. RNA Splicing
Accomplished by Spliceosome
Composed of;
Several proteins
snRNP - small nuclear
Ribonuclearproteins
each snRNP has a RNA
molecule, 150 letters
I. Modification of mRNA after transcription
3. RNA Splicing
Functions
1.Contol which introns stay in
2.Regulate passage out of nucleus
3.Splice genes to code for more than
one polypeptide
I. Modification of mRNA after transcription
3. RNA Splicing
Alternative RNA Splicing
More than one polypeptide from one
gene.
Depends on which segments are treated as exons
I. Modification of mRNA after transcription
4. Split genes facilitates protein evolutionProteins have different regions - domainsdifferent exons may code for different
domains
Presence of introns - facilitate crossing over at that point.
Creates new genes by mixing exons from different genes.
Leads to new proteins
III. Translation A. mRNA sequence to protein
Transfer RNAs place amino acids in correct order according to the
sequence of codons
III. Translation B. Transfer RNA - tRNA
Made according to genes in the nucleus
Can be used in the cytoplasm over and over
1. Action - picks up a specific amino acid
places it in correct sequence at ribosome
Returns to cytosol to get another of the same amino acid
III. Translation B. Transfer RNA - tRNA
2. Structure of tRNA
80 nucleotides
Loops back on itself for a 3D shape
At one end - attachment for a specific amino acid.
At opposite end - Anticodon
compliments and binds to the codon
for the amino acid it carries
III. Translation B. Transfer RNA - tRNA
3. Some anticodons recognize more than one codon.Why? Two reasons
- The third base in a codon includes some flexibility- called Wobble
At this Wobble position
U on anticodon can bind to
A or G in third codon position
- Some tRNA anticodons have Inosine
A modified Adenine
Can bond with U,C, or A.
III. Translation B. Transfer RNA - tRNA
4. Amino Acid joins to tRNA by
Aminoacyl-tRNA Synthetase
20 different Synthetases to match
the 20 different amino acids
Each has active sites for a specific
tRNA and A. Acid combo.
Forms a covalent bond.
III. Translation C. Ribosomes
Composed of large and small subunit
made of proteins and rRNA (Active parts)
Subunits form in the nucleolus
exit nucleus through nuclear pores
Large and small subunits only joins as a functional ribosome when they contact a mRNA molecule
Prokaryotic ribosomes differ from eukaryotic ones
Ribosome Structure
III. Translation C. Ribosomes
Binding Sites
P - tRNA with chain
A - tRNA with next
A. Acid
E - Discharges used
tRNAs.
III. Translation D. Details of the Process
1. Initiation
2. Elongation
3. Termination
All need protein aids
1 and 2 need energy
hydrolysis of GTP
D. Details of the Process
1. Initiation - Brings together
mRNA
First tRNA with A.Acid
Two Ribosome subunits
D. Details of the Process
1. Initiation
Sm. Ribosomal subunit
binds mRNA
and initiator tRNA (methionine)
Attaches to first codon
D. Details of the Process
1. Initiation
Initiation factors bring Lg subunit
Attaches so that
Initiator tRNA occupies P site
D. Details of the Process
2. Elongation
Series of 3 step cycles
Codon Recognition
A tRNA binds to mRNA codon
at the A site
Binding assisted by an
Elongation factor
Requires hydolysis of 2 GTPs
D. Details of the Process
2. Elongation
Series of 3 step cycles
Peptide Bond Formation
new A. acid in A site is bonded
to growing chain at P site
Peptide bond catalyzed by
rRNA
tRNA at P site separates from
chain
Chain now hooked to A site
2. Elongation
Series of 3 step cycles
Translocation
Ribosome moves the tRNA
at A site with chain
To the P site
This slides the mRNA in
(anti-codon still bonded)
tRNA from P site moves to E
and leaves ribosome
Fueled by hydrolysis of GTP
3. Termination
When a stop codon reaches A site
Release factor binds to stop codon
breaks the bond (hydrolyzes)
between the tRNA and chain at the
P site
Polypeptide chain is now free
Translation complex disassembles
E. Many polypeptides can be made from a mRNA at the same time.
More than one ribosome can travel down a mRNA at the same time
Polyribosomes
F. After translation
Polypeptide folds up spontaneously
Primary to ….. Tertiary … quaternary
G. Post-translational modifications
1. Additions of
sugars, lipids, phosphate groups
2. Enzymes may cleave the chains or remove sections
3. Multiple polypetides may join
Tertiary structures to Quaternary
H. Post-translational destinations of proteins
Final destinations for proteins effects how they are processed during translation
1. Two kinds of Ribosomes
Free - suspended in cytosol
translate proteins for cytosol
Bound - Attached to cytosol side
of Endoplasmic Reticulum
Synthesizes proteins for
1. The endomembrane system
2. Secretion outside cell
H. Post-translational destinations of proteins
2. All Ribosomes begin translating in cytosol
But
Whether the protein will be
1. Part of endomembrane system
or
2. For export out of cell
It has a Signal Peptide region at the leading end (synthesized first)
About 20 Amino acids
H. Post-translational destinations of proteins
Signal Recognition Particle (SRP)
Binds to the Signal Peptide
Attaches it and its Ribosome to the ER
SRP - Protein RNA combo
After binding, SRP leaves
H. Post-translational destinations of proteins
Growing protein snakes through protein pore in ER membrane into cisternal space
When complete
An enzyme cleaves off the signal peptide
Secretory proteins gather completely in cisternal space
ER Membrane proteins remain embedded
H. Post-translational destinations of proteins
3. Other kinds of Signal Peptides send proteins to
Mitochondria, Chloroplasts, Nucleus, other organelles
Acts like a postal code
Translation is completed in cytosol by free ribosomes
IV. RNA RolesA. Diverse functions based on
1. Ability of RNA to form H-bonds with other nucleic acids
2. Ability to take on a 3D shape by looping back on itself with H-bonds
(tRNA)
B. RNA more versatile than DNA
Structural
Informational
Catalytic
V. Prokaryote vs. EukaryoteA. Eukaryotic RNA polymerases differ
B. Eukaryotic RNA polymerases require transcription factors
C. Transcription termination differs
D. Ribosomes differ
E. Prokaryotes can transcribe and translate a gene at the same time
Eukaryotes, nuclear membrane separates the two processes
Additional steps needed for coordination
VI. Mutations
A. Change in one base pair = Point Mutation
1. In gametes? - to future generations
2. Example - Sickle-cell Anemia
3. Base-pair substitution
Replacement of one pair with
another
VI. MutationsA. Change in one base pair = Point Mutation
3. Base-pair substitution
Silent mutations - no effect, why?
- change results in same amino acid due to redundancy in code
- change results in new amino acid with same properties
- change in protein region where amino acid sequence is not essential
VI. Mutations
A. Change in one base pair = Point Mutation
3. Base-pair substitution
Some cause key changes
Sometimes advantageous
At protein active sites - more likely to have impact
VI. Mutations
B. Missense mutations - still code for an amino acid, but for a different one
C. Nonsense mutations - change an amino acid codon to a stop codon.
D. Insertions or Deletions
Disastrous effect
Frameshift - All nucleotides downstream will be improperly grouped into codons
VI. Mutations
E. Causes of Mutations
Country Music!
1. Errors occuring during
DNA Replication
DNA Repair
DNA Recombination
Called spontaneous mutations
VI. Mutations
E. Causes of Mutations
2. Mutagens - Chemicals or physical agents that alter DNA
(Country Music?)
Physical agents - radiation
VI. MutationsE. Causes of Mutations
2. Mutagens - Chemicals or physical agents that alter DNA
Chemical agent methods
- Some mimic DNA bases
causes incorrect pairing
- Insertion into and distortion of the double helix
- Chemical changes to bases
Connection mutagens - carcinogens
How do we define Gene ?
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