Download Ch .15 - Crestwood Local Schools

Chapter 15
From Genes to
Proteins
Question?
How
does DNA control a cell?
By controlling Protein
Synthesis.
Proteins are the link between
genotype and phenotype.
For tests:
Name(s)
of experimenters
Outline of the experiment
Result of the experiment and
its importance
1909 - Archibald Garrod
Suggested
genes control
enzymes that catalyze
chemical processes in cells.
Inherited Diseases - “inborn
errors of metabolism” where
a person can’t make an
enzyme.
Example
Alkaptonuria
- where urine
turns black after exposure to
air.
Lacks - an enzyme to
metabolize alkapton.
George Beadle and
Edward Tatum
Worked
with Neurospora and
proved the link between
genes and enzymes.
Neurospora
Pink bread mold
Experiment
Grew
Neurospora on agar.
Varied the nutrients.
Looked for mutants that
failed to grow on minimum
agar.
Results
Three
classes of mutants for
Arginine Synthesis.
Each mutant had a different
block in the Arginine
Synthesis pathway.
Conclusion
Mutations
were abnormal
genes.
Each gene dictated the
synthesis of one enzyme.
One Gene - One Enzyme
Hypothesis.
Current Hypothesis
One
Gene - One Polypeptide
Hypothesis (because of 4th
degree structure).
Central Dogma
DNA
Transcription
RNA
Translation
Polypeptide
Explanation
DNA
- the Genetic code or
genotype.
RNA - the message or
instructions.
Polypeptide - the product for
the phenotype.
Genetic Code
Sequence
of DNA bases that
describe which Amino Acid to
place in what order in a
polypeptide.
The genetic code gives the
primary protein structure.
Code Basis
If you use:
1 base = 1 amino acid
4 bases = 4 amino acids
41 = 4 combinations, which
are not enough for 20 AAs.
If you use:
2
bases = 1 amino acid
42 = 16 amino acids
Still not enough combinations.
If you use:
3
bases = 1AA
43 = 64 combinations
More than enough for 20
amino acids.
Genetic Code
Is
based on triplets of bases.
Has redundancy; some AA's
have more than 1 code.
Proof - make artificial RNA and
see what AAs are used in
protein synthesis (early 1960’s).
Codon
3-nucleotide “word” in the
Genetic Code.
64 possible codons known.
A
Codon Dictionary
Start-
AUG (Met)
Stop- UAA
UAG
UGA
60 codons for the other 19
AAs.
For Testing:
Be
able to “read” a DNA or
RNA message and give the
AA sequence.
RNA Genetic Code Table will
be provided.
Code Redundancy
Third
base in a codon shows
"wobble”.
First two bases are the most
important in reading the code
and giving the correct AA.
The third base often doesn’t
matter.
Code Evolution
The
genetic code is nearly
universal.
Ex: CCG = proline (all life)
Reason - The code must have
evolved very early. Life on
earth must share a common
ancestor.
Reading Frame and
Frame Shift
The
“reading” of the code is every
three bases (Reading Frame)
Ex: the red cat ate the rat
Frame shift – improper groupings
of the bases
Ex: thr edc ata tat her at
The “words” only make sense if
“read” in this grouping of three.
Transcription
Process
of making RNA from
a DNA template.
Transcription Steps
1.
2.
3.
4.
RNA Polymerase Binding
Initiation
Elongation
Termination
RNA Polymerase
Enzyme
for building RNA
from RNA nucleotides.
Binding
Requires
that the enzyme
find the “proper” place on the
DNA to attach and start
transcription.
Binding
Is
a complicated process
Uses Promoter Regions on
the DNA (upstream from the
information for the protein)
Requires proteins called
Transcription Factors.
Transcription Initiation
Complex
The
complete assembly of
transcription factors and RNA
Polymerase bound to the
promoter area of the DNA to
be transcribed.
Initiation
Actual
unwinding of DNA to
start RNA synthesis.
Requires Initiation Factors.
Elongation
RNA
Polymerase untwists
DNA 1 turn at a time.
Exposes 10 DNA bases for
pairing with RNA nucleotides.
Elongation
Enzyme
moves 5’
3’.
Rate is about 60 nucleotides
per second.
Comment
Each
gene can be read by
sequential RNA Polymerases
giving several copies of RNA.
Result - several copies of the
protein can be made.
Termination
DNA
sequence that tells RNA
Polymerase to stop.
Ex: AATAAA
RNA Polymerase detaches
from DNA after closing the
helix.
Final Product
Pre-mRNA
This
is a “raw” RNA that will
need processing.
Modifications of RNA
1. 5’ Cap
2. Poly-A Tail
3. Splicing
5' Cap
Modified
Guanine nucleotide
added to the 5' end.
Protects mRNA from
digestive enzymes.
Recognition sign for
ribosome attachment.
Poly-A Tail
150-200
Adenine nucleotides
added to the 3' tail
Protects mRNA from
digestive enzymes.
Aids in mRNA transport from
nucleus.
Let’s
see Transcription in
motion…
http://www.hhmi.org/biointera
ctive/media/DNAi_transcriptio
n_vo2-lg.mov
RNA Splicing
Removal
of non-protein
coding regions of RNA.
Coding regions are then
spliced back together.
Introns
Intervening
sequences.
Removed from RNA.
Exons
Expressed
sequences of
RNA.
Translated into AAs.
Spliceosome
Cut
out Introns and join
Exons together.
Made of snRNA and snRNP.
Result
Ribozymes
RNA
molecules that act as
enzymes.
Are sometimes Intron RNA
and cause splicing without a
spliceosome.
Introns - Function
Left-over
DNA (?)
Way to lengthen genetic
message.
Old virus inserts (?)
Way to create new proteins.
Final RNA Transcript
Alternative Splicing
The
RNA can be spliced into
different mRNA’s.
Each different mRNA produces a
different polypeptide.
Ex. – variable regions of
antibodies.
Another Example
– inhibits apoptosis
Bcl-XS – induces apoptosis
Bcl-XL
Two
different and opposite
effects!!
DSCAM Gene
Found
in fruit flies
Has 100 potential splicing sites.
Could produce 38,000 different
polypeptides
Many of these polypeptides have
been found
Commentary
Alternative
Splicing is going to
be a BIG topic in Biology.
About 60% of genes are
estimated to have alternative
splicing sites.
One gene does not equal one
polypeptide.
Translation
Process
by which a cell
interprets a genetic message
and builds a polypeptide.
Materials Required
tRNA
Ribosomes
mRNA
Transfer RNA = tRNA
Made
by transcription.
About 80 nucleotides long.
 Carries AA for polypeptide
synthesis.
Structure of tRNA
Has
double stranded regions
and 3 loops.
AA attachment site at the 3'
end.
1 loop serves as the
Anticodon.
Anticodon
Region
of tRNA that base
pairs to mRNA codon.
Usually is a compliment to
the mRNA bases, so reads
the same as the DNA codon.
Example
DNA
- GAC
mRNA - CUG
tRNA anticodon - GAC
Comment
"Wobble"
effect allows for 45
types of tRNA instead of 61.
Reason - in the third position,
U can pair with A or G.
Inosine (I), a modified base in
the third position can pair
with U, C, or A.
Importance
Allows
for fewer types of
tRNA.
Allows some mistakes to
code for the same AA which
gives exactly the same
polypeptide.
Aminoacyl-tRNA
Synthetases
Family
of Enzymes.
Add AAs to tRNAs.
Active site fits 1AA and 1
type of tRNA.
Uses a “secondary genetic”
code to load the correct AA to
each tRNA.
Ribosomes
Two
subunits made in the
nucleolus.
Made of rRNA (60%)and
protein (40%).
rRNA is the most abundant
type of RNA in a cell.
Large subunit
Proteins
rRNA
Both sununits
Large Subunit
Has
3 sites for tRNA.
P site: Peptidyl-tRNA site carries the growing polypeptide
chain.
 A site: Aminoacyl-tRNA site holds the tRNA carrying the next
AA to be added.
E site: Exit site
Translation Steps
1. Initiation
2. Elongation
3. Termination
Initiation
Brings
together:
mRNA
A
tRNA carrying the 1st AA
2 subunits of the ribosome
Initiation Steps:
1. Small subunit binds to the
mRNA.
2. Initiator tRNA (Met, AUG)
binds to mRNA.
3. Large subunit binds to
mRNA. Initiator tRNA is in
the P-site
Initiation
Requires
other proteins
called "Initiation Factors”.
GTP used as energy source.
Elongation Steps:
1. Codon Recognition
2. Peptide Bond Formation
3. Translocation
Codon Recognition
tRNA
anticodon matched to
mRNA codon in the A site.
Peptide Bond
Formation
A
peptide bond is formed
between the new AA and the
polypeptide chain in the
P-site.
Bond formation is by rRNA
acting as a ribozyme
After bond formation
The
polypeptide is now
transferred from the tRNA in
the P-site to the tRNA in the
A-site.
Translocation
tRNA
in P-site is released.
Ribosome advances 1 codon,
5’ 3’.
tRNA in A-site is now in the
P-site.
Process repeats with the next
codon.
Comment
Elongation
takes 60
milliseconds for each AA
added.
Termination
Triggered
by stop codons.
Release factor binds in the
A-site instead of a tRNA.
 H2O is added instead of AA,
freeing the polypeptide.
Ribosome separates.
Let’s
see Translation in
motion…
http://www.hhmi.org/biointera
ctive/media/DNAi_translation
_vo2-lg.mov
Polyribosomes
Cluster
of ribosomes all
reading the same mRNA.
Another way to make multiple
copies of a protein.
Prokaryotes
Comment
Polypeptide
usually needs to
be modified before it
becomes functional.
Examples
Sugars,
lipids, phosphate
groups added.
Some AAs removed.
Protein may be cleaved.
Join polypeptides together
(Quaternary Structure).
Signal Hypothesis
“Clue”
on the growing
polypeptide that causes
ribosome to attach to ER.
All ribosomes are “free”
ribosomes unless clued by
the polypeptide to attach to
the ER.
Result
Protein
is made directly into
the ER .
Protein targeted to desired
location (e.g. secreted protein).
“Clue” (the first 20 AAs are
removed by processing).
Mutations
Changes
in the genetic
makeup of a cell.
May be at chromosome or
DNA level
Chromosome Alterations
Deletions
Duplications
Inversions
Translocations
General Result
Loss
of genetic information.
Position effects: a gene's
expression is influenced by
its location to other genes.
Evidence of Translocation
Translocations
Cri Du Chat Syndrome
Part
of p arm of #5 has been
deleted.
Good survival.
Severe mental retardation.
Small sized heads common.
Philadelphia
Chromosome
An
abnormal chromosome
produced by a translocation
of portions of chromosomes
9 and 22.
Causes chronic myeloid
leukemia.
Mutation types - Cells
cells or body cells –
not inherited
Germ Cells or gametes inherited
Somatic
DNA or Point Mutations
Changes
in one or a few
nucleotides in the genetic
code.
Effects - none to fatal.
Types of Point
Mutations
1. Base-Pair Substitutions
2. Insertions
3. Deletions
Base-Pair Substitution
The
replacement of 1 pair of
nucleotides by another pair.
Sickle Cell Anemia
Lets
see how this mutation
will affect the cell…
http://www.hhmi.org/biointera
ctive/media/DNAi_sicklecelllg.mov
Types of Substitutions
1. Missense - altered codons,
still code for AAs but not the
right ones
2. Nonsense - changed codon
becomes a stop codon.
Question?
What
will the "Wobble" Effect
have on Missense?
If the 3rd base is changed,
the AA may still be the same
and the mutation is “silent”.
Missense Effect
Can
be none to fatal
depending on where the AA
was in the protein.
Ex: if in an active site - major
effect. If in another part of
the enzyme - no effect.
Nonsense Effect
Stops
protein synthesis.
Leads to nonfunctional
proteins unless the mutation
was near the very end of the
polypeptide.
Sense Mutations
The
changing of a stop
codon to a reading codon.
Result - longer polypeptides
which may not be functional.
Ex. “heavy” hemoglobin
Insertions & Deletions
The
addition or loss of a base
in the DNA.
Cause frame shifts and
extensive missense,
nonsense or sense
mutations.
Question?
Loss
of 3 nucleotides is often
not a problem.
Why?
Because the loss of a 3 bases
or one codon restores the
reading frame and the protein
may still be able to function.
Mutagenesis
Process
of causing
mutations or changes in the
DNA.
Mutagens
Materials
that cause DNA
changes.
1. Radiation
ex: UV light, X-rays
2. Chemicals
ex: 5-bromouracil
Spontaneous
Mutations
Random
errors during DNA
replication.
Comment
Any
material that can
chemically bond to DNA,
or is chemically similar to the
nitrogen bases, will often be
a very strong mutagen.
Summary
Know
Beadle and Tatum.
Know the central dogma.
Be able to “read” the genetic
code.
Be able to describe the
events of transcription and
translation.
Summary
Be
able to discuss RNA and
protein processing.
Be able to describe and
discuss mutations.