Chapter 17
DNA Expression
Transcription
Translation
Central Dogma –now we know it’s not totally correct since
not all RNA is translated into protein
DNA RNA  Protein
DNA DNA via replication
DNA RNA via transcription
RNA protein via translation
Beadle and Tatum’s Theory:
One gene One protein
Which is a correct statement of
central dogma?
A. ProteinRNADNA
B. RNAproteinDNA
C. DNARNAprotein
Today we know that really one gene one RNA, but
RNA is not always translated to protein
Which type of RNA is correctly
paired with its function?
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rRNA: ribozymes in ribosome
which catalyze production of
peptide bonds
tRNA: code the order of
nucleotides in a polypeptide
mRNA: deliver amino acids into
the ribosome
snRNA: prevent expression of a
gene, either by targeting its RNA
for destruction Or by forming a
double stranded DNA that can
not be translated.
siRNA & miRNA: part of a
complex that attaches to the
junctions of exons and introns
during alternative splicing
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A.
Central Dogma
transcription
translation
transcription
RNA
processing
translation
Which is NOT a difference in gene expression for
eukaryotes and prokaryotes?
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A. Prokaryotes do not splice out exons prior
to translation, but eukaryotes do.
B. Prokaryote mRNA is generated in the
cytoplasm & also translated in the
cytoplasm
C. Eukaryotes can produce several different
proteins from the same gene, according
to how its processed, but prokaryotes
can not.
D. Prokaryotes use a simpler genetic code
than eukaryotes.
What is the evidence that genes code proteins?
Beadle & Tatum: One gene  one enzyme (protein)
Yeasts able to produce all 20
amino acids via metabolic
pathways (several enzymes
working sequentially) were
treated with radiation to create
mutations. They cloned
mutated cells, then tested
them one by one to find which
could live only with amino acid
arginine added to the medium
(or one of the 2 precursor
products in the 3 step
metabolic pathway).
They concluded that one gene
codes for one enzyme; now we
generalize to one gene1
protein
Events of gene expression (in eukaryotes, each
gene is transcribed independently of each other
gene.
Antisense strand
contains promoter
sequence where RNA
polymerase binds
5’ methyl cap added,
in some polyA tail
added, alternative
splicing by snRPs
Triplet Code—mRNA codons:
sequence of 3 ribonucleotides that
control insertion of one amino acid
The genetic code is degenerate (the 3rd
base seems not as important for
defining the amino acid coded by a
codon—wobble hypothesis).
It is also redundant: 64 codons for
only 20 amino acids.The
Note Note the the methionine codon,
AUG, is the start codon, but it also
appears later in the sequence.
Stop codons are bound by tRNA’s which
don’t get charged with an amino acid;
these are termed “release factors”
because the ribosome releases the
mRNA & polypeptide at a stop codon.
promoter sequence of the gene,
then reads the 3’to 5’ oriented
strand & produces a single
stranded mRNA in the 5’ to 3’
direction.
The RNA polymerase may add a
polyA tail code if this sequence is
coded by the gene. The RNA
polymerase disassociates from the
gene at the termination site
sequence.
Open reading frame—
from promoter to
terminaor
How is transcription different from
replication?
Replication copies all of the genome (every nucleotide—
all 3 to 5 billion in each 23
chromosomes of the human
cell (so 6 to 10 billion in each cell)
Transcription only copies one
side (template strand) of
one gene in the genome
most genes are
present in one copy
Uracil instead of Thymine in RNA
3’ C or ribonucleotides has an –OH
Functional group, but in deoxyribonuCleotides does not.
Prokaryotes can have translation happening at the
same time as transcription—no nucleus,
no introns
Eukaryotes must remove introns between
transcription and translation, as well as adding a
3’ poly A tail (controls longevity) and a
5’ guanine cap (essential
for ribosome assembly)
(prok do have methyl cap & some a poly A tail)
Processed DNA is sent to the cytoplasm where
ribosome complexes assemble at the start codon
(fmet), then translation occurs.
Bacterial translation is rapid, beginning as soon as the
RNA begins separation from the DNA. Polyribosomes
(several traveling on same RNA) amplify gene
expression.
In both prokaryotes and eukaryotes, RNA is destroyed when
protein synthesis is no longer needed, by RNAase enzymes.
Allowing DNA to avoid destruction by RNAases
are several shape differences between DNA & RNA, such
that RNA is a substrate for RNAase, but DNA is not.
T in DNA vs U in RNA
Double stranded DNA vs single stranded RNA
2’ C bonded to 2 H in DNA but to an H and an -OH in RNA
Double stranded RNA does occur is found when an RNA
retrovirus infects a host, and cells have evolved to
destroy ds RNA with enzymes; this is why miRNA &
siRNA can downregulate the expression of a gene during
normal development, too.
Once processed, the triplet code (genetic code)
is used to control the order of amino acids in the
protein
The triplet code is the order of ribonucleotides that
codes one amino acid—note stop, start, “wobble”
(often, the 3’ nucleotide differs in codons coding
for the same amino acid.
Sense strand is read 3’ to 5’, starting at a
particular distance from the promoter TATAAA
box
Transcription factors bind the 5’ untranscribed region of the gene (5’
UTR), making the promoter sequence accessible to RNA polymerase,
allowing initiation of transcription. Then RNA polymerase adds
ribonucleotides (5’3’)complementary to the sense (template strand)
of the gene. At the termination signal, the RNA polymerase detaches,
then the new RNA is processed before translation.
Pre-RNA processing in the nucleus: Spliceosomes remove
introns at pallindromic sequences between introns.
Alternative splicing allows different exons to be used to make
different versions of proteins coded by the same gene.
Signals received by cells (signal molecules like
hormones and receptor proteins) convey info about
how to splice the RNA, allowing alternative splicing.
Once RNA is processed, ribosomes translate it
Initiation: Ribosome complex
“in frame”
formation: large subunit P site
attached to f-met tRNA
complementary base paired to
the start codon, AUG, & to a
small ribosomal subunit
Elongation: Ribosome translocates
(GTP energy) 5’ to 3’ one codon
at a time—new tRNA to A site,
then to P site (where it transfers
its amino acid & growing peptide
chain to the amino acid in A site,
then out the E site, until
termination when it reaches a
stop codon where a release factor
binds the stop codons UGA, UAG,
UAA.
tRNA decodes the mRNA by having its
anti-codon bind to a complementary
codon in the A site of the ribosome.
Aminoacyl transferase enzyme loads up the amino
acid—the anticodon fits into the active site at the
dame time as the correct amino acid
After translation, proteins are modified (post
translational modification) and transported.
Signal peptides (several amino acids at the amino
terminus of the protein) define the target.
Types of post-translational modifications:
Trimming off signal peptides once the peptide has
been transported into the RER.
Adding sugar chains (glycoproteins) in the RER or
golgi or lipid chains (lipoproteins) in the RER—
allows interaction with other molecules or
attachment to membranes.
e.g., cell identification molecules that extend into the
extracellular matrix are often glycoproteins
Phosphorylation—alter shape and activity or
interaction with other proteins
Chapter 17 in-class essay writing quiz—one of the
following essays was chosen at random 10/27/09.
(scored out of 10 points, % of 20 pts awarded)
1. Describe steps that occur during transcription of a
gene. Identify molecules involved, and describe
the functions of each. Include initiation,
elongation, and termination. (purple choice)
2. Describe steps of post-transcriptional regulation.
List the events, and describe the function
(importance) of each modification. (red choice)
3. List & describe steps of translation. Identify the
molecules and organelles involved. (teal choice)
Mutations
Mutations that affect the coding sequence of a
protein fall into several categories:
Point mutations: only one nucleotide changed
missense—substitute one amino acid
nonsense—create early stop codon
Insertion—add nucleotides
Deletion—remove nucleotides
Frameshift—add or remove multiples not equal to
3 (change entire remaining reading frame)
Translocation—move entire section of gene
somewhere else in the genome
Inversion—turn a section of gene backwards
Mutations can occur at the level of
DNA or RNA.
RNA isn’t proof read, so mistakes occur more
often in RNA.
Since many copies in RNA are made in each
round of transcription, then a mistake in RNA
rarely has a significant impact on the
phenotype.
Mutations can occur in the codons of a gene
(the coding region) or in the
untranscribed/translated regions (5’ & 3’ UTRs)
of a gene where regulatory proteins bind.
When do mutations affect phenotype?
When the gene is expressed
(transcribed then translated)
in the wrong cells or in
abnormal amounts or at
abnormal times
These mutations occur in
the 5’ UTR (untranslated region)
and the 3’ UTR, outside of the
coding region of the gene. They
alter transcription factor binding.
When do mutations affect phenotype?
Mutations may change the phenotype if post-transcriptional
exon splicing is altered:
i causing different proportions of the possible variants
of the protein to be made by alternative splicing
OR
ii causing errors in splicing—e.g., creating frameshift s
by inserting or deleting nucleotides near palindromes
at the exon/intron boundaries.
This type of mutation may occur outside of the coding region
of the gene, in the UTRs or inside the introns.
A mutation affects phenotype if it changes the size or shape
of a protein so that either its longevity, interaction with
regulators, or ability to function is altered
These mutations occur in coding region (exons) & affect phenotype
IF the mutation changes one or more amino acids.
Some amino acid substitutions
(missense) have greater impact
than others.
Conservative missense mutations substitute an
amino acid with similar properties, such that
3D protein shape is altered very little.
e.g., if a nonpolar amino acid is substituted
for another nonpolar amino acid of similar size
Silent mutations change a codon to a replicate
codon for the same amino acid.
Mutations often affect phenotype when a stop
codon is inserted at least several amino acids prior to
the normal stop codon since the protein is likely to
fold incorrectly after such a nonsense mutation.
Mutations that create frame shifts in the coding
region often affect phenotype since changing every 3’
codon after the mutation likely alters protein shape.
Use 30 minutes to write an answer. Due 10/29/09.
A List the types of mutations that change the coding
region of a gene. Describe how each type of
mutation alters the DNA,the RNA, & the protein.
B Explain why mutations in the untranscribed &
untranslated regions of genes can affect
phenotype.
Differential expression of DNA Lab 10/28/09
Hypothesis: Since peroxidase enzyme (catalyzes
breakdown of peroxide) is abundant in detoxifying cells,
then we expect higher peroxidase enzyme gene expression
in liver than muscle.
Compare the content of peroxidase enzyme in 1 g of macerated
(mashed) chicken liver to the amount in 1 g macerated chicken leg
muscle.
Add the tissue extract to 10 ml 1.5% peroxide, then swirl and wait 1
minute. Add 10 ml 1 M H2SO4 to denature the enzyme. Remove 5
ml of the mixture and titrate to 30s color change with 2% KMnO4 .
Record the start and stop volumes, and the volume change.
Whichever uses LESS KMnO4 contains LESS of the enzyme since the
reaction of KMnO4 with undegraded peroxide is clear—once all the
undegraded peroxide is reacted, excess purple permanganate ions
make the solution purple. Less enzyme will leave more peroxide
needing reacted.