Transcript Activation sites and enhancer proteins

The “Central Dogma”
Overview Of DNA
DNA
Transcription
mRNA
Translation
proteins
Enzymes
Replication
Structure
Movement
Hormones
Gas exchange
Amino Acid Storage
Protein Synthesis
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How does DNA control the structure and
function of the cell?
it makes proteins!
– Structure: collagen, elastin, keratin
– Enzymes: catalase, amylase, sucrase, etc
– Hormones: insulin, glucagon, etc
– Amino acid storage: albumin, ovalbumin, etc
What is similar about protein synthesis in
prokaryotes and eukaryotes? What is different?
Protein Synthesis!
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Transcription
http://www.johnkyrk.com/DNAtranscri
ption.html
Translation
http://www.johnkyrk.com/DNAtranslati
on.html
Notas – From Gene
to Protein
Metabolism teaches us about
genes
 Metabolic defects caused by
non-functional enzyme
 Studying metabolic diseases
suggested that genes
specified proteins
– PKY
– Alkaptonuria (black urine)
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Genes dictate the phenotype
1 gene – 1 enzyme hypothesis
 Beadle and Tatum – 1941
– Compared different nutritional mutants of
bread mold, Neurospora
– Created mutations by X-ray treatments Xrays break DNA)
– Wild type grows on “minimal” media (sugar)
– Mutants require different amino acids
because each mutant lacks a certain enzyme
needed to produce a certain amino acid
– Conclusion: Broken gene = non-functional
enzyme
1 gene – 1 enzyme
hypothesis
 Beadle and Tatum
– 1941
 Problems
with:
–One gene – one enzyme
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not all proteins are enzymes, and they’re
coded by genes too
–One gene – one protein
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many proteins consist of several
polypeptide, and each polypeptide has it’s
own gene
 One
gene – one polypeptide?
Defining a gene…
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“Defining a gene is problematic because small
genes can be difficult to detect, one gene can code
for several protein products, some genes code only
for RNA, two genes can overlap, and there are
many other complications.” – Elizabeth Pennisi,
Science 2003
How would YOU define a gene in your own words?
From nucleus to cytoplasm…
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Where are the genes?
in DNA on chromosomes in the nucleus
Where are proteins synthesized?
on ribosomes (free or on the ER) in the cytoplasm
How does the information get from the nucleus to
the cytoplasm?
mRNA is made in the nucleus and can travel into the
cytoplasm to the ribosomes
deoxyribose
A-T, C-G
Double
ribose
T-A, A-U, C-G
Single
Transcription!
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http://highered.mcgrawhill.com/sites/0072507470/student_vie
w0/chapter3/animation__mrna_synthe
sis__transcription___quiz_2_.html
http://www.johnkyrk.com/DNAtranscri
ption.html
Transcription Basics
Initiation
1.
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RNA polymerase binds to promoter sequence on DNA
1
2
3
2
Elongation
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3
where to start reading = Promoter (initiation site)
which strand to read = template strand
direction on DNA = reads 3’5  builds 5’  3’
RNA polymerase unwinds DNA ~20 bp at a time
Reads DNA 3’  5’
Builds RNA 5’  3’
No proofreading, about 1 error/105 bases
Many copies, short life, no problem 
Termination
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RNA polymerase stops at termination sequence
mRNA leaves nucleus through pores
promoter
Terminator
Transcription
RNA polymerase
Template
Strand
Initiation
Elongation
mRNA
Termination
Completed mRNA transcript
RNA Processing or Editing
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5’ cap
– protection
– targets mRNA for ribosome
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Poly-A tail
– protection
– leads mRNA out of nucleus
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Spliceosome
– composed of snRNPs (small nuclear ribonucleoproteins)
– introns – intervening, interrupting = removed by
spliceosome
– exons – expressed
Sliceosome
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snRNPs = small
nuclear
ribonucleoproteins
snRNPs
Other
proteins
Spliceosome
Intron
Exons
Animations
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http://www.johnkyrk.com/DNAtranscri
ption.html
Putting it Together –
Transcription to Translation
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How does mRNA code for proteins?
How can you code for 20 aa with only 4
nucleotide bases (A, U, G, C)?
How can an alphabet of 4 letters
(nucleotides) translate into an alphabet of
20 letters (aa)?!
Breaking the code
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Nirenberg and Matthaei
Determined 1st codon – amino acid match
– UUU coded for phenylalanine
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Created artificial poly(U) mRNA
Added mRNA to test tube of ribosomes and
nucleotides
– mRNA synthesized a single amino acid polypeptide chain:
phe-phe-phe-phe-phe-phe
DNA
Gene 1
DNA
3’
5’
Transcription
mRNA
5’
Translation
3’
codon
Protein
Amino acids
The CODE!!
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For ALL life!! (yes, even prokaryotes…)
– Strongest support for common origin for all life
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Code is redundant
– Several codons for each amino acid
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Start codon: AUG = methionine
Stop codons: UGA, UAA, UAG
Translation
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Ribosome reads mRNA in codons
tRNA brings in correct amino acid
tRNA matches codon of mRNA = anticodon
Amino acids assembled into polypeptide chain
tRNA Structure
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“clover leaf” structure
– anticodon on “clover leaf” end
– amino acid on 3’ end
– anticodon written 3’  5’ to match
codons which are 5’  3’
Aminoacyl tRNA synthetase
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enzyme which bonds amino acid to tRNA
– endergonic reaction (does it require energy?)
– ATP  AMP (how many phosphates do we use?)
– Energy stored in tRNA-aa bond
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Unstable
Ribosomes
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Facilitate coupling of tRNA anticodon to
mRNA codon
– Organelle or enzyme?
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Structure
– Ribosomal RNA and proteins
– 2 subunits: large and small
– A site (aminoacyl-tRNA site)
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Holds tRNA carrying next amino acid to be added to
chain
– P site (peptidyl-tRNA site)
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Holds tRNA carrying growing polypeptide chain
– E site (exit site)
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Discharged tRNA leaves ribosome from exit site
Building a Polypeptide
1.
Initiation
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2
3
Brings together mRNA, ribosome
subunits, proteins and initiator
tRNA
Elongation
Termination
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Release polypeptide
“release protein” bonds to A site
Bonds water molecule to
polypeptide chain
Polyribosomes
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Many ribosomes read single mRNA
simultaneously making many copies of a
protein!
Protein Targeting
Signal polypeptide
– ~20 aa at the beginning of the polypeptide
– Recognized by SRPs (signal recognition particles)
– SRP brings polypeptide and ribosome to ER so that
polypeptide is secreted into the ER as it’s built.
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Destinations – other signal polypeptides used to
target
–
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Secretion
Nucleus
Mitochondria
Chloroplasts
Cell membrane
Cytoplasm
Protein Targeting
Comparing!
Mutations
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Are all mutations bad?
Do all mutations lead to changes in
amino acids?
Sickle Cell Anemia - Mutation
K-ras Oncogene – Point Mutation
Mutations
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Point Mutations
– 1 base pair change
– Base-pair substitution
Silent mutation: no amino acid change because of
redundancy in code
 Missense: change amino acid
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Nonsense: change to stop
Mutations
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Insertions – adding base(s)
Deletions – losing base(s)
BOTH cause frameshift
Question
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Which mutations, point mutations or
frameshift mutations, do you think are
more harmful and why?
This weekend…
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Work on Quest! You should be able to
answer everything except the
questions about gene regulation which
we will do on Monday in class
Quest will be due Monday at midnight
Gene Regulation
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In a nutshell…
Prokaryotes regulate transcription through operons.
Area where RNA
Polymerase binds
Area where
repressor can bind
to stop binding of
RNA polymerase
Segment of DNA containing several
Regulatory Gene
genes, a promoter, and an operator
Codes for
repressor;
gene is
upstream or
downstream
from operator
2 Kinds of Feedback
Prokaryotes regulate transcription through operons.
Area where RNA
Polymerase binds
Area where repressor
can bind to stop
binding of RNA
polymerase
Segment of DNA containing several
Regulatory
genes, promoter, and operator
gene = codes
for repressor;
gene is
upstream/do
Too much product, stop;
wnstream
not enough, keep going!!
from operator
Keep going if you can!
2 Examples of Negative
Feedback
can bind to stop
Prokaryotes
regulate transcription through operons.
binding of RNA
polymerase
Segment of DNA containing several
Regulatory
genes, promoter, and operator
gene = codes
for repressor;
gene is
upstream/do
Too much product, stop; not
wnstream
from operator enough, keep going!!
•Default “on” because repressor not
bound
•Product binds to repressor to
“activate” and turn “off”
transcription when enough product
has been made
•Usually anabolic pathways (ex: trp)
Keep going if you can!
can bind to stop
Prokaryotes
regulate transcription through operons.
binding of RNA
polymerase
Segment of DNA containing several
Regulatory
genes, promoter, and operator
gene = codes
for repressor;
gene is
upstream/do
wnstream
Too much product, stop; not
from operator enough, keep going!!
•Default “on” because
repressor not bound
Keep going if you can!
•Default “off” because repressor bound
•Product binds to repressor to
“activate” and turn “off”
transcription
•Inducer binds to repressor to
“inactivate” and release the repressor
and turn “on” transcription only when
there is substrate to be broken down
•Usually anabolic pathways
(ex: trp)
•Usually catabolic pathways (ex: lac)
can bind to stop
Prokaryotes
regulate transcription through operons.
binding of RNA
polymerase
Segment of DNA containing several
Regulatory
genes, promoter, and operator
gene = codes
for repressor;
gene is
upstream/do
wnstream
Too much product, stop; not
from operator enough, keep going!!
Keep going if you can!
•Presence of activator
turns “on”
•Default “on” because
repressor not bound
•Ex: lac with lactose
and no glucose
•Default “off” because repressor
bound
•Product binds to repressor to
“activate” and turn “off”
transcription
•Product binds to repressor to
“inactivate” and release the repressor
and turn “on” transcription
•Usually anabolic pathways
(ex: trp)
•Usually catabolic pathways (ex: lac)
Summary of Prokaryotic
Gene Regulation: Operons
• Negative Feedback
– Repressible
– Inducible
• Positive Feedback
Gene Regulation
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In a nutshell…
Eukaryotes regulate gene expression pre-transcription,
during transcription, and post-transcription.
Before transcription; which
genes are “on” and “off”
•Controls access of RNA
polymerase to promoter
•Histone acetylation
•Acetylated = less bound
= easier access
•DNA methylation
•methylated = more
bound = less access
Eukaryotes regulate gene expression pre-transcription,
during transcription, and post-transcription.
•Histone acetylation
•Acetylated = less
bound = easier access
•Controls access of RNA
polymerase to promoter
•Aids in RNA
polymerase binding
•DNA methylation
•methylated = more
bound = less access
•Transcription Factors =
proteins that help RNA
poly binding at promoter
•Activation sites and
enhancer proteins =
also aid in RNA poly
binding; 1000s of bp
away
polymerase to promoter
•methylated = more
expressionbound
pre-transcription,
= less access
Eukaryotes regulate gene
during transcription, and post-transcription.
•Aids in RNA
polymerase binding
After mRNA has been made
Translational
Regulation
RNA Processing
mRNA
Degradation
•Transcription Factors =
proteins that help RNA
poly binding at promoter
•Activation sites and
enhancer proteins = also
aid in RNA poly binding;
1000s of bp away
Protein
Processing/Regulation
polymerase to promoter
•methylated = more
expressionbound
pre-transcription,
= less access
Eukaryotes regulate gene
during transcription, and post-transcription.
•Aids in RNA
polymerase binding
After mRNA has been made
Translational
Regulation
RNA Processing
•Alternate splicing
= different combos
of exons are
expressed (some
can be removed)
•Essential in making
antibodies
mRNA
Degradation
•Transcription Factors =
proteins that help RNA
poly binding at promoter
•Activation sites and
enhancer proteins = also
aid in RNA poly binding;
1000s of bp away
Protein
Processing/Regulation
polymerase to promoter
•methylated = more
expressionbound
pre-transcription,
= less access
Eukaryotes regulate gene
during transcription, and post-transcription.
•Aids in RNA
polymerase binding
After mRNA has been made
Translational
Regulation
RNA Processing
mRNA
Degradation
•Transcription Factors =
proteins that help RNA
poly binding at promoter
•Activation sites and
enhancer proteins = also
aid in RNA poly binding;
1000s of bp away
Protein
Processing/Regulation
•Poly-A tail/5’ cap
•3’ and 5’ UTR (untranslated region) = nucleotides
before the start codon (AUG) and/or after the stop
codon
•RNAi = RNA interference  small interfering
RNAs (siRNAs) and microRNAs (miRNAs) =
bind to mRNAs and prevent them from being
translated or trigger their degradation
polymerase to promoter
•methylated = more
expressionbound
pre-transcription,
= less access
Eukaryotes regulate gene
during transcription, and post-transcription.
•Aids in RNA
polymerase binding
After mRNA has been made
Translational
Regulation
RNA Processing
•Poly-A tail/5’ cap =
mRNAaffect
assembly/binding of
Degradation
ribosome
•3’ and 5’ UTR
(untranslated region)
= nucleotides before
the start codon
(AUG) and/or after
the stop codon
•Transcription Factors =
proteins that help RNA
poly binding at promoter
•Activation sites and
enhancer proteins = also
aid in RNA poly binding;
1000s of bp away
Protein
Processing/Regulation
polymerase to promoter
•methylated = more
expressionbound
pre-transcription,
= less access
Eukaryotes regulate gene
during transcription, and post-transcription.
•Aids in RNA
polymerase binding
After mRNA has been made
Translational
Regulation
RNA Processing
mRNA
Degradation
•Transcription Factors =
proteins that help RNA
poly binding at promoter
•Activation sites and
enhancer proteins = also
aid in RNA poly binding;
1000s of bp away
Protein
Processing/Regulation
•Processing= some proteins
need to get folded, spliced
(parts cut off), or have groups
added
•Degradation= all proteins
need to be “marked” for
degradation/to get broken
down; most “marked” by
ubiquitin and broken down by
proteosomes
Summary of Eukaryotic
Gene Regulation
• Pre-transcriptional
• During transcription
• Post-transcriptional