gene expression in prokaryotes

1. Regulation of Gene
Expression
AP Biology
Ch 15

2. Gene  Protein Control
• Feedback inhibition –
enough product is made
the system shuts down
– More product is made
when needed
– The product shuts down
the process
• Gene Expression –
genes are only expressed
when needed. Often
regulated at
transcription.

3. Gene Expression: Prokaryotes
• Operon – grouped genes that are transcribed together –
code for functionally similar proteins
• Key Players
– Promoter – section of DNA where RNA polymerase binds
– Operator – Controls activation of transcription
• on off switch
• between promoter and genes for proteins – structural genes
– Repressor protein – binds to operator to block RNA polymerase
and shut down transcription
• Turns off the operon
• Corepressor – keeps the repressor protein on the operator
– Trp operon
• Inducer – pulls repressor off the operator
– Turns on the operon – lactose on the lac operon
– Regulatory gene – produces the repressor protein
– Structural genes – code for proteins

4. Positive and Negative Gene Regulation
• Negative
– Repressible: usually on but
can be inhibited trp operon,
allosteric inhibition,
tryptophan present prevents
its own production.
(anabolic)
– Inducible: usually off, but
can be turned on, an inducer
(a specific small molecule,
allolactose in the lac operon)
inactivates the repressor and
allows transcription
(catabolic)
• Positive
– E. coli prefer to use glucose for
energy, they will only use lactose
when glucose is in short supply
– glucose cAMP binds to
regulatory protein “CAP” & stimulates
gene transcription
Positive gene regulation!
– The cAMP & CAP combination allow
RNA polymerase to bind to the
promoter sequence more efficiently.
– Remember cAMP is regulating the
gene expression in the bacteria

5. Trp operon: repressible,
always making tryptophan,
repressed if tryptophan is
“eaten” tryptophan is
necessary for the cell to
function

6. Lac operon: inducible,
only turned on if lactose is
“eaten” lactose is not
necessary for the cell to
function

7. Eukaryotic Chromosome
• Chromosomes – tightly coiled DNA
around proteins during cell division
• Chromatin – loosely packed DNA
around proteins
• Histones – protein which the DNA
wraps around
• Nucleosomes – grouped histones
together
– Heterochromatin – tighter packed
chromatin
• Not transcribing
– Euchromatin – looser packed chromatin
• Transcription occurring

8. Gene Expression: Eukaryotes
• Cell Differentiation –
cell specialization
• All cells contain the
same genes
• The genes that are
expressed determines
the type of cell
– Ex: Skin cell vs. a nerve
cell

9. Chromatin Regulation
• Histone acetylation –
allows transcription
factors to bind to DNA
allowing transcription to
occur
– Creates loosely packed DNA
- euchromatin
• DNA Methylation – occurs
after DNA synthesis has
occurred
– Lower transcription rates
– One X in females is highly
methylated
– Works w/ a deacetylation
enzyme in some spp.

10. Epigenetic inheritance
• Not controlled by base
sequences.
• DNA methylation
(deactivates one
homologous chromosome)
may explain abnormal or
unexpected DNA
expression as is often seen
in identical twins.
http://images.the-scientist.com/content/images/general/55342-1.jpg

11. Regulation of Transcription
• Transcription involves RNA Polymerase II and
transcription factors
• RNA polymerase II attaches to the promoter
(TATA box) sequence to begin transcription
• Control elements – non coding sequences of
DNA where the transcription factors attach

12. Regulation of Transcription
......AP Bio 15-16Genetics15_10TranscripInitiation_A.swf
• Enhancer – control element far from a gene
or intron
• Activator – bind to enhancers to turn on
transcription of a gene
• Transcription factors + enhancer + activator
+ RNA Polymerase II = transcription
initiation complex
– Needed for transcription to begin
• Repressors – inhibit gene expression
– Turn off transcription
– Block activators from binding to enhancers

13. Distal control
element
Activators
Enhancer
Promoter
Gene
TATA
box
General
transcription
factors
DNA-bending
protein
Group of
Mediator proteins
RNA
Polymerase II
RNA
Polymerase II
RNA synthesis
Transcription
Initiation complex
Chromatin changes
Transcription
RNA processing
mRNA
degradation
Translation
Protein processing
and degradation
A DNA-bending protein
brings the bound activators
closer to the promoter.
Other transcription factors,
mediator proteins, and RNA
polymerase are nearby.
2
Activator proteins bind
to distal control elements
grouped as an enhancer in
the DNA. This enhancer has
three binding sites.
1
The activators bind to
certain general transcription
factors and mediator
proteins, helping them form
an active transcription
initiation complex on the promoter.
3

14. RNA Processing Regulation
• Alternative RNA Splicing – different regions of the
pre-mRNA serve as introns or exons creating different
mRNA strands depending on what is removed &
spliced together.

15. mRNA Degredation
• Prokaryotes
– Short Life span
– Degraded in seconds
– Allows rapid response to
environmental changes
• Eukaryotes
– Survive from hours to
weeks
– Internal conditions
constant, no need for
rapid response

16. ncRNA: 1000’s of RNA’s, current
research
• miRNA’s - micro RNA hat can
degrade mRNA or block
translation
• Causes mRNA to fold on
itself and base pair to create
dsRNA which is then
digested with an enzyme
• Short interferring RNA
(siRNA) – also degrade
mRNA or block translation
(blocking by siRNA is called
RNAi, or RNA interferance)

17. Protein Degradation
18_12ProteinDegradation_A.swf
• Proteosomes – break apart proteins in to
smaller peptide units
Chromatin changes
Transcription
RNA processing
mRNA
degradation
Translation
Protein processing
and degradation
Ubiquitin
Protein to
be degraded
Ubiquinated
protein
Proteasome
Proteasome
and ubiquitin
to be recycled
Protein
fragments
(peptides)
Protein entering a
proteasome

18. Single Gene Expression
• Different cells express different genes,
therefore they make different mRNA’s
• We can detect mRNA in a cell using nucleic
acid hybridization, by pairing it to a nucleic
acid probe
• Each probe is labeled with a fluorescent tag to
allow visualization
• The technique allows us to see the mRNA in
place (in situ) in the intact organism and is
thus called in situ hybridization

19. Figure 15.16
mRNAs
cDNAs
Embryonic stages
1
cDNA synthesis
PCR amplification
Gel electrophoresis
Results
Technique
1
2
3
Primers
-globin
gene
2 3 4 5 6

20. Figure 15.15-5
Test tube containing
reverse transcriptase
and mRNA
DNA in nucleus
mRNAs in
cytoplasm
Reverse transcriptase
makes the first
DNA strand.
Reverse
transcriptase
mRNA
Poly-A tail
DNA
strand
Primer
5
3
3
5
A A A A A A
1
2
mRMA is degraded.
3
5
3
3
5
A A A A A A
DNA polymerase
synthesizes the
second strand.
DNA
polymerase
5
3
3
5
4
5
3
3
5
cDNA
cDNA carries complete
coding sequence
without introns.
5
T
T T T T T
T T T T

21. Groups of Gene Expression
• Recall Microarray assays:
• Used to pinpoint differences in gene
expression between 2 different cell types
• How it’s done:
– Sequence a genome
– Use PCR to copy the genes (verification steps
here)
– Split the genes into single strands
– Place the single stranded DNA onto microscope
slides in spots (robots & computers do all this)