Transcript Where do pumpkins come from?

Control of Genetic Systems in
Prokaryotes and Eukaryotes
• Gene Regulation – level of gene
expression can vary under different
conditions
• Constitutive genes – unregulated,
continuously needed for survival
• Regulated genes – majority;
expressed only when needed
• Regulated at genetic level
1.Metabolism
2.Response to environmental stress
3.Cell Division
• A multistep process (at
transcription, translation, posttranslation)
• Transcriptional regulation most
common in prokaryotes
• Not all gene expression results in a
protein!
Genetic Control in Prokaryotes
• Important because prokaryotes
compete for limited resources, also
typically live in changing
environments (temp, availability of
nutrients, etc.)
• Prokaryotes have two levels of metabolic
control:
• Vary the numbers of specific enzymes
made (regulation of gene expression)
– Slow, but can have a dramatic effect on
metabolic activity
• Regulate enzymatic pathways (feedback
inhibition, allosteric control)
– Rapid and can be fine-tuned, but if the enzyme
system does not have this level of control, then
it is useless
– Typically post-translational
Prokaryotes are "simple," single celled
organisms, so they have "simple"
systems
• Genes are grouped together based on
similar functions into functional units
called operons
• MANY GENES UNDER ONE CONTROL!!!
– There is one single on/off switch for the
genes
Figure 14.1
14-4
lac operon in E. coli
• Function - to produce enzymes which
break down lactose (milk sugar) lactose
is not a common sugar, so there is not
a great need for these enzymes
• when lactose is present, they turn on
and produce enzymes
Two components - repressor genes and
functional genes
• Three functional genes:
• lacZ produces B-galactosidase. This enzyme
hydrolyzes the bond between the two sugars,
glucose and galactose
• lacY produces permease. This enzyme spans the
cell membrane and brings lactose into the cell
from the outside environment. The membrane is
otherwise essentially impermeable to lactose.
• lacA produces B-galactosidase
transacetylase. The function of this enzyme is still
not known.
• Promoter (P) - aids in RNA
polymerase binding
• Operator (O) - "on/off" switch binding site for the repressor
protein
Repressor (lacI) gene
• repressor gene (lacI) - produces
repressor protein w/ two binding sites,
one for the operator and one for lactose
• The repressor protein is under allosteric
control - when not bound to lactose, the
repressor protein can bind to the
operator
• When lactose is present, an isomer of
lactose, allolactose, will also be present
in small amounts. Allolactose binds to
the allosteric site and changes the
conformation of the repressor protein so
that it is no longer capable of binding to
the operator.
Operation - If lactose is not present:
• the repressor gene produces repressor,
which binds to the operator. This blocks the
action of RNA polymerase, thereby
preventing transcription.
Operation - if lactose is present:
• the repressor gene produces repressor,
which has a site for binding with
allolactose.
• The allolactose/repressor compound is
incapable of binding w/ the operator, so
the RNA polymerase is uninhibited
• once the concentration of lactose
decreases, the repressor-allolactose
complex falls apart and transcription is
again inhibited
If lactose is present
http://vcell.ndsu.nodak.edu/animations/lacOperon/movie-flash.htm
• The lac operon is an example of an
inducible operon - it is normally
off, but when a molecule called an
inducer is present, the operon
turns on. Used in catabolic
reactions
The trp operon is an example of
a repressible operon - it is
normally on but when a molecule
called a repressor is present the
operon turns off. Used in anabolic
reactions
It Gets More Complicated - the lac
Operon Revisited
• It is not enough for lactose to be
present to induce the lac operon
• Glucose is the sugar of choice of E. coli
and if glucose is in supply, then the
bacteria will preferentially break down
glucose over lactose
• If glucose is present, the lac operon will
be repressed - how does this happen
you ask?
The role of CAP
• RNA polymerase has a low affinity for
the promoter of the lac operon unless
helped by a regulatory protein - cAMP
receptor protein (CAP)
• CAP only becomes activated if the
concentration of cyclic AMP (cAMP) is
high
Remember cAMP is a second messenger used in signaling pathways.
• Glucose inhibits the formation of
cAMP. If the concentration of
glucose is high, the concentration
of cAMP is low
• If the concentration of glucose is
low, the concentration of cAMP is
high
• Therefore, if the concentrations of glucose
and lactose are high, the concentration of
cAMP will be low, CAP will not be activated,
RNA polymerase will not be able to bind well
to the promoter, and the operon will be
operating at a very low level (i.e. almost off)
• However, if the concentrations of glucose is
low and lactose is high, the concentration of
cAMP will be high, CAP will be activated and
bind to the DNA which will promote RNA
polymerase binding and initiate transcription
CAP
CAP
The lac Operon
Figure 14.3
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
14-13
Regulatory Sequences of the Lac Operon
Diauxic Growth Curve Demonstrated
Adaptation to Lac Metabolism
trp Operon - and example of a
repressible operon
• five genes (trpA, trpB, trpC, trpD, and
trpE) involved in the production of the
amino acid tryptophan
• another gene (trpR) produces an
inactive repressor protein
• accumulation of the end product
(tryptophan) represses synthesis of
the enzymes
– tryptophan binds to the inactive
repressor protein at an allosteric site
– the conformation changes and the
repressor + tryptophan complex binds
to the operator, repressing the operon
- The tryptophan acts as a corepressor.
•Tryptophan can accumulate due
to internal production or from
external sources
• remember, E. coli is found in
the intestines of humans so if
you eat a tryptophan-rich
meal, this will accumulate in
the bacteria and turn off the
operon
• why waste resources when a
supply of this amino acid is
readily available?
http://highered.mheducation.com/olcweb/cgi/pluginpop.cgi?it=swf::
535::535::/sites/dl/free/0072437316/120080/bio26.swf::The+Trypto
phan+Repressor
In summary:
• Regulatory proteins – bind to DNA and affect
rate of transcription or one or more nearby
genes
- repressors (negative control)
- activators (positive control)
• Small effector molecules – bind to activator or
repressor to cause a conformational change
so regulatory proteins cannot bind to DNA
• - inducers
• - corepressors
• - inhibitors

Regulatory proteins have
two binding sites


One for a small effector
molecule
The other for DNA
Other ways prokaryotes can control
gene expression
• Translational regulatory proteins –
recognize sequences in mRNA and
inhibit translation (sometimes at the
start codon)
• Antisense RNA – a RNA strand that is
complementary to mRNA binds to the
mRNA and keeps it from being
translated
Antisense RNA
• Post-translational Regulation
1. Feedback inhibition
As the final molecule is made, its
concentration increases, the product can
bind to an enzyme in the pathway
(allosterically) and stop the pathway.
2. Posttranslational covalent
modification
– involved in assembly of protein so
alterations may be disulfide bond
formations, attachment of certain
groups, such as sugars or lipids,
phosphorylation, acetylation, etc.
Lab: Specific Binding of Dyes
to DNA
• This lab indicates how to determine by
using dyes if substances such as
transcription factors or certain proteins
bind to DNA.
Gel with
no DNA.
Gels
with
DNA.
Comparison of gels
w/o and with DNA
GENE REGULATION IN THE
BACTERIOPHAGE LIFE CYCLE
• Bacteriophages are viruses that infect bacteria
– Their study has greatly advanced our basic
knowledge of genetic regulation and helped to
combat viral diseases by inhibiting viral growth
• The structural genes of bacteriophages are
often in an operon arrangement
– Like bacterial operons, phage operons can be
controlled by repressor proteins or activator proteins
• To understand how this works, we will examine
the two life cycles of phage l (lambda) (infects
E. coli)
Discovery of phage Lambda λ
• Esther Lederberg (1951)
Life Cycles of Phage l


Phage l can bind to the surface of a bacterium
and inject its genetic material into the bacterial
cytoplasm
The phage will then proceed along only one of
two alternative life cycles



Lytic cycle
Lysogenic cycle
Let’s review Figure 6.9
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14-64
This
process is
termed
induction
It will undergo
the lytic cycle
Prophage can
exist in a dormant
state for a long
time
Virulent phages only
undergo a lytic cycle
Figure 6.9
Temperate phages can
follow both cycles
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
14-65

Figure 14.18 shows the genome of phage l

Inside the viral head, phage l DNA is linear


After injection into the bacterium, the two ends attach covalently
to each other forming a circle
The organization of the genes within this circular
structure reflects the two alternative life cycles of the
virus

The genes in the top center are transcribed very soon after
infection, at the beginning of either life cycle

The pattern of their expression determines which of the two cycles
prevails

The genes on the left side of the viral genome encode proteins
that are responsible for the lysogenic infection

The genes on the right side of the viral genome encode proteins
that are responsible for the lytic infection
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Transcribed right
after
infection
 Transcribed right after infection
Lysogenic
control
Figure 14.18
Lytic Control
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• Which cycle is “on” is determined by
the gene expression for certain
proteins.
• If cro accumulates, lytic cycle prevails
• If cll/clll accumulates, lysogenic
prevails
• The OR region has binding sites for
the a repressor and cro protein and
can act as a genetic switch between
the lytic and lysogenic cycles.

Genetic switches, like the one just described in
phage l, are also important in the
developmental pathways of bacteria and
eukaryotes

For example

The choice between sporulation and vegetative
growth in bacteria

Initiation of cell differentiation during development in
eukaryotes
http://media.hhmi.org/biointeractive/click/Gene_Switches/01-vid.html
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14-79
Genetic switches in human
DNA
http://www.channel4.com/news/gene-switches-reveal-what-makes-humans-tick
Gene control in Eukaryotes
Much more complex - take humans for example
•Every cell (except gametes) have the same DNA,
with the same information
•Usually, every gene has more than one gene
regulator (all of which must be on for the gene to
function)
• The latest estimates are that a
human cell, a eukaryotic cell,
contains approximately 35,000
genes.
• Some of these are expressed in all cells all the time.
These so-called housekeeping genes are responsible
for the routine metabolic functions (e.g. respiration)
common to all cells.
• Some are expressed as a cell enters a particular
pathway of differentiation.
• Some are expressed all the time in only those cells
that have differentiated for a specific job. way. For
example, a plasma cell expresses continuously the
gene for the antibody it synthesizes.
• Some are expressed only as conditions around and in
the cell change. For example, the arrival of a hormone
may turn on (or off) certain genes in that cell.
How is gene expression
regulated?
There are several methods used by eukaryotes.
•Chromatin Remodeling
•The region of the chromosome must be opened up in order
for enzymes and transcription factors to access the gene
•Transcription Control
The most common type of genetic regulation
•Post-Transcriptional Control
Regulation of the processing of a pre-mRNA into a mature
mRNA
•Translational Control
Regulation of the rate of Initiation
•Post-Tranlational Control (protein activity control)
Regulation of the modification of an immature or inactive
protein to form an active protein
Chromatin Alteration
• Chromosomes are composed of chromatin, a
complex of histone proteins and DNA
• In order to transcribe a gene, the tightly
wound chromatin must become
decondensed. One group of proteins called
chromatin-remodeling complexes reshape
chromatin.
• Methylation promotes coiling, hence no
transcription or expression
(heterochromatin)
• Acetylation promotes uncoiling, hence
transcription and expression
(euchromatin)
CPG islands
areas of methylation near the promoter; if
methylated, not transcription.
Transcriptional Control
• Like prokaryotes, the promoter is
where RNA polymerases bind (usually
RNA polymerase II) to initiate
transcription.
• Eukaryotic RNA polymerases have a
much more complex activation
mechanism than prokaryotic RNA
polymerase.
• They form initiation complexes.
Basal Transcription Factors
• such as the TATA binding protein (TBP) and
TFIID (Transcription Factor II D)
• They are found in nearly all eukaryotic
genes. They do not provide much in the way
of regulation of gene transcription, but must
be present .for transcription to occur.
Basal transcription
factors
Regulatory Transcription Factors
• proteins that bind to enhancers,
silencers, or promoter-proximal
elements. They are specific to
particular genes (or families of genes)
and are the chief regulatory
mechanisms for gene expression in
eukaryotes.
• Enhancers and silencers are far away
from the promoter while the promoterproximal elements are close.
• They have sequences that are unique
to specific genes.
• Enhancers speed up transcription
• Silencers inhibit transcription.
•Regulatory sequences which increase the rate of
transcription are called enhancers - those which
decrease the rate of transcription are called silencers
•Enhancers can function if their normal 5' -- 3' orientation
is flipped.
• Many different genes and
many different types of cells
share the same transcription
factors - not only those that
bind at the basal promoter
but even some of those that
bind upstream.
• What turns on a particular
gene in a particular cell is
probably the unique
combination of promoter
sites and the transcription
factors that are chosen – the
combinatorial effect.
Just how do proteins bind to DNA?
Protein to protein and protein to DNA can form three
structures:
• Helix to helix (loops or turns)
• Zinc fingers
• Leucine zippers
DNA footprinting
• DNA footprinting is an in
vitro technique used to
examine the binding of
proteins to specific regions
of DNA.
• This technique cleverly
exploits the fact that when a
transcription factor is
bound to DNA with a certain
affinity, the DNA is
protected from degradation
by nucleases.
• The transcription factor of
interest thus leaves its
"footprint" on the DNA.
Steroid Hormones and
Regulatory TF factors
• Steroid hormones affect gene
transcription
• Steroid hormones act as signaling
molecules that are synthesized by
endocrine glands and secreted into the
bloodstream.
• Regulatory TF factors can bind the
steroid hormone directly to it
• The cells can
respond to the
hormones in
different ways.
• EXglucocorticoid
hormones
influence
nutrient
metabolism in
most body
cells
Post-Transcriptional Control
Pre-mRNA processing
– Splicing
– Poly A tailing
– 5’ Cap
How are the pieces cut and
spliced?
• Spliceosomes consist of a variety of
proteins and small nuclear RNA
(snRNA), “snRNP’s”, that recognize
the splice sites
Did you call me?
Oh, “snurps” not smurfs!
Fig. 17-11-1
RNA transcript (pre-mRNA)
5
Exon 1
Protein
snRNA
Intron
Exon 2
Other
proteins
snRNPs
Fig. 17-11-2
RNA transcript (pre-mRNA)
5
Exon 1
Intron
Protein
snRNA
Other
proteins
snRNPs
Spliceosome
5
Exon 2
Fig. 17-11-3
RNA transcript (pre-mRNA)
5
Exon 1
Intron
Protein
snRNA
Exon 2
Other
proteins
snRNPs
Spliceosome
5
Spliceosome
components
5
mRNA
Exon 1
Exon 2
Cut-out
intron
• Ribozymes are catalytic RNA molecules
that function as enzymes and can also
splice RNA
The discovery of ribozymes
rendered obsolete the belief that
all biological catalysts were
proteins.
The Functional and Evolutionary
Importance of Introns
• Alternative RNA splicing can enable
some genes to encode more than one
kind of protein depending on which
segments are treated as exons.
• Introns allow more room for crossingover which results in exon shuffling may
result in the evolution of new proteins
mRNA stability
• For many genes RNAi limits life span or
translation rates.
RNAi: Slicing, dicing and serving your cells - Alex Dainis | TED-Ed
http://www.nature.com/nrg/multimedia/rnai/animation/index.html
RNAi
•
Involves noncoding RNAs.
•
Process used originally by cells to
fiend off viruses
http://www.teachersdomain.org/asset/lsps0
7_vid_rnai/
• RNA interference is an evolutionary
conserved mechanism of specific gene
silencing induced by double stranded
RNA homologous to the target mRNA.
• Small interfering RNAs (siRNAs) are
widely used for the control of gene
expression in molecular biology and
experimental pharmacology. Currently,
siRNAs are successfully used for the
validation of potent drug targets.
Steps of forming siRNA
• at the first stage, specific ribonuclease Dicer binds
to and cleaves long dsRNAs yielding short (21-23 nt)
siRNAs.
• at the second stage, siRNAs molecules form a
multiprotein complex (RISC – RNA-induced silencing
complex).
• One of siRNA strands undergoes cleavage and
dissociation from the complex upon RISC activation;
the other strand remains in the complex.
• Activated complex RISC* specifically binds to RNA
target and cleaves it
They can enter the cell naturally
or be injected into the cell.
http://www.pbs.org/wgbh/nova/next/body/rnai/
MicroRNA’s
• Small snippets of RNA that are made in
the nucleus and move to the dicer
complex and also recognize specific
base sequences in mRNA to cut it up.
• In humans, there are almost 2,000
distinct microRNAs, which collectively
regulate somewhere between 30 and 80
percent of human genes.
https://www.youtube.com/watch?v=gZZyxVP02UU&list=PL12F5DCE13035F497
&index=2
Translational Control
• Regulatory proteins bind to mRNA
molecules in cytoplasm making them
degraded and recycled to make more
RNA.
• This varies the amount of gene product
that is produced (as a mRNA that's
degraded quickly won't express much
protein).
Post-Translational Control
• Protein Cleavage and/or Splicing - the newly
formed protein is rarely functional as is. They
typically need to be modified (i.e. insulin)
• Chemical modification. Protein function can
be modified by addition of methyl, phosphoryl,
or glycosyl groups.
• Protein-folding by chaperone proteins
• Signal sequences direct packaging and
secretion. Some proteins have "signal
sequences" which direct their packaging in
the Golgi and movement through the
endoplasmic reticulum (ER) to be secreted.
The signal sequences usually end up cleaved
off.
• Prokaryotes exhibit transcriptional
control (as seen in regulation of
operons) and post-translational
control (protein modification).
• They do not exhibit chromatin
alteration (since they have naked
DNA). They exhibit very little posttranscriptional control and
translational control.
Genotype to Phenotype Lab
• In the experiment, you are given two
plasmids (A and B). One plasmid has a
functional gene for the enzyme ßgalactosidase. The ß-galactosidase gene in
the other plasmid is inactive because it
contains a segment of foreign DNA. Both
plasmids have a gene to code for an enzyme
that degrades ampicillin so they can live in
the presence of ampicillin, an antibiotic.
• In the first part of the exercise, students
analyze restriction digests of both plasmids
in order to determine which plasmid should
• In the first part of the exercise,
students analyze restriction digests of
both plasmids in order to determine
which plasmid should have a functional
ß-galactosidase gene (genotype).
• In the second part of the lab, the
plasmids are introduced into E. coli by
transformation and the color of the
resulting colonies (blue or white) is
then used to assess the functional
status (phenotype) of the ßgalactosidase gene.