Download VII. Some methods for studying gene expression

IV. Translation
IV. Translation
(3) Translation termination
i. When a ribosome comes to a nonsense codon (or stop
codon, usually one of UAA, UAG and UGA), translation
stops and polypeptide is released from ribosome.
ii. Stop codons do not encode an amino acid, so they have no
corresponding tRNA.
iii. Termination requires release factors (RF1 and RF2) which
recognize nonsense codon and promote the release of the
polypeptide form the tRNA and the ribosome from the
mRNA. (Fig. 2.35)
Termination of translation at a
nonsense codon
IV. Translation
5. Polycistronic mRNA
※ In bacteria and archaea, the same mRNA can encode more
than one polypeptide. Such mRNAs, called polycistronic
mRNAs, must have more than one TIR to allow simultaneous
translation of more than one sequence of the mRNA.
(1) Even if the two coding regions overlap, the two
polypeptides on an mRNA can be translated independently
by different ribosomes.
(2) Translational coupling – The translation of upstream gene is
required for the translation of the gene immediately
downstream. The secondary structure of the RNA blocks
translation of the second polypeptide unless it is disrupted
by a ribosome translating the first coding sequence.
Structure of a polycistronic mRNA
Even if the two coding regions overlap, the two polypeptides on
an mRNA can be translated independently by different ribosomes.
IV. Translation
(3) Polar effect on gene expression - Some mutations that affect
the expression of a gene in a polycistronic mRNA can have
secondary effects on the expression of downstream gene.
i. The insertion of an transcription terminator prevents the
transcription of downstream gene.
ii. The mutation changing a codon to a nonsense codon will
dissociate the ribosome from mRNA, then the translation
of downstream gene that is translationally coupled to the
upstream gene will not translated.
(4) ρ–dependent polarity (as shown in Fig.2.38)
A. Normally the rut site is masked by ribosome translating the
mRNA of gene Y.
B. If translation is blocked in gene Y by a mutation that changes
the codon CAG to UAG, the ρ factor can cause transcription
termination before the RNA polymerase reach gene Z.
C. Only the fragment of gene Y protein and mRNA are produced
and even gene Z is not even transcribed into mRNA.
Model for translational coupling in
polycistronic mRNA
Polarity in transcription of a polycistronic
mRNA transcribed from PYZ.
V. Regulation of gene
expression
1. Transcriptional regulation
(1) Genes whose products regulate the expression of other
genes are called regulatory genes. Their products can be
either activator or repressor.
(2) The set of genes regulated by the same regulatory gene
product is called a regulon. If a gene product regulates its
own expression, it is said to be autoregulated.
(3) Bacterial genes are often arranged in an operon which
consists of a promoter region, an operator region and
several structure genes. The mRNA of bacteria are made on
a number of genes whose products perform related
functions. This kind of mRNA is called polycistronic mRNA.
Transcriptional regulation
(4) There are two general types of transcriptional regulation :
i. In negative regulation, a repressor binds to an operator and
turns the operon off by preventing RNA polymerase from
using or access the promoter. An operator sequence can
be close to (up- or downstream), or even overlapping the
promoter.
ii. In positive regulation, an activator binds to the upstream of
the promoter at an upstream activator site (UAS), where it
can help RNA polymerase bind to the promoter or help
open the promoter after the RNA polymerase binds.
Two general types of
transcriptional regulation
Lac operon
A. Bacteria respond to rapidly changing environments
B. Examples:
a. Lac operon（乳醣操縱組）of E. coli（大腸桿菌）
1. promoter sequence（起動子序列）– RNA polymerase
（RNA聚合酶）
2. operator sequence（操縱子序列）– repressor
protein
3. structural genes （構造基因）– Z (betagalactosidase; 半乳醣分解酶), Y (permease; 滲透酶)
and A (transferase; 轉移酶)
b. regulator gene（調控基因）– repressor protein
(抑制蛋白質）
The regulation of gene expression of Lac operon
Operon
Operator
Regulatory
gene
Promoter
Lactose-utilization genes
DNA
mRNA
Protei repressr
RNA polymerase
cannot attach to
promoter
Active
repressor
OPERON TURNED OFF (lactose absent)
DNA
RNA polymerase
bound to promoter
mRNA
Protein
Repressor
Lactose
Inactive
repressor
Enzymes for lactose utilization
OPERON TURNED ON (lactose inactivates repressor)
The β- galactosidase reaction
The lac control region
Diauxic growth curve of E. coli
grown with a mixture of glucose and
lactose
The interaction of promoters and CAP
proteins in Lac operon
A. CAP proteins are involved in positive regulation
a. positive regulation – activator（活化子）
b. CAP – catabolite activator proteins（降解物活化蛋白質）
c. CAP binding site
d. cAMP – cyclic adenosine monophosphate（環腺嘌呤二磷酸）
e. CAP/cAMP complex – increasing the efficiency the
ability of RNA polymerase binds to promoter.
B. Catabolite repression（降解物抑制）– enabling E.
coli to use glucose (葡萄糖) preferentially for
energy even in the presence of lactose or other
complex sugar.
a. decreasing the level of cAMP
b. permease - nonfunctional
Positive Control of lac Operon
• Positive control of lac
operon by a
substance sensing
lack of glucose that
responds by
activating lac
promoter
– The concentration of
nucleotide, cyclic-AMP,
rises as the
concentration of
glucose drops
The phosphoenolpyruvate (PEP)-dependent
sugar phosphotransferase system (PTS)
• Both HPr and IIA are the components of the PTS,
which is responsible for transporting certain sugars,
including glucose.
Catabolite repression of the lac operon
• Exogenous glucose
inhibits both cAMP
synthesis and the
uptake of other
sugars, such as
lactose.
• Components of the
cascade :
- HPr, the
phosphotransferase
(for histidine protein)
transfers the
phosphate from
IIAGlc~P to sugar as
the sugar is
transported.
- IIAGlc protein has two forms
* IIAGlc~P activates adenylate
cyclase to make cAMP.
* IIAGlc inhibits sugarspecific permease that
transport sugar
Upstream activator site (UAS)
1. The αCTD (carboxyl
terminus of the α
subunits) binds to UP
element (UAS), and
αNTD binds to subunit.
(A, B)
2. Some promoters lack a -35 sequence and instead have what is
called extended -10 sequence. This sequence is recognized not
byσ4 but, rather by σ3. (C)
Hypothesis for CAP-cAMP
activation of lac transcription
Proposed CAP-cAMP Activation of
lac Transcription
• The CAP-cAMP dimer
binds to its target site
on the DNA
• The aCTD (a-carboxy
terminal domain) of
polymerase interacts
with a specific site on
CAP(ARI: activation
region I)
• Binding is
strengthened between
promoter and
polymerase
• (The α–subunit N-terminal
and C-terminal domains (αNTD and α–CTD,
respectively) fold
independently to form two
domains that are tethered
together by a flexible linker.)
V. Regulation of gene expression
2. Posttranscriptional regulation – Gene expression can be
regulated by
(1) Inhibition of the translation of the gene even after mRNA is
made (translational regulation).
(2) Degradation of mRNA as soon as it is made or before it can
be translated .
(3) The protein product may be degraded by other protein,
called protease.
(4) By feedback inhibition – The final product inhibits enzyme
activity of the first reaction in a pathway.
V. Regulation of gene expression
3. Introns and inteins:
(1) some genes have intervening sequence in the region of
DNA encoding a RNA or protein. These sequence can move
from one DNA to another. These sequences must be
spliced out of RNAs and proteins after they are made to
restore the function of RNAs or proteins.
i. The intervening sequences that splice themselves out of
RNA are called introns which are much more common in
eukaryotic cells.
ii. The intervening sequences that splice themselves out of
protein are called intein.
Feedback inhibition regulation
VI. Expression vectors
@ The cloning vectors designed to express (made) large amounts
of proteins for biochemical or structural analysis.
1. Besides the elements of cloning vectors, expression vectors
should have a promoter including operator, TIR including ATG,
SD sequence and termination codon.
2. The gene or DNA sequence inserts into cloning site must be
in-frame with ATG.
3. For easy purification of expressed protein, some affinity tags
are also include in the vectors.
(1) Histidine tag – DNA sequence encoding six histidine amino
acids
i. Histidines binds strongly to nickel, and so the protein
contains histidines will bind to a column containing nickel.
ii. Then the bound protein can be eluted by washing the
column with high concentration of imidazole, which also
binds to nickel and so will displace the Hist tag.
(2) Other tag, such as glutathione S-transferase (GST) is used
often.
VI. Expression vectors
Use pET-15b as an example.
VI. Expression vectors
Transcriptional and tranlational
fusions to express lacZ
VII. Some methods for studying gene
expression - Northern blotting
Buffer (20 X SSC) /1 L, pH 7.0 : 175.3 g of sodium
chloride; 88.2 g 0f sodium citrate
Northern Blots
• You have cloned a cDNA
– How actively is the corresponding gene expressed in
different tissues?
– Find out using a Northern Blot
• Obtain RNA from different tissues
• Run RNA on an denatureing agarose gel (usually
containing formaldehyde) and blot to membrane
• Hybridize to a labeled cDNA probe
– Northern plot tells abundance of the transcript
– Quantify using densitometer
• Cytoplasmic mRNA isolated from 8 rat
tissues probed with GPDH
(glyceraldehyde-3-phosphate
dehydrogenase)
VII. Some methods for studying gene
expression – Reverse transcription
VII. Some methods for studying gene
expression - Primer extension
• Start with in vivo
transcription, harvest
cellular RNA containing
desired transcript
• Hybridize labeled
oligonucleotide [18nt]
(primer)
• Reverse transcriptase
extends the primer to the
5’-end of transcript
• Denature the RNA-DNA
hybrid and run the mix on
a high-resolution DNA gel
• Can estimate transcript
concentration also
VII. Some methods for studying gene
expression - S1 nuclease mapping
Use S1 nuclease mapping to locate the ends of RNAs
and to determine the amount of a given RNA in cells at
a given timeLabel a ssDNA probe that can only
hybridize to transcript of interest
- Probe must span the sequence start to finish
- After hybridization, treat with S1 nuclease which
degrades ssDNA and RNA
- Transcript protects part of the probe from
degradation
- Size of protected area can be measured by gel
electrophoresis
* Amount of probe protected is proportional to
concentration of transcript, so S1 mapping can be
quantitative
S1 Mapping the 5’ End
Real-Time PCR
1. Real-time PCR quantifies the
amplification of the DNA as it occurs
2. As DNA strands separate, forward and
reverse primers anneal to DNA strand as
that in regular PCR reaction.
3. A fluorescent-tagged oligonucleotide
binds to part of one DNA strand
Fluorescent Tags in Real-Time PCR
1. This fluorescent-tagged
oligonucleotide serves as a reporter
probe
– Fluorescent tag at 5’-end
– Fluorescence quenching tag at
3’-end
2. With PCR rounds, the 5’ tag is
separated from the 3’ tag
3. Fluorescence increases with dNTPs
incorporation into DNA product
4. The whole process takes place
inside a fluorimeter that measure of
the fluorescence of tag, which is in
turn is a measure of the progress
of the PCR reaction (in real time)
5. The reaction can be coupled to RTPCR
VII. Some methods for studying gene
expression –Biochip (Microarray )
Run-Off Transcription
• DNA fragment containing gene to
transcribe is cut with restriction
enzyme in middle of transcription
region
• Transcribe the truncated fragment
in vitro using labeled nucleotides,
as polymerase reaches truncation
it “runs off” the end
• Measure length of run-off
transcript compared to location of
restriction site at 3’-end of
truncated gene
• Size of run-off transcript locates
transcription start site
• Amount of transcript reflects
efficiency of transcription
Nuclear Run-On Transcription
• Isolate nuclei from cells, allow them to extend in
vitro the transcripts already started in vivo in a
technique called run-on transcription
• RNA polymerase that has already initiated
transcription will “run-on” or continue to elongate
same RNA chains
• Effective as initiation of new RNA chains in isolated
nuclei does not generally occur, one can be fairly
confident that any transcription observed in the
isolated nuclei is simply a continuation of
transcription that was already occurring in vivo
• Therefore, the transcripts should reveal not only
transcription rates but also give an idea about which
genes are transcribed in vivo.
VII. Some methods for studying gene
expression – RNA interference (RNAi)
1. Also called cosuppression and posttrancriptional gene
silencing (PTGS)
2. RNA interference occurs when a cell encounters dsRNA from a
virus, a transposon, or a transgene (or experimentally added
dsRNA).
3. This trigger dsRNA Is degraded into 21~23-nt fragments (siRNA)
by an RNaseIII-like enzyme, Dicer.
4. The double-stranded siRNA, with Dicer and the associated
protein R2D2, constitute a complex (complex B).
5. Complex B delivers the siRNA to the RISC loading complex
(RLC), which probably separates the two strands of the siRNA
and transfers the guide strand to the RNA-induced slicing
complex (RISC), which includes a protein called
Argonaute2 (Ago2).
VII. Some methods for studying gene
expression – RNA interference (RNAi)
6. The guide strand of the siRNA then base-pairs with
the target mRNA in the active site in the PIWI
domain of Ago2, which an RNase H-like enzyme
also known as slicer.
7. Slicer cleaves the target mRNA in the middle of the
region of its base-pairing with siRNA.
8. In an ATP-dependent step, the cleaved mRNA is
ejected from the RISC, which can then accept a new
molecule of mRNA to be degraded.
RNA interference (RNAi)
RNA interference (RNAi)
shRNA (siRNA); hRluc (Renilla luciferase)