week 2 - ways to investigate gene regulation Flashcards
IN VIVO
what
Studying things in the cell
- In vivo means measuring something that has been made by the living cell (e.g. mRNA, protein)
- This may be whole the cell is still living or after the mRNA/protein has been extracted
IN VIVO
advantages
o Comes closer to what a cell actually does
IN VIVO
disadvantages
o Cells are complex; often, mechanistic details are lost
IN VIVO
methods tell us about
o What happens in a cell (induced/repressed)
o Regions important for regulation (approx.)
o Components important for regulation
o Conditional that can regulate
direct measurement
- Determination of actual mRNA or protein levels
indirect measurment
- Measurement of something that corresponds to actual mRNA or protein levels but is easier to measure
what can you measure
WT, mutant, background, and over time
IN VIVO
Promoters regulated by?
repressors and/or activators
IN VIVO
promoters - operon
- Bit the regulator protein binds to
- (in literature this is to do with repressor proteins but in the case of the course activator as well)
IN VIVO
promoters
repressor
- Promoter is on
- Repressor turns off
when bound: mRNA not made
not bound: mRNA made
IN VIVO
activators
- Promoter is off
- Activator turns on
bound: mRNA made
not bound: no mRNA made
IN VIVO
promoter probe fusion
- In vivo
- Indirect
IN VIVO
Steps to get a readout from a promoter probe fusion
- Cloning the Suspected Promoter:
A DNA fragment suspected to contain a promoter is cloned upstream (before) of a reporter gene (like lacZ, gfp, or luc).
The reporter gene lacks its own promoter, so its expression depends entirely on the inserted DNA fragment. - Fusion Construct:
This construct (promoter + reporter gene) is inserted into a plasmid or chromosome of a suitable host (often E. coli or another bacterium).
3.Expression Testing:
If the DNA fragment contains an active promoter, it will drive expression of the reporter gene.
The activity can then be measured:
lacZ → blue colonies with X-gal.
gfp → green fluorescence under UV.
luc → light emission measured with a luminometer.
- Interpreting Results:
High reporter activity means the cloned region has a strong promoter.
No activity suggests the region lacks a promoter or has a very weak one.
IN VIVO
northern blotting
technique used to detect and study specific RNA molecules in a sample. It’s mainly used to analyze gene expression.
IN VIVO
northern blotting
method
- RNA Extraction:
Total RNA is isolated from cells or tissues. - Gel Electrophoresis:
The RNA is separated by size using agarose gel electrophoresis (often with formaldehyde to denature RNA and prevent secondary structures). - Transfer to Membrane:
The separated RNA is transferred (blotted) onto a nylon or nitrocellulose membrane. - Hybridization with Probe:
The membrane is incubated with a labeled DNA or RNA probe that is complementary to the RNA sequence of interest.
The probe hybridizes (binds) specifically to the target RNA. - Detection:
The bound probe is detected via radioactivity, fluorescence, or chemiluminescence, revealing the presence, size, and abundance of the RNA transcript.
IN VIVO
northern blotting
different transcripts
- Non uncommon to see two different transcripts
- Internal promoter in operons etc
IN VIVO
interpreting data
- Need to decide a probe
- Piece of DNA that is a complementary strand to the gene
- Both B C and D have a region that should bind to the transcript
- Will all look the same on the northern blot
- The probe does not affect where the band will run (probe after separating by size)
- A will not bind as it has no regions that is complementary, B, C,D will all show a 1.5kb band
- Note size of RNA band does not depend on size of probe
- Probe needs to fully or partially overlap the transcript
need to map start point of mRNA (primer extension)
IN VIVO
primer extension
Primer extension is a laboratory technique used to analyze the sequence of nucleotides in DNA. In this process, a primer (a short nucleic acid sequence) is annealed to a single-stranded DNA template.
DNA polymerase is then used to extend the primer, synthesizing a complementary strand of DNA. The resulting extended product can be analyzed to determine the sequence of the DNA region adjacent to the primer.
Primer extension is often used to map the start sites of transcription (e.g., identifying transcription initiation points) and to study gene expression and RNA processing.
IN VIVO
primer extension
background: DNA replication needs
- DNA polymerase
- DNA template
- dNTPs
- Primer with a free 3-OH end
- Complementary primer
IN VIVO
primer extension
background: DNA replication direction
- Direction of DNA polymerase if 5’ -> 3’ am dot is necessary that the primer has a free 3’-OH end
IN VIVO
primer extension
background: If you add DNA polymerase + all four dNTPs and a primer which is complementary to the single-stranded DNA fragment in a test tube, what will happen
- DNA polymerase will copy the rest of the template
IN VIVO
primer extension
method
if mixed
- A single stranded RNA fragment
- A complementary primer
- All four dNTPs
- RNA-dep DNA polymerase (reverse transcriptase)
- The enzyme reverse transcriptase makes cDNA complementary to RNA
- It also required a primer complementary to the mRNA
And incubated them together (in a suitable buffer)
And made one of the dNTPs radioactive
And ran reaction products down a gel
And exposed the gel to x-ray film
End up with:
- A single band corresponding to a single piece of DNA (stops synthesising at 5’ end of mRNA so gives the exact size)
IN VIVO
primer extension
then what?
can use DNA sequencing to visualise products
e.g. Chain termination method
IN VIVO
chain termination method
- DNA replication can be stopped by incorporating deoxyribonucleotide triphosphate
- dATP, dCTP, dGTP, and a mixture of dTTP and ddTTP
- this will give a series of fragments, depending on when the first ddTTP is incorporated
- then can separate these fragments by size (gel electrophoresis)
smaller fragments run faster - then can do the same thing with dCTP/ddCTP, dGPT/ddGPT, dATP/ddATP and will get:
a read out of order of bases - can use this to work out DNA sequence
IN VIVO
primer extension will…
and what do you know from it
map start point of mRNA
- the cDNA will be the same size as one of the extended products from the same primer used to sequence the DNA from which the RNA is made
- by using radioactive primer or radioactive DNA precursors, the extended products can be visualised by autoradiography
what you get: read of where the start point is
Now know:
* how much RNA is produced
* The size
* The promoter
IN VIVO
adv and drawbacks
- promoter probes
Adv
- Easy and cheap
- Can investigate multiple conditions or mutations quite fast
Drawbacks
- Indirect
- No direct information about transcript start site or length
IN VIVO
adv and drawbacks
northern blots
Adv
- Gives info about size and amount of transcript
Drawbacks
- Technically harder
- Can’t determine start point accurately
IN VIVO
adv and drawbacks
primer extension
Adv
- Precise determination of start site
- Quantitative
Drawbacks
- Technically harder
- No info about overall transcript length
IN VIVO
other methods for studying RNA directly
qRT-PCR
- qRT-PCR is a molecular biology technique used to quantify gene expression levels by converting RNA into complementary DNA (cDNA) through reverse transcription, followed by quantitative PCR amplification.
IN VIVO
other methods for studying RNA directly
qRT-PCR - method
- RNA Extraction – Isolate total RNA from the sample
- Reverse Transcription – Convert RNA to cDNA using reverse transcriptase.
- qPCR Amplification – Use specific primers and a fluorescence-based detection system (e.g., SYBR Green or TaqMan probes) to amplify target cDNA.
- Quantification – Measure fluorescence intensity in real-time to determine gene expression levels.
IN VIVO
other methods for studying RNA directly
qRT-PCR - pros
- High Sensitivity & Specificity – Detects low-abundance transcripts.
- Quantitative & Reproducible – Provides precise expression data.
- Rapid & High Throughput – Faster than traditional Northern blotting.
- Wide Dynamic Range – Can quantify a broad range of gene expression levels.
IN VIVO
other methods for studying RNA directly
qRT-PCR - cons
- RNA Quality-Dependent – Degraded RNA affects results.
- Primer Design Sensitivity – Poor primer design can cause non-specific amplification.
- Normalization Issues – Requires stable reference genes for accurate results.
- Cost & Equipment – Expensive reagents and specialized qPCR machines are needed.
IN VIVO
other methods for studying RNA directly
microarrays
- Microarrays are a high-throughput technique used to analyze gene expression levels or detect genetic variations across thousands of genes simultaneously. It relies on hybridization between nucleic acids and pre-designed probes attached to a solid surface.
IN VIVO
other methods for studying RNA directly
microarrays - method
- RNA Extraction & Labeling – Extract RNA and convert it to cDNA or cRNA, labeling it with fluorescent dyes.
- Hybridization – Apply the labeled sample to a microarray chip containing immobilized DNA probes.
- Washing & Scanning – Remove unbound sequences and use a laser scanner to detect fluorescence signals.
- Data Analysis – Measure fluorescence intensity to determine gene expression levels.
IN VIVO
other methods for studying RNA directly
microarrays - pros
- High-Throughput – Analyzes thousands of genes in a single experiment.
- Efficient & Time-Saving – Faster than traditional gene expression methods.
- Comparative Analysis – Suitable for studying differential gene expression.
- Standardized & Reproducible – Commercially available chips ensure consistency.
IN VIVO
other methods for studying RNA directly
microarrays -cons
- Expensive & Data-Intensive – High sequencing costs and complex computational analysis
- Bioinformatics Expertise Required – Requires specialized software and pipelines.
- Library Prep Variability – Biases can be introduced during cDNA synthesis.
- Higher Error Rate in Some Platforms – Long-read sequencing methods may have higher error rates.
IN VIVO
other methods for studying RNA directly
RNA-seq
- RNA-Seq is a high-throughput sequencing technique used to analyze transcriptomes, providing insights into gene expression, alternative splicing, and novel transcripts. It uses next-generation sequencing (NGS) to capture a comprehensive view of RNA molecules in a sample.
IN VIVO
other methods for studying RNA directly
RNA-seq - method
- RNA Extraction & Quality Control – Isolate total RNA and assess quality.
- Library Preparation – Convert RNA to cDNA, fragment, and add adapters.
- Sequencing – Use high-throughput platforms (e.g., Illumina, PacBio) to sequence cDNA.
- Data Processing & Analysis – Align reads to a reference genome or assemble de novo, then quantify gene expression.
IN VIVO
other methods for studying RNA directly
RNA-seq - pros
- High Sensitivity & Accuracy – Detects low-abundance transcripts.
- Unbiased & Comprehensive – Identifies known, novel, and alternative transcripts.
- No Probe Dependency – Unlike microarrays, it does not require predefined sequences.
- Wide Dynamic Range – Captures both highly and lowly expressed genes.
IN VIVO
other methods for studying RNA directly
RNA-seq - cons
- Expensive & Data-Intensive – High sequencing costs and complex computational analysis
- Bioinformatics Expertise Required – Requires specialized software and pipelines.
- Library Prep Variability – Biases can be introduced during cDNA synthesis.
- Higher Error Rate in Some Platforms – Long-read sequencing methods may have higher error rates.
IN VITRO
- ## reproducing some aspects involved in the expression of mRNA or protein in the test tube, using purified components (e.g. DNA, RNA polymerase, mRNA precursors)
IN VITRO
- adv
- can control components, making it possible to investigate fine details of mechanism
IN VITRO
- disadvantages
- may not be what happens in the cell/may be overly simplified
IN VITRO
- can tell us
- details of regulation (mechanism)
- details of regulatory regions
IN VITRO
- allows us to make what predictions
in vivo approaches allow us to make predictions about how genes are regulated and in vitro methods allow direct tests of these predictions with purified components
- can use in vitro to look at subsets of individual components or reconsitiute the whole system
- some components of system may be mutation – results should correspond with results in vivio
IN VITRO
electrophoretic mobility shift assay (ESMA)
- Electrophoretic mobility shift assay
Aka. Gel retardation or gel shift assay - Run off transcription
IN VITRO
electrophoretic mobility shift assay (ESMA)
how does it work
- A protein-DNA complex moves more slowly through a gel than the DNA alone
- If a protein binds to DNA the DNA now had a different charge distribution
- (it is not just that the protein-DNA complex is bigger)
IN VITRO
electrophoretic mobility shift assay (ESMA)
what is mutated DNA (binding site mutated + protein) for?
if this is the site of binding
when mutated should no longer bind
therefore should not see complex
can confirm the specific site protein binds
IN VITRO
electrophoretic mobility shift assay (ESMA)
extent of bindings depends on…
the amount of protein and tightness of binding
- Can use this to compare (for example) the strength of binding of a WT and a mutated protein to the same operator
- Can give quite precise measurement of strength of binding
IN VITRO
what if want to map more precisely where a protein binds
DNase foot printing
IN VITRO
DNase footprinting
DNase I footprinting is used to identify the specific DNA sequences bound by a protein, such as a transcription factor. It tells you where a protein binds on a DNA fragment.
Works on basis that bound protein will protect DNA from digestion with DNase
IN VITRO
DNase footprinting
method
- isolate DNA fragment containing predicted protein binding site
- add protein of interest
- cleave DNA
- run on denaturing polyacrylamide gel
The protein-bound sample shows a “footprint”—a region with no bands where the protein protected the DNA from DNase
IN VITRO
DNase footprinting
what does it show
- DNase footprint showing binding of regulatory protein (FNR) to a specific region of a DNA fragment
IN VITRO
DNase footprinting
what does it show
why are some bands stronger in presence of FNR
o Suggests more exposure of parts of DNA due to change in conformation
FNR senses transitions into anaerobic conditions
IN VITRO
DNase footprinting
control?
Need to always include control
* So know what protein looks like when DNA is not there
IN VITRO
DNase footprinting
starts?
When binds at one point –> change shape of DNA?
* This effect regulation
IN VITRO
EMSA and DNase footprinting
what do they look at?
individual components
IN VITRO
method to reconstitute the whole system
run off transcription
IN VITRO
run off transcription
Run-off transcription is an in vitro technique used to study promoter activity and to map the transcription start site (TSS) of a gene.
It measures RNA synthesized from a known DNA template until RNA polymerase reaches the end of the template—hence the term “run-off.”
IN VITRO
run off transcription
method
- DNA-containing promoter of interest
A DNA fragment containing the promoter and transcription start site is cloned.
The DNA is linearized at a known point downstream (usually by restriction enzyme digestion). - add purified RNA polymerase, radiolabelled rNTPs, and buffer (stabalises)
Transcription starts at the promoter and continues until the polymerase runs off the end of the DNA. - RNA Analysis:
The resulting RNA is analyzed (usually by gel electrophoresis).
The length of the RNA corresponds to the distance from the TSS to the cut site, allowing precise mapping of the start site.
IN VITRO
run off transcription
what do you get?
Analyzing a run-off transcription gel is all about interpreting the size of the RNA products to learn about promoter activity and the transcription start site (TSS).
- Identify the Run-Off Transcript
Look for a single band in your sample lane.
The band’s position (size) should correspond to the expected distance from the promoter start site to the restriction site (the end of your template). - Determine the TSS
If the transcript is, say, 120 nucleotides long, and you know the restriction site is 120 bp downstream of your cloned promoter, the TSS is right where you’d expect transcription to start.
If you see unexpected transcript sizes, this might mean:
- The promoter is not functioning as predicted.
- There are alternative start sites.
- There’s premature termination or degradation. - Compare Experimental Conditions (if applicable)
If testing different promoters or regulatory proteins, compare the intensity and size of the bands:
- Stronger bands = higher transcriptional activity.
- Multiple bands = multiple start sites or processing events.
IN VITRO
run off transcription
what components can you add
multiple
see notes for table
what is the best approach to studying gene expression
- Best approaches combine in vivo and in vitro methods
IN VIVO
summary
- (Indirect) promoter probes
- (direct) mRNA measurement
- Northern blots
- Primer extension
- Q-RT-PCR / micro-arrays / RNAseq
IN VITRO
summary
- DNA-protein interactions:
- EMSA
- DNase foot printing
- Run off transcription
After Promoter Probe Fusion:
(chatgpt)
Use site-directed mutagenesis to test specific regulatory elements.
Combine with EMSA to confirm loss of regulator binding.
After Northern Blot:
(chatgpt)
Follow up with primer extension to locate the transcription start site.
Use qRT-PCR for more quantitative data.
After Primer Extension:
(chatgpt)
Compare extension products from WT and mutant to see changes in start site.
Use RNA-seq to explore global changes or alternative splicing.
After Run-Off Transcription:
(chatgpt)
Add different purified proteins to test their effects on initiation.
Use mutant promoter DNA to map essential sequences.
IN VITRO METHODS TELL US
* EMSA
Shifts or supershifts can reveal the presence of a complex, its specificity, and binding affinity (compare mutant vs WT).
IN VITRO METHODS TELL US
* DNase Footprinting
Protected regions identify the exact binding site. If mutations abolish protection, it confirms that region is essential.
IN VITRO METHODS TELL US
* Run-off transcription
: Lower transcript levels with mutant promoters suggest defects in initiation; presence of alternative band sizes suggests cryptic or alternative TSS.
