BIO230 Lecture 2 Flashcards
Genomes are made out of ______, but _____ for viruses.
- DNA
2. RNA
Define: Genome
Information to make and maintain an organism
Genome expression
Release of biological info stored in genome
First product of genome expression
Transcriptome
Transcriptome
RNA molecules present present in a cell at a particular time
DNA microarray
Gives snapshot of transcriptome
Shows differences in mRNA expression in different cells
How is the transcriptome maintained?
Transcription
DNA -> RNA synthesis
What is the second product of genome expression?
Proteome
Define: Proteome
- Collection of proteins in a cell
- Define biochemical functions of the cell
What does 2D GEL ELECTROPHORESIS do?
- Gives snapshot of the proteome
- Allows us to visualize differences b/t two protein samples
- Separates based on size (y-axis) and charge (x-axis)
What do the red and blue spots in 2D gel electrophoresis readings mean?
Red = same proteins are in both samples
Blue = different proteins are the samples
Define: isoelectric point
The PH where a protein has no charge
Describe the method of DNA microarray (simple)
mRNA islation followed by hybridization to DNA microarrays
How is the proteome maintained?
Translation
RNA -> protein
What is the “central dogma” of molecular biology?
Genome (DNA) -> transcriptome (RNA) -> proteome (protein)
Do different cells of the same multicellular organism contain the same or different genome?
Same
How are different cell types produced? (e.g. liver cell, brain cell)
Differences in genome expression
Same DNA, but choose to express certain aspects resulting in different behaviour.
What percentage of the human genome is expressed at any one time?
30~60%
The bacterial toxin cyclohexamide inhibits eukaryotic translation. Which of the following would you expect to be most affected in human cells treated with cyclohexamide?
A) Genome
B) Transcriptome
C) Proteome
C) Proteome
translation = RNA to proteins -> proteome would be affected because none would be made
Where can eukaryotic gene expression be controlled?
- Transcriptional control
- RNA processing control
- RNA transport & localization control
- Translation control
- mRNA degradation control
- Protein activity control
Regulation of gene expression is crucial because…
- Response properly to extracellular stimuli (for both multicellular & unicellular organisms)
- A cell’s individuality; defining cell types (only multicellular organisms)
What enzyme is responsible for transcribing DNA -> RNA?
RNA Polymerase
In what direction does transcription occur?
3’ -> 5’
What direction does the newly formed RNA come out of the RNA polymerase during transcription?
5’ comes out of the RNA channel first
What is used to form mRNA during DNA transcription?
Ribonucleoside triphosphates
Where does the ribonucleoside triphosphate go into the RNA polymerase during transcription?
Ribonucleoside triphosphate uptake channel
What is the RNA polymerase holoenzyme composed of?
sigma factor + RNA polymerase
Outline prokaryotic transcription
- DNA has promotor section (that may not be the transcription start site) that positions the start site
- Sigma factor binds to promotor, then RNA polymerase binds -> forms holoenzyme
- Holoenzyme unwinds DNA
- Transcription begins
- Sigma factor released after ~10 nucleotides synthesized
- Transcription elongation
- Transcription termination (stem loop/hairpin structure usually forms from the RNA) -> RNA released
Can genes be transcribed at different speeds?
Yes, some are faster than others.
What regulates gene expression in both prokaryotes and eukaryotes?
Gene Regulatory Proteins (transcription factors)
Where do Gene Regulatory Proteins bind to?
Regulatory regions of DNA (cis elements)
Can be found in/before/after promotor, or in coding sequence, or anywhere.
What are activators?
Gene regulatory proteins that can turn genes on; positive regulators
What are repressors?
Gene regulatory proteins that can turn genes off; negative regulators
Give one example of a repressor.
Tryptophan (Trp) operon
How were gene regulatory proteins discovered?
Bacterial genetics
Who was responsible for discovering gene regulating proteins?
Francois Jacob
Andre Lwoff
Jacques Monod
What conditions do e.coli follow in terms of transcription regulation?
Food availability.
Change regulation by food availability.
List some traits of E. coli.
- unicellular prokaryote
- one chromosome of circular DNA
- encodes ~4300 proteins
Define: operon
Multiple genes can be transcribed into a single mRNA.
Allow gene expression to be coordinately controlled.
Are operons in eukaryotes or prokaryotes, or both?
Only prokaryotes
How many genes does the tryptophan operon have?
5
Is the Trp operon an activator or repressor?
Repressor
What does the Trp operon do?
encode enzymes for tryptophan biosynthesis
How is the Trp operon regulated?
Transcriptionally regulated by a single promotor
In a diagram of the promoter region, where does the sigma factor bind to?
The “yellow” areas (two blocks near each end of the promoter area)
-35 and -10
How many protein-bound states does the Trp operon promoter have?
*protein-bound = how many types proteins can bind to the promoter?
2
What are the protein-bound states of the Trp operon promoter?
- Bound by RNA polymerase
2. Bound by tryptophan repressor protein
When the Trp operon promotor is bound by RNA polymerase, what is the gene expression like?
Tryptophan gene expression ON -> make 5 genes and tryptophan
When the Trp operon promotor is bound by a tryptophan repressor protein, what is the gene expression like?
Tryptophan gene expression OFF -> tryptophan repressor protein bind to operator (specific area of promotor sequence that prevents RNA polymerase from binding)
What happens when Trp levels is high in a cell?
The tryptophan repressor protein binds to the operator section of the promotor area and prevents more tryptophan from being made.
What happens when Trp levels are low in a cell?
RNA polymerase binds to -35 and -10 of the promotor area and makes more tryptophan.
How is tryptophan repressor regulated?
2 molecules of tryptophan is needed to activate the repressor (proving that there is an excess)
What happens when Trp levels drop while Trp repressor protein is bound?
Trp repressor protein releases the 2 Trp required to activate it -> becomes inactive -> allow RNA poly to bind
What type of DNA binding motif does Tryptophan repressor contain?
Helix-Turn-Helix
What is the most common DNA-binding motif?
Helix-Turn-Helix
Where does the Helix-Turn-Helix bind to?
Major groove of DNA double helix.
Contacts the edges of base pairs.
Define: recognition helix
C-terminal alpha helix of the Helix-Turn-Helix structure.
What is the recognition sequence responsible for?
Sequence specific recognition of DNA.
What is the N-terminal of the Helix-Turn-Helix structure responsible for?
Structural component.
Positions recognition sequence.
When tryptophan binds to the Trp repressor, what happens?
Conformation change of Trp repressor occurs -> bind the DNA sequence (Helix-Turn-Helix) -> block transcription of genes that encode enzyme to make Trp operon -> genes turned off
Where does the Trp repressor fit on the DNA?
The major groove of the operator section.
Define: dual regulation
Both positive and negative control
What organism is the Lac operon found in?
E. coli
What is the Lac operon code?
3 genes that transport lactose into cell for catabolism (breakdown into simpler materials)
The Lac operon can ______ regulate.
Dual
What does the Lac operon allow the cell to do when there is a lack of glucose?
Use lactose for energy
What conditions MUST be met in order for a cell to use lactose as an energy source?
Glucose low AND lactose high.
Lac operon will be on -> E. coli use lactose for energy
What acts as the ACTIVATOR for the Lac operon?
Catabolite Activator Protein (CAP)
Promotes Lac expression (low glucose & high lactose)
What acts as the REPRESSOR for the Lac operon?
Lac Repressor Protein
Inhibits Lac expression (low lactose and/or high glucose)
What is the first gene of the Lac operon?
LacZ
What is LacZ responsible for?
Codes for beta galactosidase
What is beta galactosidase responsible for?
Break down lactose -> glucose & galactose
Where does the catabolite activator protein (CAP) bind?
CAP-binding site
blue section before the promotor
Where does the RNA polymerase bind?
RNA-polymerase binding site (promotor)
Where does the Lac repressor bind?
Operator (to the right of where the sigma factor binds, but still inside the promotor)
When is the Lac repressor bound to the operator?
Lactose levels are low.
Does Lac operon gene expression turn off?
No, but very very low.
What are 2 conditions that have to be met in order for the Lac operon to be on?
- Lac repressor not bounded
2. CAP bounded.
What happens when there is high glucose and high lactose?
CAP is not bound.
Lac repressor not bound.
What happens when there is high glucose and low lactose?
CAP not bound.
Lac repressor bound.
What type of relationship does lactose and allolactose have?
Positively correlated relationship:
More lactose = more allolactose
Less lactose = less allolactose
Define: allolactose
Isomer of lactose
What is required in order for lactose -> allolactose?
Beta galactosidase
How does increased lactose remove Lac repressor from the operator?
- When allolactose is high, it binds to Lac repressor
- Lac repressor undergoes conformational change
- Conformational change decreases DNA-binding activity of Lac repressor
- Lac repressor releases from the operator
Why is an activator needed in order to transcribe the Lac operon?
- RNA pol inefficiently binds to Lac promotor
- CAP needs to be bound (RNA pol likes CAP, therefore will bind efficiently)
What structural feature does CAP contain?
Helix-turn-Helix DNA binding domain
How is CAP DNA binding activated?
Low glucose
What is the relationship between glucose and cyclic AMP (cAMP)?
Negative relationship:
More glucose = less cAMP
Less glucose = more cAMP
What does cAMP do to the CAP protein?
- Conformational change
- CAP increase DNA binding activity -> bind to DNA
- RNA pol bind to CAP binding site
Under conditions where both glucose and lactose levels are low, you would expect the expression of the Lac operon to be…
Off (very low)
Why is important that expression of the Lac operon is always slightly on?
A) E. coli needs B-galactosidase encoded by Lac operon to use glucose
B) E. coli needs B-galactosidase encoded by Lac operon to respond to lactose
C) RNA polymerase requires B-galactosidease ended by Lac operon to bind DNA
D) Lac can be completely shut down without consequence
B
B-galactosidase break down lactose -> allolactose -> bind to repressor -> repressor does not bind to RNA
