WEEK 4 (Control of gene expression) Flashcards
What does an organism’s DNA encode?
All of the RNA and protein molecules that are needed to make its cells
How is cell differentiation achieved?
By changes in gene expression
Hundreds of different cell types carry out a range of specialised functions that depend on what?
Genes that are switched on in that cell type but not in most others
What is gene expression?
A complex process by which cells selectively direct the synthesis of the many thousands of proteins and RNAs encoded in their genome
Describe the evidence that cells have the ability to change which genes they express without altering the nucleotide sequence of their DNA
Experiments where the genome from a differentiated cell is made to direct the development of a complete organism
example experiment:
Nucleus from skin cell in adult frog and injected into frog egg which nucleus has been removed -> Doctored egg will develop into normal tadpole -> Nucleus from transplanted skin cell didn’t lose any critical DNA sequences
The various cell types of an organism differ not because they contain different genes, but because _______________________________
They express them differently
How can a cell control the proteins it contains?
1) Controlling WHEN and HOW OFTEN a gene is TRANSCRIBED
2) Controlling how an RNA transcript is SPLICED/PROCESSED
3) SELECTING which mRNAs are exported from the nucleus to the cytosol
4) REGULATING how quickly certain mRNA molecules are DEGRADED
5) SELECTING which mRNAs are TRANSLATED into protein by ribosomes
6) REGULATING how rapidly specific proteins are destroyed after they have been made & ACTIVITY of individual proteins once synthesised
What is the main site of gene expression control for most genes?
STEP 1: TRANSCRIPTION OF A DNA SEQUENCE INTO RNA
What are transcription of individual genes switched on and off by?
Transcription regulators/factors
What are transcription regulators/factors?
Proteins that bind to short stretches of DNA called REGULATORY DNA SEQUENCES
What is the difference between transcription factors in prokaryotes and eukaryotes?
PROKARYOTES = transcription factors bind to REGULATORY DNA SEQUENCES close to where RNA POLYMERASE binds. Binding can either ACTIVATE or REPRESS transcription of a gene.
EUKARYOTES = REGULATORY DNA SEQUENCES are often separated from the PROMOTER by thousands of NUCLEOTIDE PAIRS
How can eukaryotic transcription regulators act?
- Directly affect ASSEMBLY PROCESS that requires RNA Polymerase and the GENERAL TRANSCRIPTION FACTORS at the promoter
- Modify the CHROMATIN STRUCTURE of promoter regions
What is the function of the Promoter region?
- Binds the enzyme RNA polymerase
- Correctly orient the enzyme to make an RNA copy of the gene
What do both promoters of bacterial and eukaryotic genes contain?
- Transcription initiation site
- Nearby sequences that contain RECOGNITION SITES for proteins that associate with RNA Polymerase (bacteria = sigma factor, eukaryotes = general transcription factors)
Where does RNA synthesis begin?
Transcription initiation site
What does eukaryotic RNA Polymerase II require to begin transcription?
A set of general transcription factors
Most eukaryotic promoters contain a DNA sequence called TATA box. What is the TATA box recognised by?
A subunit of the general transcription factor TFIID called TATA-binding protein (TBP)
What is the role of the sigma factor in Bacterial RNA Polymerase?
- Recognises the promoter of a gene
- Once transcription begins, sigma factor is released
Describe the difference between regulatory DNA in prokaryotes and in eukaryotes
- PROKARYOTES = sequences are SHORTER and act as simple REGULATORY SWITCHES
- EUKARYOTES = sequences are VERY LONG and integrate variety of signals into a command that determines how often transcription of the gene is initiated
What acts as a switch to control transcription?
The binding of a transcription regulator to a regulatory DNA sequence
Describe the interaction between a transcription regulator and a regulatory DNA sequence
Protein inserts into the major groove of the DNA double helix and makes a series of INTIMATE, NON-COVALENT molecular contacts with the NUCLEOTIDE PAIRS within the groove -> Although each individual contact is weak, the 10-20 contacts that form at the PROTEIN-DNA INTERFACE combine to ensure interaction is HIGHLY SPECIFIC and VERY STRONG
Why do many transcription regulators bind to the DNA helix as dimers?
Dimerisation doubles the AREA OF CONTACT with the DNA which greatly increases the POTENTIAL STRENGTH and SPECIFICITY of the protein-DNA interaction
Give an example of bacteria regulating the expression of their genes according to food sources available in their environment
In E.Coli 5 genes code for enzymes that manufacture TRYPTOPHAN when this amino acid is scarce -> Genes are arranged in a CLUSTER on the chromosome and are TRANSCRIBED from a single promoter as one long mRNA molecule (transcribed clusters are called OPERONS) -> mRNA is translated to produce a full set of BIOSYNTHETIC ENZYMES which work in tandem to synthesise the amino acid
What happens when Tryptophan is abundant in the cell?
Transcription regulator binds to the operator which blocks access of RNA Polymerase to the promoter -> Prevents transcription of the operon -> Prevents production of tryptophan-synthesising enzymes
[Repressor can bind to DNA only if it is bound to tryptophan]
How is the bacteria cell adapted to fluctuating tryptophan levels?
Gene that encodes TRYPTOPHAN REPRESSOR PROTEIN is continuously transcribed at a LOW LEVEL so small amount of repressor protein is always being made -> Protein is always present in cell -> Bacteria can respond very RAPIDLY to increases and decreases in tryptophan concentration
What does RNA Polymerase need to initiate transcription?
An activator protein
In bacteria, the binding of the activator to DNA is controlled by what?
The interaction of a metabolite or other small molecule with the activator protein
What do repressors and activators do?
REPRESSORS = TURN GENES OFF
ACTIVATORS = TURN GENES ON
What does the Lac operon encode?
Proteins required to import and digest the disaccharide lactose
Describe how the Lac Operon is controlled by an activator and a repressor
1) When Lactose is absent, Lac repressor bind to Lac Operator and shuts off expression of the operon
2) Addition of lactose increases intracellular concentration of ALLOLACTOSE. This binds to LAC REPRESSOR causing it to undergo a CONFORMATIONAL CHANGE that releases its grip on the operator DNA
3) When glucose is absent, cyclic AMP is produced by the cell and CAP binds to DNA
[for the operon to be transcribed, glucose must be absent and lactose must be present]
What are Enhancers?
The DNA sites to which eukaryotic gene activators bind
[called ENHANCERS since their presence dramatically enhances the rate of transcription]
What is the importance of eukaryotic activator proteins?
They can enhance transcription even from a distance (even thousands of nucleotide pairs away) of the gene’s promoter
(Eukaryotic repressor proteins do the OPPOSITE - decrease transcription by PREVENTING assembly of this complex)
What forms a Transcription initiation complex?
- General transcription factors
- RNA Polymerase at the promoter
What does a broken stretch of DNA signify?
The segment of DNA between the enhancer and the start of transcription varies in length
What does an activator protein bound to a distant enhancer do?
Attracts RNA Polymerase and the general transcription factors to the promoter
What does looping of the intervening DNA allow?
Contact between the activator and the transcription initiation complex bound to the promoter
How can chromatin structure be altered?
- Chromatin-remodeling complexes
- Enzymes that covalently modify the histone proteins that form the core of the nucleosome
Describe how transcription is initiated by Chromatin-modifying proteins
1) HISTONE-MODIFYING ENZYMES such as HISTONE ACETYLTRANSFERASES add ACETYL GROUPS to specific histones which serve as binding sites for proteins that STIMULATE transcription initiation
2) CHROMATIN-REMODELING COMPLEXES make DNA packaged in nucleosomes more ACCESSIBLE to other proteins in the cell including those for TRANSCRIPTION INITIATION
What is Combinatorial control?
The process by which groups of transcription regulators work together to determine the expression of a single gene
What do transcription regulators direct the assembly of?
- Mediator
- Chromatin-remodeling complexes
- Histonemodifying enzymes
- General transcription factors
- RNA Polymerase
What is the difference between General Transcription Factors and Transcription Regulators?
- General transcription factors = assemble at the promoter are the same for all genes transcribed by RNA Polymerase
- Transcription regulators = locations of their DNA binding sites relative to the promoters are different for different genes
The effects of multiple transcription regulators combine to determine the final rate of what?
Transcription initiation
The ___________ DNA sequences that separate the regulatory DNA sequences are not recognised by any transcription regulators
Spacer
Describe how combinations of a few transcription regulators can generate many cell types during development
A decision to make a new transcription regulator is made after each cell division -> Repetition of this can generate EIGHT cell types using only three transcription regulators -> Each of these hypothetical cell types would express different sets of genes
What are the two types of Transcription factors?
- GENERAL TRANSCRIPTION FACTORS that bind at core promoter sites in association with RNA Polymerase
- SEQUENCE-SPECIFIC TRANSCRIPTION FACTORS that bind to various regulatory sites of particular genes (can act as activators/repressors)
What are the properties of gene transcription?
- Complex
- Regulated by the presence of multiple binding sites for transcription factors and the binding affinity for these transcription factors to each sequence
- Transcription factors have preferred binding sites
- A region UPSTREAM might contain one or more PREFERRED, HIGH AFFINITY SITES or sites with a slightly different sequence that bind with LOWER AFFINITY
What can direct the formation of an entire organ?
A master transcription regulator
How do researchers learn which sites in the genome interact with a particular transcription factor?
- Deletion mapping
- DNA foot printing
Describe what happens in Deletion mapping
1) DNA molecules are prepared that contain DELETIONS of various parts of the gene’s promoter
2) Altered DNAs are introduced into cells and the ability of the deletion mutants to INITIATE TRANSCRIPTION is measured
3) Usually, deletion of a few nucleotides has no effect on transcription of an adjacent gene. However,
If deletion falls WITHIN A REGION that PREVENTS binding of transcription factors that activate transcription = Transcription will DECREASE
If deletion falls WITHIN A REGION that inhibits transcription = Transcription will INCREASE
What is DNA footprinting?
An in-vitro molecular technique used to identify protein binding regions on a DNA molecule
What is DNA foot printing mainly used to identify?
- Transcription factors that bind to promoter, enhancer or silencer region of gene to regulate its expression
- The regulation of transcript ion of a gene
Describe how DNA foot printing works
1) When a transcription factor binds to a DNA sequence, the presence of the protein protects the DNA sequence from digestion by nucleases
2) Chromatin is isolated from cells and treated with DNA-digesting enzymes
3) Regions that are not protected by proteins are digested whereas those with bound proteins are protected
4) Once chromatin has been digested, bound protein is removed and protected DNA sequences are identified
What are the principles of DNA footprinting?
- Nucleases like DNAse I is used which will degrade DNA molecule
- Nucleases cannot degrade DNA if it is bounded by a protein (region is protected from degradation by nucleases)
- Protected DNA region is called the FOOTPRINT
