Lecture 1 Flashcards
why are organisms different?
Because they inherit different instructions for building cells and body plans.
What is the heritable material in all organisms (except viruses)?
DNA
How do we know that DNA is the heritable material?
Based on a famous experiment performed by Avery and MacLeod in 1944:
- working with a bacterium that caused pneumonia
- when grown on petri dishes, the pathogenic bacteria form smooth colonies= S strain
- every now and then there would be a random mutation producing a colony called the R strain (rough due to rough edges of colony)
- R strain were non pathogenic
- Why??
- If you grew R strain in the presence of either heat-killed S strain or cell-free extract of S strain some R strain cells were transformed to S strain cells, whose daughters were pathogenic and caused pneumonia
= conclusion: something IN the pathogenic bacteria that could transform the non pathogenic bacteria into a pathogenic state / Molecules that can carry heritable information are present in the S strain cells
- fractionated S strain cells into different components and were able to purify the cell-free extract into classes of purified molecules: RNA, protein, DNA, lipid, and carbohydrate
- incubated non-pathogenic R strain cells with each of these components and looked to see which one caused a transformation in the S strain > DNA caused this transformation so they therefore concluded that this was the molecule that contained the heritable information
How can different cells be produced in an organism if all cells share the same DNA?
Differentiation can cause dramatic differences in cell morphology and function. These differences are due to changes in gene expression - producing different RNAs and proteins. With the exception of some lymphocytes this is done without altering the DNA sequence.
What is the central dogma of cell biology?
DNA is transcribed to form RNA, which is translated to form a protein.
How do we know that cells generally change gene expression patterns without altering the nucleotide sequence of the DNA itself?
Cloning! All of the instructions to regulate correct cell division and differentiation patterns of an oocyte into a frog are contained within the nucleus of a differentiated cell.
e.g. you take skin cells from an adult frog and grow them in a culture dish. You take a nucleus from one and inject it into an oocyte cell that has had its nucleus destroyed by UV light. With a little ‘trickery’ (electric shock) you can cause the cell to start dividing and it will develop into a normal embryo/tadpole.
This therefore means that ALL the instructions for any cell in the body/the process of development were contained in the nucleus of the fully differentiated skin cell thus telling us that differentiation does not change the underlying nucleotide sequence of the DNA.
In general, what can we say about the translation of a protein for which lots of RNA has been transcribed?
More transcription > more translation
How is transcription controlled?
By proteins binding to regulatory DNA sequences. Eukaryotic RNA polymerase requires general transcription.
Upstream of the start site of transcription/the area to be transcribed is a region called the ‘TATA box’, or the promoter of the gene. Promoter regions are control regions and they regulate how much transcription is going to occur from the start site.
In order for transcription to begin a bunch of proteins need to bind to this promoter region. TBP (Tata binding protein) binds to the TATA box, recruits a whole bunch of other factors, General Transcription Factors, that get loaded onto the promoter.
RNA polymerase starts the transcription itself.
General Transcription Factors (and their respective binding sites) are found in most cells so what causes cell/tissue specific gene expression?
Gene transcription is controlled by the binding of tissue-specific activator proteins (regulatory or transcription factors) to gene promoters and their interaction with RNA polymerase.
Gene transcription can be controlled by the presence/absence of thse regulatory factors in specific tissues or even by the ability/inability of them to enter the nucleus (or by other molecules binding to the regulatory factors.)
What is the difference between the enhancer and the promoter region?
The promoter is the region just adjacent to the start site of transcription.
Gene regulatory/activator proteins bind to the enhancer region: this is often not close to the start site of transcription, could be up to kB away. The DNA will loop around to allow the activator protein to come in contact with GTFs/the mediator protein etc. Transcription initiates when all the proteins come together.
Why do Gene activator proteins often have a modular structure?
Because they need to bind to the DNA at one site and to the GTFs either directly or indirectly through a mediator.
How can we experimentally demonstrate the modular structure of GAPs?
Through genetic engineering form a chimeric protein e.g. Gal-4 activation domain with LexA DNA-binding domain:
- put the recognition sequence upstream of LacZ gene so that it can be visualised when the gene is switched on (blue colour)
- have one with the recognition sequence for Gal4 and one with the recognition sequence for LexA
- LacZ not produced in cell with Gal4 recognition sequence but is produced in cell with recognition sequence for LexA therefore indicating that the two binding domains are separate/have separate purposes and the protein’s modular structure.
- activation domain can still interact with RNA polymerase even though it came from the Gal4 protein (normally activates transcription of gene for galactokinase) because activation domains are very similar therefore the DNA binding domain is really where the selectivity comes from
How do GAPs often work?
Synergistically: usually many enhancer sites and many enhancer proteins for one gene. E.g. one GAP will incite 1 unit of transcription, another 2 units, but both together 100 units. Acting together they rapidly influence the rate of transcription.
Control is often a complex event.
Might require many GAPs to get efficient transcription of the gene.
How do GAPs cause local changes in chromatin structure?
- histone-modifying enzyme creating a specific pattern of histone modification e.g. acetylation
- Recruit a chromatin remodelling complex that:
a. remodels nucleosomes
b. removes histones
c. replaces histones
i. e. a loosening of the packaging
How do repressor proteins antagonise activator proteins?
- competitive DNA binding
- masking the activation surface
- direct interaction with the general transcription factors
- recruitment of chromatin remodelling complexes
- recruitment of histone deacetylases
- recruitment of histone methyl transferase
