Gene expression Flashcards
Why must gene expression be regulated?
Every cell has the same genome, but have very different functions.
Differentiation depends on changes in gene expression.
Levels of gene control?
Transcription
RNA processing
RNA transport control
mRNA degradation control
Translational control
Protein activity control
Why is it a good idea to regulate transcription?
Top of the hierarchy so don’t waste time manufacturing mRNA.
In prokaryotes, simultaneous translation occurs so not much point trying to regulate translation!!
How is transcription controlled?
Gene regulatory proteins recognise and bind to specific DNA sequences in gene regulatory regions.
DNA binding proteins distort the structure of DNA, often causing bending,
Features of DNA binding proteins?
Contain structural motifs that can “read” DNA sequences, the recognition sites are usually short.
Specific nucleotide sequence creates a pattern of structural features on the surface of the double helix.
The simplest DNA binding motif is the helix turn helix motif found in eukaryotes and prokaryotes. consists of two helices held at a fixed angle.
Carboxyl terminal helix is the recognition helix, fits into the major groove. Amino acid side chains recognise specific DNA binding sequence.
DNA binding proteins regulating transcription are generally known as transcription factors.
How do we measure DNA/protein interaction?
Gel electrophoresis.
Run DNA fragments on a gel, the proteins make the fragment longer so it runs slower.
Example of inhibitive genetic switch?
Tryptophan repressor in E.Coli
Tryptophan can be made in the cell or taken up from the environment. When it’s available in the medium, there’s no point making it in the cell.
Trp operon – when trp is present in the medium, it enters the cell and the trp operon is switched off. The operator contains a short recognition sequence for the trp repressor, a helix-turn-helix motif protein.
The repressor is activated to bind DNA by the presence of trp. 2 molecules of trp bind to the repressor and tilt the h-t-h motif so that it can bind to the major groove.
The repressor then competes with RNA polymerase for DNA access, transcription is therefore blocked.
Example of activator genetic switch?
Lac operon and catabolite activator protein (CAP).
In bacteria there are proteins which increase the efficiency of transcription initiation, usually bind to a nearby site.
CAP enables lactose to be utilised instead of glucose when glucose isn’t available.
Low glucose leads to high cAMP which binds to CAP and this complex binds to CAP site.
When cAMP-CAP and RNA polymerase bind to the lac control region simultaneously they form a complex and stimulate each other’s binding – cooperativity.
CAP is also a helix-turn-helix protein.
Both trp repressor and CAP require small cofactors to bind to DNA but affect transcription via RNA polymerase in different ways.
Lac repressor shuts off lac operon in absence of lactose whilst CAP activates the operon in absence of glucose.
Allows for the integration of two signals and so there has to be no glucose, but lactose must be present in order for the operon to be switched on, and RNA made.
Gene expression in eukaryotes?
Same basic strategies but more complex switches. Need to integrate a much larger number of switches.
Why are transcription factors needed?
RNA Polymerase II can’t initiate transcription on its own.
General transcription factors are required for an assembly process which provides an important site for the integration of control pathways.
Transcription factors can even act when bound to DNA kbs away from the RNA polymerase binding site.
How do general transcription factors assemble at promoters?
- TBP (TATA binding protein) subunit of Transcription Factor II D (TFIID) binds to TAFA box
- TFIID enters complex
- Polymerase II enters complex, escorted by TFIID
- TFIIE and TFIIH then assemble into complex
- In presence of ATP, TFIIH phosphorylates Pol II C – terminal domain, releasing the polymerase so it can initiate transcription
This mechanism is highly conserved in eukaryotes
What are enhancers?
Operate at a distance from promoters (several kb)
Can also be found downstream of genes
Intervening DNA between promoter and enhancer loops out to allow proteins bound to enhancer to interact directly with general transcription factors or RNA polymerase. DNA acts as a tether between the two proteins
What are gene repressor proteins?
In eukaryotes, these don’t directly compete with RNA polymerase for access to DNA (like in bacteria).
Mechanisms aren’t well understood.
What are regulatory proteins?
May act as activator in some complexes and repressors in others.
Function depends on final assembly of all components.
What are developmental genes?
Combinatorial gene control provide the complexity of gene expression required during development.
More than 10,000 cell types can be specified by only 25 different gene regulatory proteins.
Different levels of DNA compaction?
Nucleosomes (DNA wrapped around histone proteins)
Nucleosomes packed into 30nm filaments
Higher order packing in heterochromatin (hyper condensed)
What is a nucleosome?
A fundamental repeating unit of chromatin.
What can’t assemble on chromatin?
General transcription factors seem unable to assemble onto promoters which are packaged into chromatin.
Activation of a gene requires what changes in the state of chromatin?
Structure of chromatin permits localised de-condensation and repackaging of DNA to facilitate the processes of replication, transcription and repair.
Chromatin and a host of enzymatic molecular machines have evolved to play a major role in the control of gene expression.
Chromatin remodelling is an active process - general process of inducing changes in chromatin structure.
Evidence for role of chromatin structure in regulating gene expression?
Cleavage by micrococcal nuclease is preferential in linker DNA between nucleosomes.
DNA in the nucleosome is protected from cleavage.
Active genes have an altered chromatin structure showed by the fact that genes are more sensitive to nuclease digestion in tissues where they are transcribed.
Describe active chromatin?
-depletion and/or phosphorylation of H1-type linker histones
+causes depletion from chromatin or might facilitate binding of other regulatory factors to DNA
-increased core histone acetylation
+acetylation of lysine residues in core histones neutralises the positive charge, altering DNA-histone interactions. Also influences nucleosome-nucleosome interactions involved in higher order folding as well as the association of non-histone regulatory proteins with chromatin
-increased incorporation of specific histone variants
+affects nucleosome structure, higher-order packing and association of non-histone factors
Describe inactive chromatin?
- dephosphorylation of H1 histones
- deacetylation of core histones
- diminishment of histone variants
- sub-nuclear localisation of genes can be important in repression e.g. silent genes are sometimes organised to be near the nuclear periphery
- DNA methylation – this may be related directly to decreased histone acetylation via methyl-binding protein recruitment. Histone methylation can also affect chromatin states
- identification of histone acetyltransferases and histone deacetylases has provided the clearest links between chromatin remodelling and gene expression. HATs transfer acetyl groups from acetyl CoA onto histones or other substrates. Transcriptional coactivators may have HAT activities that acetylate the tails of nucleosomal histones
What is long range gene silencing?
- DNA in some special forms of chromatin is inaccessible to transcription factors. This includes heterochromatin
- often large areas of the genome are silenced, sometimes irreversibly. Some forms of modified chromatin can spread along a chromosomal DNA molecule. “Barrier” sequences block the spread of chromatin modifying complexes, thereby separating chromatin domains
~silencing may be position dependent, maybe telomeres
Position-dependent transcriptional silencing:
Sir2 protein is targeted to chromatin through interaction with sir3 and sir4. Sir proteins also involved in silencing at other loci, proteins conserved throughout eukaryotes
~sometimes groups of related genes are co-regulated by changes in chromatin structure
~genomic imprinting also involves changes in chromatin structure
This refers to the unequal expression of autosomal genes based on the parent of origin (non mendelian). Mammals are therefore hemizygous for all imprinted genes with an increased genetic risk. Example: Igf2 is only expressed from the paternally contributed gene. Often, imprinted genes function to regulate fetal growth. Often clustered in the genome.
~locus control region (LCR) control of mammalian globin genes
globin gene cluster only transcribed in red blood cells. Each gene within cluster is controlled independently during development by local interactions with different transcription factors but the LCR maintains global control over whether the whole cluster is active
What happens in X inactivation?
Permanent inactivation of one X chromosome in female mammals. Random whether maternal or paternal chromosome affected.
Chromosome becomes highly condensed into heterochromatin (Barr body), form of epigenetic inheritance as the cell memory is based on an inherited protein structure rather than a change in DNA sequence.
Inactivation spreads along the X chromosome from a discrete nucleation site (inactivation centre), condensed X is re-activated during germ cell formation.
Properties of inactive X chromatin:
late replicating during S phase
extensively cytosine methylates at CpG dinucleotides
enriched in histone variants
histones H3 and H4 hypo-acetylated
certain loci escape silencing, e.g. XIST which is essential for X-inactivation
