Gene expression

Question 1

Why must gene expression be regulated?

Answer 1

Every cell has the same genome, but have very different functions.

Differentiation depends on changes in gene expression.

Question 2

Levels of gene control?

Answer 2

Transcription

RNA processing

RNA transport control

mRNA degradation control

Translational control

Protein activity control

Question 3

Why is it a good idea to regulate transcription?

Answer 3

Top of the hierarchy so don’t waste time manufacturing mRNA.

In prokaryotes, simultaneous translation occurs so not much point trying to regulate translation!!

Question 4

How is transcription controlled?

Answer 4

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,

Question 5

Features of DNA binding proteins?

Answer 5

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.

Question 6

How do we measure DNA/protein interaction?

Answer 6

Gel electrophoresis.

Run DNA fragments on a gel, the proteins make the fragment longer so it runs slower.

Question 7

Example of inhibitive genetic switch?

Answer 7

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.

Question 8

Example of activator genetic switch?

Answer 8

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.

Question 9

Gene expression in eukaryotes?

Answer 9

Same basic strategies but more complex switches. Need to integrate a much larger number of switches.

Question 10

Why are transcription factors needed?

Answer 10

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.

Question 11

How do general transcription factors assemble at promoters?

Answer 11

1. TBP (TATA binding protein) subunit of Transcription Factor II D (TFIID) binds to TAFA box
2. TFIID enters complex
3. Polymerase II enters complex, escorted by TFIID
4. TFIIE and TFIIH then assemble into complex
5. 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

Question 12

What are enhancers?

Answer 12

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

Question 13

What are gene repressor proteins?

Answer 13

In eukaryotes, these don’t directly compete with RNA polymerase for access to DNA (like in bacteria).

Mechanisms aren’t well understood.

Question 14

What are regulatory proteins?

Answer 14

May act as activator in some complexes and repressors in others.

Function depends on final assembly of all components.

Question 15

What are developmental genes?

Answer 15

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.

Question 16

Different levels of DNA compaction?

Answer 16

Nucleosomes (DNA wrapped around histone proteins)

Nucleosomes packed into 30nm filaments

Higher order packing in heterochromatin (hyper condensed)

Question 17

What is a nucleosome?

Answer 17

A fundamental repeating unit of chromatin.

Question 18

What can’t assemble on chromatin?

Answer 18

General transcription factors seem unable to assemble onto promoters which are packaged into chromatin.

Question 19

Activation of a gene requires what changes in the state of chromatin?

Answer 19

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.

Question 20

Evidence for role of chromatin structure in regulating gene expression?

Answer 20

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.

Question 21

Describe active chromatin?

Answer 21

-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

Question 22

Describe inactive chromatin?

Answer 22

• 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

Question 23

What is long range gene silencing?

Answer 23

• 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

Question 24

What happens in X inactivation?

Answer 24

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

Question 25

Describe DNA methylation?

Answer 25

Inactive chromatin tends to be extensively cytosine methylated at CpG.

CpG islands are unmethylated and associated with functional promoters - mark active genes.

Methylation causes repression:

1. Transcriptional factors mostly unable to bind to methylated DNA.
2. Methylation may influence chromatin structure directly by changing the way that histone H1 binds to DNA
3. The most common way is via a family of methyl CpG binding proteins

Question 26

Biggest difference in gene expression control in prokaryotes and eukaryotes?

Answer 26

Spatial and temporal separation of transcription and translation in eukaryotes.

Question 27

What is promoter proximal pausing of RNA polymerase II?

Answer 27

• occurs in some rapidly induced genes, like heat shock genes
• RNA polymerase II pauses after transcribing 25 nucleotides
• heat shock induces activation of the heat-shock transcription factor (HSTF) which binds to the promoter-proximal region of hsp70, this stimulates the paused polymerase to continue chain elongation
• this mechanism allows a rapid response. No time is required to allow the assembly of transcription-initiation complexes as RNA synthesis is already primed
• genes are generally involved in saving the cell from trouble

Question 28

What processing occurs to RNA molecules?

Answer 28

• capping, cleavage and polyadenylation, and splicing all happen in the nucleus
• nascent pre-mRNA transcripts are associated with a class of abundant RNA-binding proteins called hnRNP proteins, which assist in the processing and subsequent transport of mRNAs.

Question 29

What is RNA capping?

Answer 29

the 7-methylguanosine cap is added to the 5’ end of a newly synthesised by a capping enzyme that associates with the phosphorylated terminal domain of RNA polymerase II shortly after transcription initiation

the cap is required or efficient nuclear export and subsequent translation, prevents exoribonucleases from digesting the 5’ end

Question 30

What is RNA cleavage and polyadenylation?

Answer 30

conserved polyadenylation signal lies ~20 nucleotides upstream from a poly(A) site where cleavage and polyadenylation will occur

a multiprotein complex including PAP carries out cleavage and polyadenylation. A poly(A) binding protein then stimulates addition of A residues by PAP and stops addition once poly(A) tail is 200 residues long

polyadenylation = addition of a poly(A) tail to a pre-mRNA molecule

Regulation can be invested at this level: uncommon
cleaves transcripts without poly(A) tail are very unstable
autoregulation can occur by U1A protein. Blocks polyadenylation but not cleavage

U1A protein inhibits polyadenylation of its pre-mRNA by binding to two identical sites upstream of the poly(A) site in U1A pre-mRNA
no functional mRNA is produced and expression of U1A protein is repressed
binding blocks polyadenylation, the transcript is then rapidly degraded by an exonuclease

Question 31

Describe splicing of RNA?

Answer 31

RNA splicing is carried out by a large ribonucleoprotein complex – the spliceosome

spliceosome catalyses two transesterification reactions that join the exons and remove the intron as a lariat structure (subsequently degraded)

group 2 introns are self-splicing – found in chloroplast/mitochondrial genes of plants/fungi, exhibit a largely conserved secondary structure essential for self-splicing

Alternative splicing: Optional exons/introns, a way of making different polypeptide chains from the same gene so could generate different versions of a protein in different cell types

Example: Src regulation – Exon sequence A is only included in nerve cells. Provides an extra site for phosphorylation of the src tryrosine kinase in the neural form.

Question 32

Describe the export of mRNA to the cytoplasm?

Answer 32

For export, mRNA must be capped and tailed, as well as dissociated from the spliceosome

Therefore junk RNA fragments such as introns are kept out of the cytoplasm

Mechanisms which block completion of splicing could block export by preventing dissociation of spliceosome

Some mRNAs may be functional in some cell types but in others fail to get delivered to the cytoplasm
– selective degradation in nucleus or exit from nucleus could be selectively blocked

Certain viruses can over-ride the control which prevents export of un-spliced mRNA:

In HIV infection: maximises viral replication, involves Rev protein encoded by HIV retroviral genome.

Rev binds to un-spliced HIV mRNA, Rev is able to overcome the block to RNA export normally imposed by association with the spliceosomes by interacting with nuclear pore complex proteins

Question 33

How are mRNAs directed to their location in the cell?

Answer 33

some mRNAs are directed to specific intracellular localisations by signals in the mRNA sequence itself

signals are typically located in the 3’ untranslated region (UTR) of the mRNA molecule

-example: localisation of bicoid mRNA in Drosophila egg – creates a gradient of bicoid protein which is crucial for development. The UTR sequences targets for mRNA binding proteins that associate with molecular motor proteins which transport mRNAs by moving along cytoskeleton actin filaments or microtubule

Question 34

How is mRNA stability regulated?

Answer 34

half-life of mRNAs can vary from minutes to hours

some proteins are required for only short periods so they are synthesised in short bursts – cytokines involved in an immune response

poly(A) tail influences stability of mRNA

unstable mRNAs contain recognition sites in their 3’ UTR for endonucleases that cleave the mRNA
degradation can occur in nucleus and cytoplasm

Question 35

Role of siRNAs?

Answer 35

Cause mRNA degradation

Question 36

Role of miRNAs?

Answer 36

Repress translation.

Question 37

Main translational controls?

Answer 37

1. Elongation controls
2. Capping
3. Cleavage/PolyA
4. Pre-mRNA splicing
5. Nuclear export of mRNA
6. Cytoplasmic localisation of mRNA
7. mRNA stability
8. Translational controls