17 - Regulation of Transcription in Eukaryotes Flashcards
trc reg is more complex in euk than prok
TFs = proetins, made by ribosomes in cytoplasm, have to enter the nucleus to do their job
the tight packing of DNA makes it less availible for Rpol
highly expressed genes are often found closely associated with the nuclear pores that allow mRNA to exit the nucleus.
Specific TFs
regulate protein-encoding genes
generally share 4 properties:
1) respond to a stimulus which signals that one or more genes should be turned on
2) unlike most proteins, TFs are capable of entering the nucleus
3) rec and bind to a specific seq on DNA
4) make contact with transcription apparatus, either directly or indirectly
TFs of higher organisms are more separated from the original signal molecule than in prok, so they are not directly bound by their signallong mplecules.
TFs have at least 2 domains (DNA binding (binds DNA) and activation (interacts with trc apparatus)).
the mediator complex
transmits info to Rpol
general TFs recognize and bind to the promoter. activators grant Rpol permission to proceed from the promoter.
Important activators: TFIIB, TFIID, TFIIH. However, this is not enough to initiate trc.
mediator = protein complex that transmits the signal from TFs to the Rpol in euks. Sits on top of Rpol. Provides site of contact for activators (esp those bound to enhancer seq).
mediator basically combines signals from multiple activators and/or repressors and sends the result to Rpol.
One role of the mediator is to recruit and stable the Rpol at the promoter. the mediator kinase needs to be removed before trc starts.
Enhancers and insulator seq segregate DNA functionally
insulator = a dna seq that shields promoters from the action of enhancers and also prevents the spread of heterochromatin
the looping mechanisms of enhancers make them able to control many genes in their neighborhood.
Insulators prevent enhancers from working on genes outside their neigborhood. They also form loops. An enhancer is prevented from working on a gene if there is an insulator between them.
Insulator also prevents heterochromatin (the silencing chromatin) from spreading.
insulator-binding protein (IBP) = protein that binds to insulator seq and is necessary for the insulator to function. Zinc-fingers.
the insulator-loops can bring genes that are far away on the chromosome into close proximity, and thus can stimulate gene expression on some occations.
an example of an IBP is CTCF (CCCTC-binding factor).
Insulators are CG rich (CG regions may be methylated). Methylation of insulators means it no longer functions, thus allowing enhancers outside the loop to act on genes inside the loop.
Matrix attachment regions
MARs = matric attachment regions = site on euk DNA that binds to proteins of the nuclear matrix or of the chromosomal scaffold - same as SAR site.
nuclear matrix = a mesh of filamentous proteins found on the inside of the nuclear membrane and udes in anchoring DNA
SAR = scaffold attachment region = site on euk DNA that binds to proteins of the chromasomal scaffold or the nulear matrix, same as a MAR site
MAR/SAR = AT rich (70%). DNA segments with multiple A runs are bent (as we know from chap 4). The nuclear proteins that bind MAR recognize the bent shape rather than the seq.
MAR allows DNA looping.
Negative regulation of trc in euks
simple repressors are common in proks, but rare in euks. However, negative regulation is still common. The neg reg generally acts by hindering activator proteins or Rpol itself in some manner.
the obvious way to obstruct an activator is to bind to its rec site on the DNA
CAAT box = seq often found in the upstream region of euk promoters that binds the TFs
MyoD = euk TF that takes part in muscle cell differentiation.
HLH = helix loop helix
Heterochromatin blocka access to DNA in euks
densly packed DNA = heterochromatin
normally packed DNA = euchromatin.
heterochromatin cannot be transcribed as it cannot be accessed by RNA pol.
The linker histone (H1) has two arms extending from its central domain. the central part binds to its own nucleosome, the arms are thought to bind to the nucleosomes on either side.
The histones of the nucleosome core has a body of ca 80 and tail of caa 20 AA on the N-termial end (faces outwards from the core). The tails contain several lysine, which can have acetyl added or removed. ALl four of the core histomes may be acetylated (H3 and H4 most often modified). higher acetylation = more trc.
HATs = histone acetyl transferase. HDACs = histone deacetylase
several co-activators are actually HATs. Several co-repressors are similarly HDACs.
chromatin remodeling complexed = protein assembly that rearranges the histones of chromatin in order to allow trc.
ISWI (“imitation switch” complex) = smaller type of chromatin remodeling complex. Can slide nucleosomes.
Swi/Snf (switch sniff) complex = larger type of chromatin remodeling complex. Can both slide and remodel nucleosomes.
chromatin remodeling complex carry out 2 main types of remodeling:
1) slide nucleosomes along the DNA molecule, so exposing seq for trc.
2) rearrange histones, so remodeling nucleosomes into looser structures that allow access to the DNA. ATP is used to provide energy for this remodeling.
The precise order in which trc factors, histone acetyl transfarases, and chromatin-remodeling complexes bind appears to vary from promoter to promoter, but a generalizes seq of events is:
1) A TF binds to DNA
2) A HAT binds to the TF
3) the HAT acetylates the histones in the vecinity and the association of the nucleosomes is loosened
4) the chromatin-remodeling complex slides or rearranges the nucleosomes, allowing acces to the DNA
5) further TFs bind
6) Rpol binds
7) initiation requires a positive signal to be transmitted via the mediator compplec from one or more specific TFs.
Histone modification
- modifies chromatin structure, affecting the binding of TFs (ant other proteins) to DNA
- the histone tails face outwards, and these tails (particularly the lysine residues) are the modification sites.
Modifiactions:
1) Acetylation. Opens up chromatin. Acetyl from Acetyl-CoA. Their availability links histone mid with state of metabolism
2) Methylation of lysine res at pos 4 and 36 in H3 promotes trc activation, but methylation of H3s 9 and 27 promotes repression
3) Phosphorylation = promotion or repression, depending on location
4) Ubiquitin (see phosphorylation). Induced by DNA damage, promoting the binding of DNA repair proteins
5) rare modifications include ADP ribosylation, sumoylation, and biotinylation (not well understood)
Polycomb group (PcG) proteins = a large protein complex that controls developmental expression of genes important for growth by methylation histones.
Methylation
in proks: distinguish own DNA
in euks: control gene expression. Often used as a marker for genes involved in tissue differentiation.
CG seq are often methylated
mantenance methylase = enzyme that adds a second methyl group to the non-methylated strand after DNA replication (old strand keeps methy group, new strand needs one).
de novo methylases = add new methyl groups
demethylases = remove methyl groups
methylation generally silences genes in euks.
methyl groups project into the major groove of DNA (hindres binding of TFs)
methylcytosine binding proteins (MeCPs) = protein in euks that recognizes methylated CG islands. These are rec by proteins that remove acetyls, resulting in condensation of DNA -> heterochromatin
Genetic imprinting
imprinting = when the expressoin of a particular allele depends on whether it originally came from the father or the mother (imprinting is a rare exception to the normal rules of genetic dominance)
occurs if the methylation patterns in the gametes survive the zygote formation (normally erased).
A way of ensuring that only one ogf a pair of genes is expressed in a diploid organism.
Usually the paternal gene is expressed (not always)
during meiosis, there is a 50% chance of the nonmethylated version to be inherited. In males, if the methylated one is inherited, the methyl groups are removed. Conversely, in females the nonmethylated one is methylated. However, mistakes here could lead to a baby with both unmethylated (= disease)
