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Topic 8-2. 2. Repression of TranscriptionCells also possess negative regulatory elementsMechanisms:Binding to promoter elementsBlocking assembly of the preinitiation complexInhibiting binding or functioning of transcriptional activatorsModifying DNA and its interaction with nucleosomesSome tr
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1. Topic 8-2 1
2. Topic 8-2 2 Repression of Transcription
Cells also possess negative regulatory elements
Mechanisms:
Binding to promoter elements
Blocking assembly of the preinitiation complex
Inhibiting binding or functioning of transcriptional activators
Modifying DNA and its interaction with nucleosomes
Some transcription factors activate some genes and repress others
3. Topic 8-2 3 Repression of Transcription - Mechanisms:
Binding to promoter elements
Blocking assembly of the preinitiation complex
4. Topic 8-2 4 Repression of Transcription - Mechanisms:
Inhibiting binding or functioning of transcriptional activators
5. Topic 8-2 5 Repression of Transcription
DNA Methylation
Methyl groups may be attached to cytosine (C5 position)
Methyltransferases
Methyl groups provide a tag
In mammals always part of a symmetrical sequence
Concentrated in CG-rich domains
Often in promoter regions
Methylation of promoter DNA highly correlated with gene repression
6. Topic 8-2 6 Repression of Transcription
DNA Methylation
Maintains a gene in inactive state rather than initiating gene repression Example:
Inactivation of genes of one X chromosome in female mammals occurs prior to a wave of methylation
Shifts throughout life in DNA-methylation levels
Early Zygote most methylation tags removed
Implantation a new wave of methylation occurs
Important example Genomic Imprinting
7. Topic 8-2 7 Repression of Transcription
DNA Methylation Genomic Imprinting
Certain genes are active or inactive during early development
Depending on whether they are paternal or maternal genes
Eg IGF-2 is only active in the gene from the male parent
The gene is imprinted according to parental origin
Mammalian genome has > 100 imprinted genes in clusters
Imprinted due to selective methylation of one of the alleles
8. Topic 8-2 8 Repression of Transcription
DNA Methylation Genomic Imprinting
In the early embryo the waves of demethylation and new methylation do not affect the methylation of imprinted genes
Thus the same alleles are affected in the zygote through to the adult stage in the individual
9. Topic 8-2 9 Repression of Transcription
Chromatin structure and transcription
DNA is not naked but wrapped around histone complexes to form nucleosomes
How are transcription factors and RNA polymerases able to interact with DNA tightly associated with histones?
Apparently nucleosome structure does inhibit initiation of transcription
Initiation of transcription requires assembly of large complexes and nucleosomes block assembly at the core promoter
10. Topic 8-2 10 Repression of Transcription
Chromatin structure role of acetylation
Genes which are actively transcribed are bound by histones which are acetylated
Each of the histones has a flexible N-terminal tail
Extends outside the core particle and the DNA helix
Acetyl groups are added to lysine residues by enzymes
Histone acetyl transferases (HATs)
Acetylation has two functions
Neutralize the positive charge on the lysine residues
Destabilize interactions between histone tails and structural proteins
11. Topic 8-2 11 Repression of Transcription
Chromatin structure role of acetylation
Some coactivators have HAT activity
Links histone acetylation, chromatin structure and gene activation
HAT activity of coactivator acetylates core histones bound to promoter DNA causing
release of nucleosome core particles or loosening of histone-DNA interaction
Subsequent binding of transcription factors and RNA polymerase
Once transcription is initiated RNA polymerase is able to transcribe DNA packaged into nucleosomes
Acetylation is dynamic enzymes also remove acetyl groups
12. Topic 8-2 12 Repression of Transcription
Chromatin structure role of deacetylation
Removal of acetyl groups
Histone deacetylases (HDACs)
HDACs associated with transcriptional repression
HDACs are subunits of larger complexes corepressors
HDACs guided to regions of DNA by methylation patterns
Example:
Inactive X chromosome of female
Largely deacetylated histones
Active X chromosome has a normal level of histone acetylation
13. Topic 8-2 13 Repression of Transcription
Chromatin structure Acetylation / Deacetylation
14. Topic 8-2 14 Repression of Transcription
Chromatin structure Acetylation / Deacetylation
15. Topic 8-2 15 Processing-Level Control
Recall that the formation of multigene families is a mechanism that generates protein diversity
Protein diversity also generated via alternate splicing
Regulates gene expression at the level of RNA processing
A mechanism by which a single gene can encode two or more related proteins
Most genes (and their primary transcripts) contain multiple introns and exons
Often more than one pathway for processing of primary transcript
16. Topic 8-2 16 Processing-Level Control
Transcripts from approx 35% of human genes may be subjected to alternate splicing
Simplest case a specific segment either spliced out or retained Example:
Fibronectin:
Synthesized by fibroblasts two additional peptides compared to that synthesized by liver
Extra peptides encoded by pre-mRNA retained in fibroblast
17. Topic 8-2 17 Translational-Level Control
Wide variety of mechanisms affecting mRNA previously transported from the nucleus
Subjects include:
Localization of mRNA in the cell
mRNA translation
Half-life of mRNA
Mediated via interactions between mRNA and cytosolic proteins
18. Topic 8-2 18 Translational-Level Control
mRNA noncoding segments untranslated regions (UTRs)
5 UTR from methylguanosine cap to AUG initiation codon
3 UTR from termination codon to end of poly(A) tail
UTRs contain nucleotide sequences which mediate translational-level control
19. Topic 8-2 19 Translational-Level Control
Cytoplasmic localization of mRNAs Example ferritin
Translation regulated by iron regulatory protein (IRP)
Activity of IRP dependent on cellular iron concentration
At low iron concentration IRP binds the 5 UTR
Bound IRP interferes physically with the binding of a ribosome to the 5 end of the mRNA
At high iron concentration the IRP changes conformation and looses affinity for the 5 UTR
20. Topic 8-2 20 Translational-Level Control
Control of mRNA stability
Half-life of mRNA is variable 10 minutes to 24 hours
Specific mRNAs are recognized in the cytoplasm and treated differentially
mRNAs lacking the poly(A) tail are rapidly degraded
Poly(A) tail is not naked mRNA but bound by the poly(A) binding protein (PABP)
Each PABP bound to about 30 adenosine residues
21. Topic 8-2 21 Translational-Level Control
Control of mRNA stability
PABP protects poly(A) tail from general nuclease activity
But increases its sensitivity to poly(A) ribonuclease
mRNA in cytoplasm is gradually reduced in length by poly(A) ribonuclease
When the tail is reduced to approx 30 residues
mRNA is rapidly degraded
Degradation occurs from the 5 end
Suggests two ends held in close proximity