| How is transcription regulated? | Chromatin structure
RNA polymerase binding
Additional binding |
| Why is gene regulation so important? | Major control point for tissue-/site specific expression. → Determines the phenotype!!! |
| Regulation of chromatin structure | Expressed genes are found in “open” chromatin. Genes within highly packed heterochromatin are usually not expressed. Chromatin can move within the nucleus to alter gene expression. |
| The histone code hypothesis | Proposes that specific combinations of chemical modifications to histones and DNA of chromatin help determine chromatin configuration and influence transcription. |
| Histone modifications | In histone acetylation, acetyl groups are attached to positively charged lysines in histone tails. This process loosens chromatin structure, thereby promoting the initiation of transcription. The addition of methyl groups (methylation) can condense chromatin. The addition of phosphate groups (phosphorylation) next to a methylated amino acid can loosen chromatin. |
| Transcribed regions move to different positions in nucleus | Initiation: N-tail is acetylated and attached to the lysines.
Inactive: N-tail gets methylated (centre of nucleus)
Active: Addition of phosphate groups (around the edge) |
| Transcribed regions move to different positions in nucleus | Relatively scarce, about 0.001% of total cell protein. Highly complex, at least 10 subunits. Three different enzymes, functionally and biochemically distinct.
Pol II: Transcribes most genes, including all those that encode proteins. But eukaryotic RNA Pol II requires many additional proteins- general transcription factors |
| RNA Pol II initiation process | RNA Pol II + TNFs. TATA box is the site for nucleation of initiation. Pol II promoters have a TATA box at -25 relative to the transcriptional start. Prokaryotes: TATAAT at position -10. |
| The general TNFs needed | TBP = recognises TATA box (causes dna to bend for access to other TFs)
TAF = regulates dna binding
TFIIB = positions rna pol
TFIIF = stabilises rna pol
TFIIH = unwinds dna and phosphorylates Ser5 |
| Transcription complexes | Upstream activators enhance speed of assembly. Additional factors mediate contact between upstream activators and basal factors |
| The roles of transcription factors | To initiate transcription, eukaryotic RNA polymerase requires the assistance of proteins called TFs. General transcription factors are essential for the transcription of all protein-coding genes. High levels of transcription of particular genes depend on control elements interacting with specific transcription factors |
| Organization of a typical eukaryotic gene | Proximal control elements are located close to the promoter.
Distal control elements, groups of which are called enhancers, may be far away from a gene or even located in an intron.
An activator is a protein that binds to an enhancer and stimulates transcription of a gene
Bound activators cause mediator proteins to interact with proteins at the TATA box |
| Leucine Zipper Transcription factors | Leucine zipper mediates both DNA and protein binding. Can bind to DNA as homo or heterodimers
Expands potential regulatory repertoire. |
| Can be activators or repressors | transcriptional activators and repressors are modular proteins, composed of distinct functional domains: DNA binding domain and Activation/repression domain. |
| Post-transcriptional regulation of gene expression | Cleavage, Capping, Polyadenylation, Splicing. These are eukaryotic specialities because they are important in: mRNA stability, translation and protein function. |
| Transcription includes modification of 5’ and 3’ end of mRNA | CTD phosphorylation of RNA Pol II is crucial for mRNA processing proteins: capping factors jump on the pol II tail when transcription starts and then jump again from the tail to the start of the pre-mRNA sequence |
| 5’ capping | Modifications distinguishes mRNA from other RNA molecules. Helps mRNA to be properly processed
and exported to cytosol. Stabilises mRNA. Required for efficient translation |
| PolyA addition - 3’ Polyadenylation - Crucial proteins | CPSF (Cleavage and polyadenylation specificity factor)
CstF (Cleavage stimulation factor F)
PAP (PolyA polymerase)
PABP (PolyA binding protein) |
| PolyA addition - 3’ Polyadenylation | 1.Polyadenylation starts when RNA Pol II has transcribed the end of the gene –marked by AAUAAA
2.AAUAAA bound by CPSF
3.The CA sequence is bound by CFII
4.GU-rich sequence beyond the cleavage site is bound by CstF
Cleavage follows: AAUAAA - PAP bond breaks, Add ATP and remove the GU sequence, ends up with AAAOH3, Add PABII (Adds A in between A and OH), Add more ATP to add more As |
| RNA splicing | We need components that recognise: 5‘ end splice site, 3‘ end splice site and a Branch point (A) in the intron sequence. Splicing is a two-step transesterification |
| Principle of splicing | Splicing must be precise, to the exact nucleotide. Small nuclear ribonucleoprotein particles (snRNPs). The Spliceosome is very complex: 5 snRNPs and more than 200 proteins. Only when splicing is complete does the mature mRNA exit the nucleus. |
| Splicing Mechanism | 1. U1 snRNP forms p[airs with 5' splice junction
2. BBP binds with U2AF at the branch point
3. U2 snRNP displaces BBP and U2AF and binds a the the branching point
4. U4/U6/U5 enter
5.U4 and U6 bind tightly together by bps interactions
6.After RNA-RNA arrangements the bond breaks
7. U6 displaces U1 at 5' splice junction
8. Forms an active site for 2nd reaction |
| The Exon Definition Hypothesis | Cell do not only rely on snRNPs for splicing: Recruitment of SR proteins (rich in S and R; mark exo boundaries) and hnRNPs (heterogenous nuclear ribonucleoproton; mark and package introns). |
| Alternative Splicing | 1. Normal adult b-globin primary rna transcript. 2. some single nucleotide changes that destroy a normal splice site cause exon skipping 3. activate cryptic splice sites 4. new exons to be incorporated. |
| What happens to the processed RNA transcript? | RNA editing, Ribozymes – catalytic RNAs, Regulation of nuclear export of mRNAs, miRNA |
| RNA editing | RNA editing alters the sequences of some pre-mRNAs. A very unexpected process by which the sequences of mRNAs are altered co- or post-transcriptionally. RNA editing occurs in mammals and other eukaryotes. |
| RNA editing - Mechanisms | Base insertions – usually Uracil
Cytosine deamination to Uracil
Adenine deamination to Inosine |
| Base insertion | 1. Guide RNA molecules transcribed separately.
2. Guide RNA molecules find regions of complementarity
3. Donate Us from their poly U 3’ end to regions of mRNA transcript that mispair with the guide RNA
4. Editing cascade |
| Cytosine deamination to Uracil | It can alter protein sequences → Alteration of protein function.
1. A cytidine deaminase activity is involved – APOBEC (ApoB mRNA editing enzyme catalytic subunit)
2. Another protein, ACF (APOBEC Complementation Factor) is also required
3. Both recognize sequences flanking the C to be edited |
| Adenine deamination to Inosine | ADAR - Adenosine Deaminase Acting on RNA. A-to-I editing affects >1000 genes in humans
For example, it can change Glu to Arg that changes Ca2+ permeability in the brain and die in seizure, infancy. |
| Why RNA editing? | Proteins from edited mRNA are involved in CNS function eg. Glutamate receptors, Serotonin receptors, DNA repair enzymes etc. Possible explanations for evolvement of RNA editing: System to revise mistakes in transcription, Enhancement of genome/transcriptome plasticity, Once evolved as defence system to inactivate retroviral mRNA/retrotransposons |
| Ribozymes | Ribonucleic acid enzymes. RNA molecules that possess catalytic / enzyme-like activities are called ribozymes. RNA can be truly catalytic. Has revolutionised our view of biological catalysis. |
| Nuclear transport of RNA | Only completely processed mRNAs are exported to the cytosol. Export of mRNAs is an energy-dependent and active process. Poly-As is an exported feature. |
| Small RNAs | Often expressed in the “junk”. MicroRNA (miRNA) was considered unimportant, it can stifle the production of a protein by interacting with the true mRNA, thereby preventing its translation. |
| What are small RNAs? | Small noncoding RNA molecules (20-30 nt). Found in all eukaryotes (except yeast). >60% of human protein-coding genes contain at least one conserved miRNA-binding site, plus other most protein-coding genes may be under the control of miRNAs.
3 Different key types of small RNA: miRNA (22nt, transaltional repression and mRNA degradation), siRNA (21nt, RNA cleavage) and piRNA (24-30nt, transcriptional repression of transposons) |
| What are microRNAs? | miRNAs are small noncoding RNA molecules that can regulate eukaryotic gene expression - degradation and translatability. miRNA function in gene knock out or knock down. |
| How are small RNAs made? | Key Players: Drosha and DGCR8 (Together cleave the “legs” from the pri-miRNA transcript).
Dicer: cleaves pre-miRNA near the terminal loop, RNase III-type endonuclease, region between the PAZ and RNase III domains functions as a ‘molecular ruler’.
Argonaut: Form the RNA-Induced Silencing Complex (RISC), Passenger strand is removed in pre-RISC, miRNA loading is energy-dependent and mediated by HSP70-HSP90, Argonaut is highly conserved. |
| Processes regulated by small RNAs | Disturbance or misactivation of small RNA activities can affect development, support diseases and behaviour.
Numbl – gene involved in asymmetric division in neural stem cells.
Pax7 – regulates muscle precursor cell proliferation
Mef2c – regulate GMP proliferation and differentiation |
| MicroRNAs appear to be tissue-specific and developmental-specific | Titrate the levels of key regulatory proteins → important in early development, cell proliferation, cell death and neurodevelopment.
Dicer is deliberately disrupted in fertilised eggs → block the generation of all miRNAs at this developmental stage.
If Dicer is deliberately inactivated in development in specific tissues, severe growth defects are seen
→ miRNAs are able to fine tune protein levels |
