Wk1: Eukaryotic Gene Expression and Regulation, RNA processing

CTD tail

• C Terminal Domain Tail

• feature of eukaryotic RNA pol II

• Phosphorylated and dephosphorylated (activated and deactivated) at different parts of the transcription process

• Involved in 5’ capping and Poly-Adenylation

General Transcription factors

Protein complexes which bind to short recognizable sequences near the promoter of a gene and help to bind and position RNA Pol II, pull apart DNA strands, and release RNA Pol II from the promoter region to prompt elongation

• labeled like ‘TFIIA’ meaning: Transcription Factor (for RNA Pol) II, letter denotes identity

TSS

Transcription start site, where RNA Pol II machinery LANDS, starting transcription

TATA box

sequence 30bp upstream from TSS, where RNA pol II binds with the help of TBP (TATA binding protein)

TATA binding protein (TBP)

Protein complex that attaches to TATA, inducing a 80 degree kink in the helix in preparation for initiation

Transcription Initiation

• TFIID, which contains TBP subunit, binds at TATA box and induces kink

• TFIIB binds at BRE element

• TFIIF brings in RNA pol II, attaching it to bound TFs

• TFIIH, which contains helicase and kinase, unwinds DNA and phosphorylates CTD tail to promote elongation

Promoter Escape

Transition from Initiation to elongation

• TFIIH unwinds DNA

• RNA pol II synthesizes short lenghts of RNA

• Ser 5 in CTD tails are phosphorylated by TFIIH causing RNA pol II to disengage from GTFs after a short abortive period

• GTFs are released

Basal Apparatus/Transcription Initiation complex

Complex of proteins at the promoter region which are composed of RNA Pol II and 6 General Transcription factors

Activators and Repressors

Transcription factors which interact with template DNA upstream of promoter region and control when/how much of a gene gets transcribed by interacting with chromatin structure

Activators

• Bind at promoter region, enhancer region, and/or distal sites

• Recruit chromatin remodeling complex to

Co-activators

Instead of binding directly to DNA, they bridge DNA-bound activators. Often play an important role in changing chromatin structure

Repressor modes of action

• competitive DNA binding: bind to activator binding site, disabling recruitment of chromatin remodeling complex

• activator masking: binds to DNA sequence nearby, and interacts with activator, rendering it unable to carry out its functions

• direct GTF contact: binds to DNA and also to Basal Apparatus, preventing it from moving into elongation

• independent actions: compress nucleosomes, recruit methylases and de-acetylases to make genes inaccessible

Mediator complex

20+ subunit complex that acts like a big coactivator which connects activators to GTFs

Interferon example

During a viral infection, TLR signal transduction pathways result in the release of 3 activators: NFkB, IRF-3/7, and Jun/ATF. These facilitate transcription of IFN-B, which is secreted to signal nearby cells to enter a non-reproductive state, so that they won’t transcribe viral DNA.

Enhanceosome

sequence of DNA-activator complexes that work cooperatively to position RNA pol II. This is verified in that ALL activators are necessary for RNA transcription, which has the effect of reserving transcription for extremely specific conditions.

DNA footprinting assay

• used to identify the target DNA sequence that a known protein binds to

1. Amplify the DNA that contains the target sequence (make many copies), and label one end with a radioactive marker

2. Combine with an excess of your protein (every target sequence is complexed)

3. Combine with cleaving enzymes (cleave at all possible sites, resulting in different lengths of DNA attached to marker)

4. Run gel electrophoresis and observe where the missing length is (this means that the area was covered by the bound enzyme)

Electrophoretic mobility shift assay (EMSA)

• Identify which part of a DNA probe is responsible for binding by mutating different parts of the sequence

• Run gel with:

1. DNA probe only (should see labelled probe band)

2. Excess DNA probe + binding protein (should see probe band and higher band)

3. Competitive DNA + binding protein + teeny probe (should only see faint far band because most of the DNA is complexed = higher and unlabelled = invisible)

4. Excess mutant + probe + protein (if the target sequence is mutated, only probe will bind to protein, and high band will show up. If not, the mutant will bind to the protein and the probe will show a low band)

5. Antibody + Protein + probe (Supershift upwards due to extra bulk of antibody)

Chromatin Immuno-precipitation (ChIP)

• Identify multiple DNA sequences bound by the same protein (ie. in an enhancer/activator region)

1. In cells, Cross-link protein-DNA complex together (covalently) with formaldehyde

2. Lyse cells

3. Add enzymes to break down non-cross-linked DNA

4. Coagulate proteins together using a good antibody

5. Reverse formaldehyde crosslinks and remove protein

6. Amplify DNA fragments via PCR to identify which sequences complex with the protein

CTD tail

52 repeats of a 7 amino acid motif

• recruits capping enzyme for 5’ cap of mRNA (beginning)

• recruits CPSF and CstF for final cleavage and Poly-Adenylation

mRNA capping process

After recruitment of capping enzyme by CTD tail

5’ nucleotide triphosphate is cleaved

2) (nucleophilic?) attack on Guanosine phosphate end, connected 5’ to 5’

3) Guanosine cap is methylated to prevent nuclear degradation

• needed for export from nucleus

• promotes mRNA splicing

• recruits ribosome

• only for RNA pol II

Cleavage and Polyadenylation

• CTD tail recruits CPSF and CstF

• extruding mRNA AAUAAA sequence at the end of the gene is recognized by CPSF and CstF, prompting them to bind and detach from CTD tail

• CTD tail, CPSF and CSTF recruit cleavage factors and PAP (Poly-A binding protein)

• RNA is cleaved, PAP adds around 200 adenine nucleotides

• Disengagement of PAP is not fully known, but its possible that As get degraded (torpedo model)

Splicing overview

1. A, an internal “branch site” nucleotide’s 2’ OH performs back side attack on 5’ exon end to form loop

2. 5’ exon’s 3’ end does a back side attack on 3’ exon end, attaching to the exon and discarding intron lariat

• splice sites are conserved and can be recognized by ribonuceloprotein complexes ie. spliceosome

snRNPs

Small nuclear ribonucleic proteins:

U1/UG: bind to 5’ splice site

U2/BBP: bind to 3’ splice site

Splicing Process

1. U1 binds to 5’ splice site

2. BBP and U2AF bind to 3’ splice site, with BBP attaching to A “branch point”

3. U2 displaces BBP, attaching to the outer edges of branch point area, making the branch point (A) bulge out

4. U4,5,6 connect U1 and U2 making a big bulge, and triggering disengagement of U2AF

5. U1 leaves, replaced by U6

6. U4 disengages, connecting U6 (5’ splice sites) to U2 (3’ splice site), A attacks 5’ splice site forming loop

7. U5 uses ATP to trigger 5’ exon end to attack 3’ exon end and splice together

Splicing evolution

• some introns code for their own splicing out

• probably splicing machinery originated from that

Alternative splicing

• environment and local signals determine which exons join together

• happens in 90% of genes

• multiple proteins can arise from one single gene

SR proteins

serine and arginine rich proteins which bind to exons and recruit spliceosome elements (ESEs = Exonic splicing enhancers)

hnRNPs

heterogenous ribonucleoproteins which bind to RNA splice zones and inhibit splicing (ESS/ISS = Intronic/Exonic splicing silencer)

Splicing repressors and activators

Block or stabilize splicing machinery

General alternative splicing

different exons are joined

Alternative last exons

Different lengths/content of 3’ UTR region

Single nucleotide changes at splice sites

• Exon skipping: nucleotide changes to Splice site causes an exon to be spliced out as an intron

• Cryptic splicing: due to nucleotide changes in a normal exon splice site, a different splice site is used, incorporating new intronic DNA

• Create a new splice site that causes a new exon to be incorporated

STAT-1

• STAT-1 deficiencies cause disruption to interferon response = lethal bacterial and viral infections

• Sequencing of STAT-1 gene and PCR showed: STAT-1 of patient was shorter and missing an exon

• Resulted from a mutation at 5’ splice site of exon 3 (causing it NOT to be spliced in)

miRNAs (microRNAs)

Small ~22nt RNA strands that bind to 3’ UTR regions of mRNA transcripts and cause degradation by AGO RISC complex

miRNA synthesis process

• RNA pol II transcribes Pri-miRNA from introns or exons (due to complimentarity, folds back on itself in a loop

• DROSHA cleaves capped and poly-A ends, turning it into 60-70nt Pre-miRNA

• Exports out of nucleus, taken up by DICER in cytoplasm

• DICER cleaves loop part making it a miRNA duplex

• miRNA complexes with AGO to form RISC, becomes single stranded

• RISC complex binds to mRNA 3’ UTR, recruits other degradation proteins, cleaving enzymes, and translation inhibition structures