Regulatory Mechanisms Flashcards
What is the TBP?
TATA Binding Protein
binds to TATA sequence in the minor groove of DNA. Upon binding it causes a kink in DNA and the structural change helps for preinitiation complex to bind
What proteins are involved in the initiation of transcription? (5)
Transcription Factors (TFs) IID, IIB, IIF, IIE, and IIH
Explain the process of transcription initiation
- TBP (TFIID) binds to TATA.
- Two-second general TF (TFIIB and TFIIA) bind to TBP to form a TBP-TFIIB-TFIIA complex at the promoter.
- TFIIA stabilises TBP DNA binding
- TFIIB serves as bridge to pol II-TFIIF
- TFIIF Stabilises interactions between pol and other factors (TBP) and helps attract TFIIE and TFIIH
- TFIIE attracts and regulates TFIIH
- TFIIH unwinds DNA, and phosphorylates Pol II tail. Phosphorylation is thought to release polymerase from its association with initiation and proceed along the template
Explain the process of transitioning from initiation into elongation
- TFIIH - DNA helicase hydrolyses ATP and unwinds DNA surrounding start site
- RNA pol II synthesises short lengths of RNA
- Period of abortive initiation - refers to the repetitive synthesis and release of short nascent RNAs by RNA polymerase before complex leaves promoter.
- Shifts to elongation when phosphate groups are added to the tail of RNA pol II - CTD (TFIIH phosphorylates Ser5 in the tail)
- Disengages from GTFs
- Acquires more proteins helping transcribe
- Most of GTFs are released - available to initiate another round of transcription
What is a mediator complex?
They act as a ‘mediator’ between transcriptional activators and pol II. Binds pol II and its initiation factors and can enhance recruitment of pol II to activator bound DNA
How does DNA footprinting work?
Agents are used to degrade DNA in the presence of proteins, regions that are protected by proteins are not susceptible to chemical/enzymatic degradation
1. Synthesize or amplify your DNA of interest
2. Label it - radioactivity or fluorescence
3. Incubate with protein
4. Use agents to cleave it - DNAse or Hydroxyl radical
5. Visualize the resulting pattern alongside a sequencing ladder
Determine DNA regulatory sequence to which transcriptional activators/repressors bind by determine the missing length of DNA
How does Electrophoretic Mobility Shift Assay work?
Protein binding to a protein should change the mobility of the DNA on a gel.
If protein binds to the probe, there will be a shift in the location but there will still be some free probes
Radioactive probe is outnumbered by non-labeled competitor so the probe line is thick and there is still binding.
Mutate the DNA where we think it interacts and thus see a shift.
Antibody added to shift even farther (super shift). Validation of binding and location of binding
How does Chromatin Immunoprecipitation work?
Determine all regulatory sequences occupied by a given transcription regulator (under set of conditions/cell types). Can also be used to determine positions along genome bound by modified histones.
1. Cross link protein to DNA so that it will stay covalently attached 2. Lyse cell 3. Break DNA into small fragments (to ensure we all extract DNA that is bound) 4. Precipitate DNA using antibodies against protein. 5. Get rid of crosslink (reverse formaldehyde)and get rid of protein. 6. Amplify the precipitated DNA by PCR
DNA corresponding to those positions in the genome that were occupied by gene regulatory protein in the cells
How are 5’ methylated caps made?
Adding a 5’ cap to mRNA. After 20-40 nucleotides are synthesized, three sequential reactions occur:
1. The 5’ triphosphate of the primary transcript is cleaved
2. Guanosine residue is added via a 5’-5’ linkage
3. Cap guanosine is methylated
Added by enzyme guanyl transferase
What is the purpose of 5’ Caps?
They protect RNA from degradation (by 5’-3’ exonuclease) and promote pre-mRNA splicing. It is needed for export from the nucleus
Acts as quality control of mRNA because with caps mRNA cannot be translated (no recruitment of ribosomes)
can be phosphorylated and dephosphorylated to mediate recruitment
Explain polyadenylation and cleavage of mRNA
CTD tail recruits enzymes necessary for polyadenylation:
CPSF and CstF
RNA will be cut and the position of cut is defined by the polyA signal that’s encoded but the polyA tail is not encoded in the genome. It is added by polyA polymerase and then bound by polyA binding protein
Explain the process of splicing (2)
It is a two-step catalytic process involving the formation of intron lariat. Two transesterification reactions occur:
- Adenine (A) residues’ 2’ OH attacks the phosphodiester bond at 5’ splice site. Cuts the RNA chain and forms 5’-to-2’ bonds (INTRON LARIAT)
- Exposed 3’ - OH upstream of the exon attacks the phosphodiester bond at the 3’ splice site. Joins the two exons
How is the splice site recognised?
Through the partnership between proteisn using guide RNAs (ribonucleoprotein complex)
RNA guides are called U1, U2, U4, U5 and U6. These are called snRNPs (small nuclear ribonucleic proteins). Recognise 5’ splice site and branch site and help catalyse RNA cleavage and joining reactions
Explain how RNA guides recognise splice sites (6)
- U1 Recognises the 5’ splice site
- BBP and U2AF bind branch point on 3’ splice site
- U2 displaces BBP, binds to branch point, makes the “A” bulge
- U4, U5 and U6 join the complex and U6 displaces U1
- U4 is displaced, active site is formed and catalysis happens - catalysis refers to the first two transesterification reactions
- U5 brings 2 exons together, final reaction
What is alternative splicing?
process of selecting different splicing sites to form different combinations of exons.
Can be constitutive where gene locus normally produces multiple protein
Can be also used a regulatory mechanism
