Bonev - Protein complexes Flashcards
What is the motif found at promoter regions upstream of the coding gene?
TATA box
There are three pathways following the initial binding of the trnacription factor to the promoter region, what are they?
- Binding of a secondary TF protein to the initial TF
- Binding of a secondary TF protein to DNA adjacent to the promoter region
- Secondary TF binds to a region further away from the promoter region, possibly at -75. This can cause the DNA to bend.
RNA Pol2 requires TFII, where does TFII bind at?
-10 position promoter site
How is the TFII able to recognise the TATA box?
It contains TBP (TATA box Binding Proteins)
What do these TBPs do?
Bend and unwind the protein to allow further proteins to act on the DNA
How does this bending of DNA help TFII(D)?
The bending allows the TFIID to come into closer proximity with the specific TF found at the enhancer and this causes a protein-protein interaction to initiate transcription
TFIID, where does it recognise DNA?
In the minor groove
Why is this important?
It leaves the major groove available for other proteins to act on the DNA
Which fits into the minor groove of DNA, beta sheets or alpha helix?
Beta sheets
TFIID contains Phenylalanine, what is the role of this aromatic amino acid?
Phe intercalates between specific bases of DNA and forms ring-ring hydrophobic interactions. This stabilises the interaction between the DNA promoter region and TFIID. Bending at about 110º
What does the binding of TFIID to the minor groove help RNA Pol2 to do?
RNA Pol2 can then bind to DNA at the major groove
Which two transcription factors are involved in the switching of lyosegnic state to lytic state in the E.coli when it has been infected by a bacteriophage?
The lambda repressor and Cro
How does lambda repressor transcription factor work?
It binds to the operator sites of the lambda DNA which prevents the expression of genes necessary for the lytic cycle. It blocks the promoter and prevents the RNA Polymerase from working. Therefore, it remains in the lysogenic cycle.
How does cro work?
Cro protein binds to the same operator sites as the lambda repressor and outcompetes it and disrupts its binding. This means RNA Polymerase can initiate transcription and the lytic cycle can start properly.
How does lambda repressor bind to DNA?
Using the helix-turn-helix motif
How is the stability of the HTH motif maintained?
Hydrophobic interactions between the surfaces of the two helices between each other
Where is the binding to the DNA by the HTH motif?
At the major groove. Alpha helices is 12º in size, major groove is 12º in width, making it a good fit.
How is the HTH motif able to recognise and be specific to the major groove?
Hydrogen bonding between the sidechains of residues in the helix, and the exposed portions of base pairs found at the major groove DNA
What is the approximate distance between the helices when they bind at the major groove?
34 angstroms to match the pitch of the DNA.
One helices will be directly specific and bind to the bases. The other will bind to the backbone and provide stability. This will allow the incorporation of other transcription factors.
Which operator regions do the cro and lambda repressor have affinity for respectively?
cro = OR3
lambda = OR1
Are zinc fingers more commonly found in eukaryotes or prokaryotes?
Eukaryotes
Which two amino acid residues coordinate the Zn2+ ion in zinc finger motifs?
2 cysteines and 2 histidines
How does the zinc finger bind to the DNA?
Alpha helix within its structure bind to the major groove
How do the beta sheets and loop connect to the alpha helix?
Through the Zn2+ ion connecting 2 His and 2 Cys
Arg and Lys interact with the phosphates to create more stability to allow the protein to slide down the DNA until it finds the correct binding
In yeast, what is GCN4 TF an example of?
Leucine zipper
In yeast, what is GCN4 TF an example of?
Leucine zipper
What is its role?
To shift the metabolism so that yeast can survive in amino acid deprived environments
GCN4 binds to TFIID at the TATA box to initiate transcription
What is the structure of the leucine zipper?
Heptad organisation, an a-helical coiled coil. Has a pair of DNA binding domains.
How often is leucine present in the structure across 35 residues?
n, n+7. When facing each other, they provide a hydrophobic interface between helices.
How is the overall stability and compactness of the coiled coil structure maintained by specific residues?
Asn-Asn, His-His, Thr-Thr, form hydrogen bonds between each other on opposite helices. Thr-Thr can form a bond with a water molecule.
Which two amino acid residues are able to bind to the phosphates of DNA to increase binding?
Argenine and Lysine
cAMP binds to protein kinase A (PKA) which is able to bind to transcription factors. CREB (cAMP-response element binding protein) contain leucine zipper motifs. CREB binds to the target DNA sequence and dimerises through the leucine zipper. CREB can bind to DNA sequences once phosphorylated, these sequecnes are CREs (cAMP response elements).
How many helices are present in the homeodomain?
3 helices. Helix 1+2 are antiparallel, helix 3 is perpendicular to both. The hydrophobic face of helix 3 packs against helix 1+2 to form the interior of the protein.
What is the role of the N-terminal in the homeodomain?
It binds to the minor groove
What are the three amino acid residues present at the N-terminal?
RGR = Arginine, Glycine, Arginine
What is the role of these residues?
Arginines engage with both strands of the DNA by interacting with the phosphates. The gylcine allows flexibility for the arginines to find the phosphates at the minor groove.
Which helix is the recognition helix?
Helix 3
Which base does arginine have two hydrogen bonds with?
Guanine. Also has hdrophobic contacts with thymine.
Which based does glutamine bind to in lambda repressor / DNA complex?
Adenine. A second glutamine binds to a phosphate.
Which tRNA is used as the first one for translation?
Formyl-methionine (tRNAfmet)
Why?
Becuase it is able to distinguish between itself as the start codon and the internal methionine
What is co-precipitation of RNA with specific RNA-binding proteins?
Antibodies specific to the RBPs or other affinity-based methods. This allows the isolation of any RBPs and RNA molecules associated with it.
What is UV crosslinking of RNA-binding proteins?
UV irradiation forms a covalent bond between RNA and RBPs. Undergo mass spectrometry to determine mRNA interactions.
What can lysine and arginine residues in the transcription factors do to help with transcription?
They are positively charged amino acids which can bind to the phosphate backbone via electrostatic interactions which increase the stability of the TF-DNA complex
