MBB 267 Week 8: Mitchel 3 Flashcards
How do homeodomains fold?
catabolite activator protein interact with DNA through a helix-turn-helix motif. The two a-helices are orientated in such a way that the C-terminal recognition helix specifically interacts with nucleotides within the major groove of DNA through ionic and van der Waals interactions, while the N-terminal helix stabilizes this interaction.
What are HoxBox genes?
The homeodomain box (Hox) genes play a key role in anterior- posterior axis development. Hox genes are clustered and their order correlates with their spatial and temporal expression.
What is a leucine zipper?
Leucine zipper domains are extended amphipathic a-helical regions, where one side of the a-helix is polar and the other side is hydrophobic. The hydrophobic surfaces interact with one another to generate a coiled coil structure, where the a-helices are wrapped around each other like the strands of a rope. Leucine zippers contain a leucine residue at every 7th position along the amphipathic helix, the leucine residues being aligned in the three-dimensional structure along the hydrophobic surface of the a-helix
What are basic leucine zipper proteins?
Basic leucine zipper (bZIP) proteins are a large sTF family that comprise of a leucine zipper to allow dimerisation and a region rich in basic residues to allow DNA binding. The extended a-helices interact with nucleotides within the major groove of the DNA.
What are zinc fingers?
Zinc finger protein domains are folded around a central zinc ion through contacts with four amino acid residues, typically either two cysteines and two histidines (C2H2) or four cysteines (C4). Amino acid residues at four specific positions along the recognition helix (-1, 2, 3 and 6 relative to the start of the a-helix) make contacts with nucleotides in a predictable manner. The predictable binding specificity of zinc finger domains allows proteins to be designed that can recognize specific nucleotide sequences. These can then be fused to catalytic domains to target desirable activities (e.g. nuclease, methylase, transcription activation or repression domain) to specific sites within the genome.
How is DNA and RNA interactions with proteins assayed?
DNA (and RNA) binding activity can be assayed using a gel shift or electromobility shift assay (EMSA). Radioactively labelled DNA or RNA is mixed with cell extract or purified protein and resolved by gel electrophoresis – binding of protein to nucleic acid reduces the mobility of the DNA through the gel and is visible as a “gel shift”. A protein in a cell extract that induces a band shift can be identified by incubating the reaction mixture with a protein-specific antibody. Binding of the antibody to the protein/DNA complex generates a larger complex that has an even slower mobility, giving rise to a “supershift”. Band Shift assays can be used to test for biological activity in an extract, to purify a nucleic acid binding protein and to determine the specificity and affinity of a given interaction.
What can RNA-binding proteins impact in a cell?
RBPs can alter;
- RNA processing
- export
- degradation
- localisation
- translation
What is the modular structure of RNPs?
A characteristic of typical RNA-binding proteins is that they contain multiple copies of one or more types of RNA-binding domain. The individual RNA binding domains act cooperatively to provide the specificity and affinity for RNA binding. The contribution of individual domains to RNA binding can be independently regulated, allowing the protein/RNA interaction to be responsive to a wide range of signals.
What is RRM?
-How is RRM work?
The RNA Recognition Motif is one of the most common eukaryotic protein domains.
-The RRM fold comprises of a four-stranded b sheet supported by 2 α helices. RNA binding is mediated by two conserved aromatic residues within the central β sheets that engage with the RNA through base stacking interactions, together with a basic residue that forms a salt bridge with the phosphodiester backbone.
What are KH domains?
-How do KH domains work?
The KH domain is an Ancient ubiquitous RNA Binding Domain
-KH (hnRNP K homology) domains bind both single stranded RNA or DNA. The domain consists of 3 alpha helices and 3 beta sheets, a1 and a2 being separated by the conserved GXXG loop.Four extended nucleotide residues are recognized by electrostatic interactions and shape complementarity (not stacking).
What is synergetic binding in modular RNA-Binding domains?
-Give an example of how it works?
When an RNA binds to a domain, and this interaction catalyses another interaction of an RNA to a domain.
-Puf (Pumilio family) domain proteins regulate mRNA stability & translation by binding U-rich sequences within the 3’ UTR. Puf proteins contain 8 copies of the a-helical Puf domain that stack to form a curved structure.Three hydrophobic, acidic and basic residues within each repeat form base stacking and ionic interactions with each nucleotide.
