RNA binding proteins Flashcards
____ and ___ are both techniques used to analyse targets of RBPs
CLIP and CRAC
Zero-length crosslinking is
Non chemical crosslinking used to analyse RNA binding sequences. Induced by UV radiation
How are the cDNA transcripts from CRAC analysed?
cross linked nucleotides in cDNA prevents Reverse Transcriptase from progressing, ending the cDNA read. Can also be skipped appearing as sequence errors.
How does the modular structure affect the activity of an RBP?
Combination of RNA binding domains and catalytic domains allow for regulation of enzyme activity. Allows dynamic assembly and disassembly of the RNP complexes.
What is the general structure of RNA Recognition Motif?
RRM has 4 stranded beta sheet and 2 supportive alpha helices. Beta sheet is interaction face.
Discuss RNA binding by the RNA recognition motif
3 or 4 nucleotides are recognized. Mediated by stacking hydrophobic residues, including two conserved hydrophobic residues. Hydrogen bonding between basic residues and phophodiester backbone. Forms salt bridge.
Describe the structure and binding of KH domain
Has 3 alpha helices, 3 beta sheets and central cleft. conserved gxxg loop. 4 nucleotides recognized in the cleft by electrostatic interactions and shape complimentarity.
Describe the interactions between RNA and double stranded RNA binding domain
interacts with dsRNA in base independent manner. IN contact with Phosphate backbone and 2’ OH on ribosyl. Shape complimentarity across 2 minor grooves and major groove.
How is specificity achieved in double stranded RNA binding domain (DRBD)
mutual arrangement of multiple DRBDs in presence of N terminal helix. Binds irregular stem loops in RNA
How many zinc fingers do RNA binding domains have? Which type of finger?
9 C2H2 zinc finger domains
How do the zinc finger domains of TF2A interact specifically with RNA?
Identical residues in the 5th zinc finger H bond with major groove bases of RNA.
How do Zinc Fingers of Ts2d interact?
Bind A/U rich regions of mRNA with H bonding
What does the pumilio family of RNA binding proteins contain?
Multiple tandem puf domains.
What do puf domains contain
3 amino acids, allows recognition of one nucleotide.
