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We suggest that this region imposes a flattened curvature on the protein; hence, the requirement for the additional nucleotide.

The principles of FBF/RNA recognition suggest a general mechanism by which PUF proteins recognize distinct families of RNAs yet exploit very nearly identical atomic contacts in doing so. Elements in 3′-UTRs are recognized by regulatory proteins that activate, repress, stabilize, and destroy target m RNAs.

In human PUMILIO1 (PUM1) bound to a nanos response element (NRE) RNA, each PUF repeat interacts with a single RNA base along an extended concave surface (19).

In a typical PUF repeat, the second α-helix provides 3 conserved amino acids that interact with an RNA base.

A crystal structure of the RNA-binding domain of FBF-2 in complex with a 9-nt consensus FBF binding element (FBE), 5′-UGUACUAUA-3′ (20, 28), was determined by single wavelength anomalous diffraction (SAD) and refined to a resolution of 2.2 Å.

The initial experimental electron density map revealed density for all 9 RNA bases [supporting information (SI) Fig. As a convention, we designate the 5′-UGU as positions 1–3 in the recognition sequences. The RNA is colored by atom type (gray, carbon; red, oxygen; blue, nitrogen; orange, phosphorus; yellow, sulfur). ( The lengths of PUF repeats vary in FBF from 38–56 residues.

Two residues contact the edge of the base via hydrogen bonding or van der Waals interactions, and a third often is sandwiched between 2 bases to form stacking interactions.

The identities of the residues contacting the edge of the bases play a key role in selecting the RNA base (19–22).

The PUF protein family includes members throughout eukaryotes.The PUF proteins, FBF-1 and FBF-2, provide a special opportunity to dissect the structural basis of PUF–RNA interactions in a biological context.FBF-1 and FBF-2 are 91% identical in amino acid sequence, have similar RNA-binding specificity, and overlap functionally (8, 23–27); they are referred to collectively as FBF.PUF proteins contain a conserved RNA-binding domain, known as the Pumilio-homology domain or PUF domain.

This domain comprises 8 PUF repeats, flanked by pseudorepeats (8–11).

To date, PUF proteins invariably bind to elements in 3′-UTRs (1).