Modelo Probit en la Determinación de los Salarios de Eficiencia

D 1:1 RREIIBTR : ZNF28 (15% cobalt, 85% zinc folded)

pH was adjusted and maintained at ~ 7.

The orange boxes highlight the most affected (paramagnetic relaxation broadened) peaks on titration. The G70 resonance is marked with a * since it is a tentative assignment. Figure 3.23 RREIIBTR – ZNF28 (100% cobalt folded). RNA concentration is 100 µM A. Free RREIIBTR, B. 1:1 RREIIBTR : ZNF28 (100% zinc folded), C. 1:1 RREIIBTR : ZNF28 (100% cobalt folded), D. 1:1 RREIIBTR : Cobalt(II) chloride. The pH was adjusted and maintained at ~ 7.

Figure 3.25 provides a better perspective by highlighting the cobalt center in the protein and the bases that show broadening in the imino spectrum (Figure 3.25).

Tertiary structural changes in ZNF on binding RREIIBTR

We have examined the changes in the protein amide backbone on RREIIBTR binding by 15N HSQC NMR spectra. The 15N HSQC for the complex (Figure 3.26B) has broader peaks than its free counterpart (Figure 3.26A), as

Figure 3.25 RREIIBTR - ZNF29G29R binding model.

(A and B) Different perspectives of the RREIIBTR – ZNF29G29R binding model.

This model is based on the data from Figure 3.24. The RNA backbone is in light grey. The RNA residues affected by paramagnetic relaxation, which have been identified in Figure 3.24, are marked in red on the RNA backbone. RNA residues that donot show any effects, and could be identified unambiguously, are marked in lime green on the RNA backbone. The G70 resonance is marked with a * since it is a tentative assignment. The coordinated cobalt is displayed as a blue sphere. This graphic was rendered in iMol (14).

expected. This occurs due to increased rotational correlation time of the RNA bound ZNF, resulting in larger linewidth. An overlay of the free and bound forms of the ZNF is displayed in Figure 3.27. As can be seen, there are no drastic

changes in the spectra, but changes that do occur are mostly limited to the " - helix, which spans residues 17 – 29. The residues in the ! sheet have very minimal changes if any. The only other residue of interest that has changes in its chemical shift is N15 which occupies the tip of the zinc finger. Residues 5, 10,

B Figure 3.26 Protein amide backbone A. 15N HSQC spectra of free ZNF29G29R (fully 15_{N labeled).Assignme-}

nts have been published previously (13).

B. 15N HSQC spectra of ZNF29G29R (fully 15N labeled) bound to unlabeled RNA. Both spectra were acquired by 2D HSQC pulse sequence using 3- 9-19 pulse sequence with gradients (12, 16) A

14, 20, 23, 27 consitute the hydrophobic core of the ZNF. Residues 5, 10, 14 and 20 maintain identical chemical shifts in their free and bound forms while 23 and 27 show minor changes.

Figure 3.27 15N HSQC overlay of free and RREIIBTR bound ZNF29G29R.

Boxes (dashed) mark resonances with large chemical shifts. Black filled triangles mark the residues that constitute the hydrophobic core. Circles (dashed) mark residues with minor chemical shift changes.

Conclusion

Our ZNF proteins bind RREIIBTR at its bulge region, the same location that Rev utilizes (2). However the imino spectrum of RREIIBTR - Rev and RREIIBTR - ZNF29G29R is markedly different indicating a different mode of binding. We observe the changes in chemical shift of U66 and U45 on ZNF binding and note that the phosphates of these nucleotides border the entrance to the major groove of the RNA, at the bulge (Figure 3.29). RREIIBTR – ZNF29G29R binding was found to be specific and had 1:1 stoichiometry. In contrast to other peptides (7) that have been designed to bind RREIIBTR, these

ZNF’s bind only when folded in their !!" motif. The aforementioned designed peptide is a random coin in solution (7) and adopts a secondary (helical) structure only after binding RREIIBTR. We have also established that our ZNF’s utilize amino acids constituting the " helix and the tip of the finger in its molecular interaction with the RNA (Figure 3.28). This is akin to other zinc finger proteins involved in RNA binding. In fact, the starting scaffold for phage display that ultimately resulted in the ZNF’s that we are currently studying (8), the 4th zinc finger of TFIIIA, also uses the same elements of its secondary structure in binding RNA (9,10).

Figure 3.29 RNA phosphate backbone.

(left) RREIIBTR RNA (pdb id 1g70) phosphate backbone at the bulge with residues (U66, U45) that are at the entrance to the major groove (wite arrow). This graphic was rendered in iMol(14).

Figure 3.28 ZNF29G29R residues affected by RNA binding.

ZNF29G29R tertiary structure (free protein pdb) displayed with the amino acids displaying large changes in amide (15N, 1H) chemical shift marked in red, moderate changes in chemical shift marked in blue and no changes in chemical shift in grey. The amino acids whose amide resonances could not be identified in Figure 3.27 are marked in black. The

coordinated zinc atom is in yellow.This graphic was rendered in iMol (14).

U66

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