Structure of HIV TAR in complex with a Lab-Evolved RRM provides insight into duplex RNA recognition and synthesis of a constrained peptide that impairs transcription.
Belashov IA, Crawford DW, Cavender CE, Dai P, Beardslee PC, Mathews DH, Pentelute BL, McNaughton BR, Wedekind JE
Nucleic Acids Res. 2018 Jul 27;46(13):6401-6415. doi: 10.1093/nar/gky529.
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Plasmids from Article
| ID | Plasmid | Purpose |
|---|---|---|
| 112720 | pET28a_6HT-TBP6.7(WT) | Wild-type TBP6.7 was one of the first evolved TAR-binding proteins to bind with exceptional affinity. It was this protein that was used to obtain co-crystals with WT TAR RNA. |
| 112721 | pET28a_6HT-TBP6.7(P46A) | This mutation (P46A) marks the first residue within the evolved region of U1A. |
| 112722 | pET28a_6HT-TBP6.7(R47A) | Arg47 within TBP6.7 is the most critical residue in terms of forming interactions with WT TAR. Mutating this amino acid to Ala results in ~600-fold reduction in Kd. |
| 112723 | pET28a_6HT-TBP6.7(R47K) | Arg47 of TBP6.7 interacts with Hoogsteen edge of TAR's Gua26. Mutating this amino acid to Lys reduces binding to the WT TAR RNA ~330-fold. |
| 112724 | pET28a_6HT-TBP6.7(Q48A) | Gln48 contacts the backbone of TAR RNA as well as mediates intramoecular interaction to stabilize the conformation of beta2-beta3 loop within TBP6.7. |
| 112725 | pET28a_6HT-TBP6.7(Q48T) | This mutant of TBP6.7 binds to TAR very similarly to TBP6.7(WT), with only 1.4-fold reduced Kd. |
| 112726 | pET28a_6HT-TBP6.7(R49A) | Arg49 of TBP6.7 and other TBPs is the only arginine that was lab-evolved from wild-type U1A to play a significant role in TAR RNA recognition. Mutation results in ~230-fold reduction in Kd. |
| 112727 | pET28a_6HT-TBP6.7(T50A) | Lav-evolved amino acid T50 within TBPs is important in engaging intramolecularly to steer also lab-evolved R49 into conformation compatible binding to RNA. |
| 112728 | pET28a_6HT-TBP6.7(P51A) | Similar to P46, P51 was selected and is present in all evolved TBPs. Mutating this position into Ala has a modest effect on binding. |
| 112729 | pET28a_6HT-TBP6.7(R52A) | 52nd position is Arg in both wild-type U1A and TBPs, but the RNA recognition by this residue in both types of proteins is fundamentally different. |
| 112730 | pET28a_6HT-TBP6.7(Q54A) | Although not evolved, this residue within TBPs participates in stabilizing the polypeptide loop responsible for RNA recognition. |
| 112731 | pET28a_6HT-TBP6.7(delta C-term) | This mutant of TBP6.7 is lacking residues 91-98 at C-terminus, and was used to test the involvement of those residues in TAR recognition. |