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Structure-based design of a dimeric RNA-peptide complex - PubMed

  • ️Mon Jan 01 2001

Structure-based design of a dimeric RNA-peptide complex

D M Campisi et al. EMBO J. 2001.

Abstract

The arginine-rich RNA-binding domain of bovine immunodeficiency virus (BIV) Tat adopts a beta-hairpin conformation upon binding to the major groove of BIV TAR. Based on its NMR structure, we modeled dimeric arrangements in which two adjacent TAR sites might be recognized with high affinity by a dimeric peptide. Some dimeric RNAs efficiently bound two unlinked BIV Tat peptides in vitro, but could not bind even one monomeric peptide in vivo, as monitored by transcriptional activation of human immunodeficiency virus long terminal repeat reporters. Results with additional reporters suggest that extending the RNA helix in the dimeric arrangements inhibits peptide binding by decreasing major groove accessibility. In contrast, a dimeric peptide efficiently bound an optimally arranged dimeric TAR in vivo, and bound with an affinity at least 10-fold higher than the monomeric peptide in vitro. Mutating specific nucleotides in each RNA 'half-site' or specific amino acids in each beta-hairpin of the dimeric peptide substantially decreased binding affinity, providing evidence for the modeled dimer-dimer interaction. These studies provide a starting point for identifying dimeric RNA-protein interactions with even higher binding affinities and specificities.

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Figures

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Fig. 1. Modeled structures of D0, D1 and D2 dimeric TAR RNAs with bound BIV Tat peptides, based on a single calculated NMR structure of the BIV Tat peptide–TAR complex (Puglisi et al., 1995) (shown on the left). The Cα atoms of Arg70 and Ile79, to be bridged by a linker in the dimeric peptides, are shown as balls. Corresponding RNA secondary structures are also shown, where the boxed regions correspond to the minimal BIV TAR-binding site and important nucleotides defined by mutagenesis (Chen et al., 1994), and the numbers indicate the critical G14:C23 base pair. The BIV Tat peptide sequence used in the NMR structure is shown on the bottom left, with important amino acids defined by mutagenesis (Chen et al., 1995) highlighted in bold.

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Fig. 2. Binding of the BIV Tat(65–81) peptide to dimeric BIV TAR RNAs in vitro. (A) Gel shift assays were performed with 0.02 nM RNA at the peptide concentrations indicated (nM). The 2bTAR RNA consists of two TAR hairpins in which the two lower stems have different sequences and the hairpins are separated by five uracils to help minimize the propensity to form alternative secondary structures. (B) Quantitation of the binding data derived from (A), fit to standard binding isotherms. The fraction of bound RNAs was estimated by measuring the disappearance of the unbound band, as described in Materials and methods.

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Fig. 2. Binding of the BIV Tat(65–81) peptide to dimeric BIV TAR RNAs in vitro. (A) Gel shift assays were performed with 0.02 nM RNA at the peptide concentrations indicated (nM). The 2bTAR RNA consists of two TAR hairpins in which the two lower stems have different sequences and the hairpins are separated by five uracils to help minimize the propensity to form alternative secondary structures. (B) Quantitation of the binding data derived from (A), fit to standard binding isotherms. The fraction of bound RNAs was estimated by measuring the disappearance of the unbound band, as described in Materials and methods.

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Fig. 3. Activation of HIV LTR–CAT reporters containing dimeric BIV TAR RNA sites by the HIV Tat(1–48)–BIV Tat(65–81) fusion protein. (A) The HIV-1 LTR–CAT reporter was constructed with various RNAs in place of HIV-1 TAR located at the 5′ end of the transcript (+1). The reporter RNAs differ slightly from those shown in Figure 1 in that three additional base pairs were inserted into the lower stems to ensure stable hairpin formation in vivo. A schematic of the HIV–BIV Tat fusion protein containing the HIV-1 Tat activation domain fused to the BIV Tat RNA-binding domain is shown. (B) CAT assays with the HIV–BIV Tat fusion protein and various BIV TAR reporters. HeLa cells were co-transfected with 10 ng of the Tat expressor plasmid and 50 ng of each reporter plasmid, and CAT activity was measured after 44 h. The inset shows the raw CAT assay data, with unreacted chloramphenicol (Cm) and acetylated forms of chloramphenicol (Ac) indicated. CAT activity with the bTAR reporter is beyond the linear range of the assay and was repeated with an appropriate amount of extract for quantitation. Fold activation is the level of activity with Tat (+) normalized to the activity of each reporter plasmid alone (–).

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Fig. 4. Activation of HIV LTR–CAT reporters containing RNA sites with extended upper stems by the HIV Tat(1–48)–BIV Tat(65–81) fusion protein. Activity was determined as in Figure 3B.

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Fig. 5. Binding of the BIV Tat(65–81) peptide to BIV TAR RNAs with extended upper stems in vitro. (A) Gel shift assays were performed with 0.02 nM RNA at the peptide concentrations indicated (nM). (B) Quantitation of the binding data in (A), as described in Figure 2. Similar apparent dissociation constants were obtained at 4 and 25°C (data not shown). The RNAs used for the in vitro binding experiments were 3 bp shorter in the lower stem than those used in the in vivo experiments shown in Figure 4.

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Fig. 6. Activation of HIV LTR–CAT reporters containing unpaired nucleotides in the extended BIV TAR stems by the HIV Tat(1–48)–BIV Tat(65–81) fusion protein. The fully paired bTAR5 RNA contains 9 bp in the upper stem. Variable numbers of uridines were inserted at the ‘X’ and ‘Y’ positions, and adenines were inserted at the ‘Z’ position intwo of the RNAs. RNAs are designated (X/Y/Z) by the number of nucleotides inserted at each position. Experiments were performed as described in Figure 3.

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Fig. 7. Binding of the BIV Tat(65–81), BIVdimer(G6) and BIVdimer peptides to dimeric TAR RNAs and RNA mutants in vitro. Gel shift assays were performed with 0.02 nM RNA at the peptide concentrations indicated (nM). D0a contains the G14:C23 to C:G mutation (see Figure 1; Chen et al., 1994) in the upper binding site, D0b contains the mutation in the lower site and D0ab contains the mutation in both sites.

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Fig. 8. Activation of HIV LTR–CAT reporters containing dimeric BIV TAR sites by Tat fusion proteins containing the activation domain of HIV Tat (residues 1–49) fused to monomeric or dimeric BIV Tat peptides. The sequences of the BIV Tat peptides and positions of Arg73 to lysine mutations are shown. Activation using the dimeric reporters indicated below was measured as described in Figure 3, except that 3 ng of each Tat expression plasmid were used in these transfections.

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References

    1. Aboul-ela F., Karn,J. and Varani,G. (1995) The structure of the human immunodeficiency virus type-1 TAR RNA reveals principles of RNA recognition by Tat protein. J. Mol. Biol., 253, 313–332. - PubMed
    1. Battiste J.L., Mao,H., Rao,N.S., Tan,R., Muhandiram,D.R., Kay,L.E., Frankel,A.D. and Williamson,J.R. (1996) α helix major groove recognition in an HIV-1 Rev peptide RRE–RNA complex. Science, 273, 1547–1551. - PubMed
    1. Beerli R.R., Dreier,B. and Barbas,C.F.,III (2000) Positive and negative regulation of endogenous genes by designed transcription factors. Proc. Natl Acad. Sci. USA, 97, 1495–1500. - PMC - PubMed
    1. Bogerd H.P., Wiegand,H.L., Bieniasz,P.D. and Cullen,B.R. (2000) Functional differences between human and bovine immunodeficiency virus Tat transcription factors. J. Virol., 74, 4666–4671. - PMC - PubMed
    1. Cai Z., Gorin,A., Frederick,R., Ye,X., Hu,W., Majumdar,A., Kettani,A. and Patel,D.J. (1998) Solution structure of P22 transcriptional antitermination N peptide–box B RNA complex. Nature Struct. Biol., 5, 203–212. - PubMed

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