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Analysis of Arginine-Rich Peptides from the HIV Tat Protein Reveals Unusual Features of RNA-Protein Recognition

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Analysis of Arginine-Rich Peptides from the HIV Tat Protein Reveals Unusual Features of RNA-Protein Recognition Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press Analysis of arginine-rich peptides from the HIV Tat protein reveals unusual features of RNA-protein recognition Barbara J. Calnan/ Sara Biancalana/ Derek Hudson,^ and Alan D. Frankel^'^ 'whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142 USA; ^MilliGen/Biosearch, Novate, California 94949 USA Arginine-rich sequences are found in many RNA-binding proteins and have been proposed to mediate specific RNA recognition. Fragments of the HIV-1 Tat protein that contain the arginine-rich region of Tat bind specifically to a 3-nucleotide bulge in TAR RNA. To determine the amino acid requirements for specific RNA recognition, we synthesized a series of mutant Tat peptides spanning this domain (YGRKKRRQRRRP) and measured their affinity and specificity for TAR RNA. Several corresponding mutations were introduced into the full-length Tat protein, and trans-activation activity was measured. Systematic substitution of arginine residues with alanines or lysines suggested that overall charge density is important but did not point to any specific residues as being essential for binding. A glutamine-to-alanine substitution had no effect on binding. Remarkably, peptides with scrambled or reversed sequences showed the same affinity and specificity for TAR RNA as the wild-type peptide. Trans-activation activity of the mutant Tat proteins correlated with RNA binding. Arginine-rich peptides from SIV Tat and from HIV-1 Rev, which can functionally substitute for the basic region of HIV-1 Tat, also bound specifically to TAR. Circular dichroism spectra suggest that the arginine-rich region of Tat is unstructured in the absence of RNA, becomes partially or fully structured upon binding, and induces a conformational change in the RNA. These results suggest that arginine-rich RNA-binding domains have considerable sequence flexibility, reminiscent of acidic domains found in transcriptional activators, and that RNA structure may provide much of the specificity for the interaction. [Key Words: HIV Tat; viral trans-activator-, RNA-binding protein; TAR RNA; arginine-rich motif; peptide structure] Received October 31, 1990; revised version accepted December 4, 1990. The Tat protein from human immunodeficiency virus recent in vitro trans-activation experiments (Marciniak (HIV) is a potent viral trans-activator (Sodroski et al. et al. 1990a). 1985a) that is essential for viral replication (Dayton et al. Trans-activation by Tat is dependent on a region near 1986; Fisher et al. 1986). Tat increases the rate of tran­ the start of transcription in the viral LTR called the scription from the HIV long terminal repeat (LTR) trans-acting responsive (TAR) element (Rosen et al. (Cullen 1986; Peterlin et al. 1986; Wright et al. 1986; 1985). TAR RNA forms a stable stem-loop structure Hauber et al. 1987; Muesing et al. 1987; Rice and (Muesing et al. 1987), and maintaining this structure is Mathews 1988; Laspia et al. 1989) and has also been pro­ important for the Tat response (Feng and Holland 1988; posed to increase translational efficiency (Cullen 1986; Hauber and Cullen 1988; Jakobovits et al. 1988; Feinberg et al. 1986; Rosen et al. 1986; Wright et al. Berkhout and Jeang 1989; Garcia et al. 1989; Selby et al. 1986; Muesing et al. 1987; Braddock et al. 1989; Roy et 1989; Roy et al. 1990c). Several experiments support the al. 1990b). Several experiments suggest that a major ef­ idea that TAR RNA, and not DNA, is essential for Tat fect of Tat is to increase the efficiency of transcriptional activation (Berkhout et al. 1989; Braddock et al. 1989). elongation (Kao et al. 1987; Laspia et al. 1989; Selby et al. TAR contains a 6-nucleotide loop and a 3-nucleotide py- 1989), and a recent study using intact HIV suggests that rimidine bulge that are essential for Tat activity. It ap­ this mechanism may account for the entire effect of Tat pears that cellular factors bind to the loop sequence within the virus (M.B. Feinberg, D. Baltimore, and A.D. (Gatignol et al. 1989; Gaynor et al. 1989; Marciniak et al. Frankel, in prep.). The elongation model is consistent 1990b) and that Tat binds to the bulge (Dingwall et al. with the observation that Tat acts on nascent RNA tran­ 1989; Muller et al. 1990; Roy et al. 1990a; Weeks et al. scripts (Berkhout et al. 1989) and is strongly supported by 1990). Tat is 86 amino acids long and contains a highly con­ ^Corresponding author. served cysteine-rich region (with 7 cysteines in 16 resi- GENES & DEVELOPMENT 5:201-210 © 1991 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/91 $1.00 201 Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press Calnan et al. +310 G+34 1 10 20 C A C G Met Glu Pro Val Asp Pro Arg Leu Glu Pro Trp Lys His Pro Gly Ser Gin Pro Lys Thr G C 21 30 40 A U Ala Cys Thr Asn Cys Tyr Cys Lys Lys Cys Cys Phe His Cys Gin Val Cys Phe lie Thr c" I Figure 1. Sequence of the HIV-1 Tat pro- o • J T- A D • T-l, U • i^ *^ JJ- 55 55-, ^° +23 AU tern and TAR site. The basic region or Tat, Lys Ala LOU Gly Ilo Ser |Tyr Cly Arg Lys Lys Arg Arg Gin Arg Arg Arg Pro| Pro Gin G C corresponding to the Tat 47-58 peptide, is A U C G highlighted. The numbering shown for the Gly Ser Gin Thr His Gin Val Ser Leu Ser Lys Gin Pro Thr Ser Gin Ser Arg Gly Asp +18;C' G^ TAR sequence is relative to the start of g^ gg G C transcription from the HIV LTR. pro rhr ciy Pro Lys GIU G C dues) and a highly conserved basic region (with 2 lysines sociation constant of 6 nM for the complex. This binding and 6 arginines in 9 residues) (Arya et al. 1985; Sodroski constant was confirmed by titrating the peptide at sev­ et al. 1985b). The cysteine-rich region is essential for Tat eral RNA concentrations (Fig. 2). Clearly, at an RNA function (Garcia et al. 1988; Kubota et al. 1988; Sadaie et concentration above the K^ (10 nM), the fraction of bound al. 1988; Kuppuswamy et al. 1989; Ruben et al. 1989; RNA was greater than at a concentration below the K^ (2 Rice and Carlotti 1990) and mediates the formation of nM). At high RNA concentrations virtually all of the metal-linked dimers in vitro (Frankel et al. 1988a,b). The RNA was bound at a 1 : 1 peptide/TAR stoichiometry basic region is important for nuclear localization (Dang (data not shown), suggesting that one peptide binds per and Lee 1989; Endo et al. 1989; Hauber et al. 1989; TAR molecule. An unrelated arginine-rich peptide, prot­ Ruben et al. 1989; Siomi et al. 1990) and mediates spe­ amine, caused the RNA to precipitate in the wells of the cific binding to TAR RNA (Weeks et al. 1990). Here we gel and did not give a gel shift at any concentration show that the amino acid requirements for specific RNA tested (data not shown). The dissociation constant of the binding are surprisingly flexible, reminiscent of acidic Tat 47-58/TAR complex and the binding stoichiometry activation domains of transcription factors, and that the are similar to values reported by Weeks et al. (1990). arginine-rich RNA-binding domain of Tat is unstruc­ The specificity of Tat 47-58 for TAR was assessed by tured in solution and becomes partially or fully struc­ comparing binding to wild-type and mutant TAR RNAs. tured upon interaction with RNA. We discuss the impli­ Genetic experiments have shown that the 3-nucleotide cations for RNA recognition by arginine-rich domains. pyrimidine bulge in TAR is important for trans-activa­ tion (Berkhout et al. 1989; Roy et al. 1990b), and both purified Tat protein and Tat fragments bind specifically Results to this region (Roy et al. 1990a; Weeks et al. 1990). As A 12-amino-acid Tat peptide binds specifically to TAR shown in Figure 3, Tat 47-58 also shows specificity for RNA the TAR bulge, with at least 20-fold higher specificity for wild-type TAR than for mutant TAR RNAs that con­ The TAR site is an RNA stem-loop structure that is tained either a deletion of the 3-nucleotide bulge or a located just 3' to the start of viral transcription and is single-nucleotide substitution within the bulge. A 4-nu- necessary for Tat trans-activation (see Fig. 1). Recently, it has been shown that the HIV-1 Tat protein and frag­ ments of Tat containing the basic region bind specifi­ cally to TAR RNA (Dingwall et al. 1989; Muller et al. 0) 1990; Roy et al. 1990a; Weeks et al. 1990). We had ini­ t 47-58/TAR tially examined the binding of a set of synthetic peptides, 1 g 1 2 3 5 10 used previously to define the regions of Tat required for trans-activation (Frankel et al. 1989), and found that res­ idues 38-58 were sufficient for specific binding to a 57- 2nM nucleotide TAR RNA (data not shown). From these re­ sults and the results of Weeks et al. (1990), it was clear that RNA binding resided in the basic region. We syn­ thesized a peptide. Tat 47-58, that contained only the lOnM basic region of Tat (Fig. 1) and tested it for binding to a 31 nucleotide TAR RNA (Fig. 1). This top part of the TAR Figure 2. TAR RNA binding by the Tat 47-58 peptide.
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