analysis of arginine-rich peptides from the hiv tat protein reveals

Analysis of arginine-rich peptides from the HIV Tat protein reveals unusual features of RNA-protein recognition Barbara J. C a l n a n / Sara Bianca lana/ 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 (HIV) is a potent viral trans-activator (Sodroski et al. 1985a) that is essential for viral replication (Dayton et al. 1986; Fisher et al. 1986). Tat increases the rate of transcription from the HIV long terminal repeat (LTR) (Cullen 1986; Peterlin et al. 1986; Wright et al. 1986; Hauber et al. 1987; Muesing et al. 1987; Rice and Mathews 1988; Laspia et al. 1989) and has also been proposed to increase translational efficiency (Cullen 1986; Feinberg et al. 1986; Rosen et al. 1986; Wright et al. 1986; Muesing et al. 1987; Braddock et al. 1989; Roy et al. 1990b). Several experiments suggest that a major effect of Tat is to increase the efficiency of transcriptional elongation (Kao et al. 1987; Laspia et al. 1989; Selby et al. 1989), and a recent study using intact HIV suggests that this mechanism may account for the entire effect of Tat within the virus (M.B. Feinberg, D. Baltimore, and A.D. Frankel, in prep.). The elongation model is consistent with the observation that Tat acts on nascent RNA transcripts (Berkhout et al. 1989) and is strongly supported by

^Corresponding author.

recent in vitro trans-activation experiments (Marciniak et al. 1990a).

Trans-activation by Tat is dependent on a region near the start of transcription in the viral LTR called the trans-acting responsive (TAR) element (Rosen et al. 1985). TAR RNA forms a stable stem-loop structure (Muesing et al. 1987), and maintaining this structure is important for the Tat response (Feng and Holland 1988; Hauber and Cullen 1988; Jakobovits et al. 1988; Berkhout and Jeang 1989; Garcia et al. 1989; Selby et al. 1989; Roy et al. 1990c). Several experiments support the idea that TAR RNA, and not DNA, is essential for Tat activation (Berkhout et al. 1989; Braddock et al. 1989). TAR contains a 6-nucleotide loop and a 3-nucleotide py-rimidine bulge that are essential for Tat activity. It appears that cellular factors bind to the loop sequence (Gatignol et al. 1989; Gaynor et al. 1989; Marciniak et al. 1990b) and that Tat binds to the bulge (Dingwall et al. 1989; Muller et al. 1990; Roy et al. 1990a; Weeks et al. 1990).

Tat is 86 amino acids long and contains a highly conserved cysteine-rich region (with 7 cysteines in 16 resi-

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Calnan et al.

1 10 20 Met Glu Pro Val Asp Pro Arg Leu Glu Pro Trp Lys His Pro Gly Ser Gin Pro Lys Thr

21 30 40 Ala Cys Thr Asn Cys Tyr Cys Lys Lys Cys Cys Phe His Cys Gin Val Cys Phe lie Thr

Figure 1. Sequence of the HIV-1 Tat pro-• J T- A D • T-l, U • i ^ *^ JJ- 55 55-, ^°

t e r n a n d T A R s i t e . T h e b a s i c r e g i o n or T a t , Lys Ala LOU Gly I lo Ser |Tyr Cly Arg Lys Lys Arg Arg Gin Arg Arg Arg Pro| Pro Gin

corresponding to the Tat 47-58 peptide, is 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 TAR sequence is relative to the start of g gg transcription from the HIV LTR. pro rhr ciy Pro Lys GIU

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dues) and a highly conserved basic region (with 2 lysines and 6 arginines in 9 residues) (Arya et al. 1985; Sodroski et al. 1985b). The cysteine-rich region is essential for Tat function (Garcia et al. 1988; Kubota et al. 1988; Sadaie et al. 1988; Kuppuswamy et al. 1989; Ruben et al. 1989; Rice and Carlotti 1990) and mediates the formation of metal-linked dimers in vitro (Frankel et al. 1988a,b). The basic region is important for nuclear localization (Dang and Lee 1989; Endo et al. 1989; Hauber et al. 1989; Ruben et al. 1989; Siomi et al. 1990) and mediates specific binding to TAR RNA (Weeks et al. 1990). Here we show that the amino acid requirements for specific RNA binding are surprisingly flexible, reminiscent of acidic activation domains of transcription factors, and that the arginine-rich RNA-binding domain of Tat is unstructured in solution and becomes partially or fully structured upon interaction with RNA. We discuss the implications for RNA recognition by arginine-rich domains.

Results

A 12-amino-acid Tat peptide binds specifically to TAR RNA

The TAR site is an RNA stem-loop structure that is located just 3 ' to the start of viral transcription and is necessary for Tat trans-activation (see Fig. 1). Recently, it has been shown that the HIV-1 Tat protein and fragments of Tat containing the basic region bind specifically to TAR RNA (Dingwall et al. 1989; Muller et al. 1990; Roy et al. 1990a; Weeks et al. 1990). We had initially examined the binding of a set of synthetic peptides, used previously to define the regions of Tat required for trans-activation (Frankel et al. 1989), and found that residues 38-58 were sufficient for specific binding to a 57-nucleotide TAR RNA (data not shown). From these results and the results of Weeks et al. (1990), it was clear that RNA binding resided in the basic region. We synthesized a peptide. Tat 47-58, that contained only the 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 stem-loop has been shown by genetic analyses to be sufficient for the Tat response (Hauber and Cullen 1988; Jakobovits et al. 1988). A single band representing the RNA-peptide complex was observed as the concentration of Tat 47-58 in the binding reaction was increased (Fig. 2). By analyzing binding curves we calculated a dis

sociation constant of 6 nM for the complex. This binding constant was confirmed by titrating the peptide at several RNA concentrations (Fig. 2). Clearly, at an RNA concentration above the K^ (10 nM), the fraction of bound RNA was greater than at a concentration below the K^ (2 nM). At high RNA concentrations virtually all of the RNA was bound at a 1 : 1 peptide/TAR stoichiometry (data not shown), suggesting that one peptide binds per TAR molecule. An unrelated arginine-rich peptide, protamine, caused the RNA to precipitate in the wells of the gel and did not give a gel shift at any concentration tested (data not shown). The dissociation constant of the Tat 47-58/TAR complex and the binding stoichiometry are similar to values reported by Weeks et al. (1990).

The specificity of Tat 47-58 for TAR was assessed by comparing binding to wild-type and mutant TAR RNAs. Genetic experiments have shown that the 3-nucleotide pyrimidine bulge in TAR is important for trans-activation (Berkhout et al. 1989; Roy et al. 1990b), and both purified Tat protein and Tat fragments bind specifically to this region (Roy et al. 1990a; Weeks et al. 1990). As shown in Figure 3, Tat 47-58 also shows specificity for the TAR bulge, with at least 20-fold higher specificity for wild-type TAR than for mutant TAR RNAs that contained either a deletion of the 3-nucleotide bulge or a single-nucleotide substitution within the bulge. A 4-nu-

0)

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Figure 2. TAR RNA binding by the Tat 47-58 peptide. Complexes were formed at the indicated peptide/TAR molar ratios and analyzed on 10% polyacrylamide gels as described in Materials and methods. Binding reactions were carried out at two TAR RNA concentrations (2 and 10 nM). The differences in the fraction of RNA bound at these concentrations show that the binding constant falls between these values. Unbovmd TAR RNA is also shown (no peptide).

202 GENES & DEVELOPMENT




RNA recognition by peptides

G G G G

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Figure 3. Binding of Tat 47-58 to TAR RNA mutants. TAR RNAs were transcribed in vitro, and binding to Tat 47-58 was measured by gel shift analysis at the peptide/RNA ratios indicated. Mutants shown are a 4-nucleotide substitution in the loop, which has little effect on binding, and a single-nucleotide substitution and 3-nucleotide deletion of the bulge, which both decrease binding. Unbound TAR RNAs are shown in all cases (0 peptide).

cleotide substitution in the loop had a slight effect on binding (Fig. 3). Changes in the loop have been shown previously to have little effect on the binding of full-length Tat (Dingwall et al. 1989; Roy et al. 1990a) or of other Tat fragments (Weeks et al. 1990) to TAR. Competition experiments using unlabeled wild-type and mutant TAR RNAs provided further evidence for a specific interaction (Fig. 4). Wild-type TAR RNA competed with the peptide-TAR complex at about a fivefold lower concentration than did TAR with the 3-nucleotide bulge deletion. Competition experiments with the single-nucleotide bulge substitution or 4-nucleotide loop substitution confirmed the specificity. Completely unrelated RNAs, such as tRNA, did not compete for Tat 47-58 binding even at a 200-fold excess (data not shown). Thus, Tat 47-58 specifically binds to the bulge in TAR RNA and shows specificity similar to the intact protein (Roy et al. 1990a). A 9-amino acid peptide containing only the basic residues (Tat 49-57) gave identical results (data not shown).

competitor Q 0 G G

3 1* '2 5 15 50 "2 5 15 50'

Figure 4. Specific competition of Tat 47-58 binding by wild-type TAR RNA. Tat 47-58 was bound to 2 nM wild-type ^^P-labeled TAR RNA at a 3 : 1 peptide/TAR stoichiometry (lane marked 47-58). Increasing amounts of unlabeled wild-type or 3 nucleotide bulge deletion TAR RNAs (at the ratios indicated) were added and analyzed on a 10% polyacrylamide gel.

RNA binding of mutant peptides

To define specific amino acids within the basic region that might be important for recognition of TAR, we synthesized a series of mutant Tat 47-58 peptides and measured their affinities and specificities for TAR (Table 1; Fig. 5). We focused primarily on arginine residues because HIV-1 Tat, simian immunodeficiency virus (SIV) Tat, and HIV-1 Rev all contain a basic region characterized by a cluster of arginine residues. It is known that SIV Tat can trans-activate the HIV-1 promoter (Viglianti and Mullins 1988) and that the basic region of Rev can functionally substitute for the Tat basic region (Endo et al. 1989; Subramanian et al. 1990).

Single arginine residues were substituted with alanine residues at positions 52, 53, 55, and 56, and each was found to reduce binding approximately twofold (Table 1; Fig. 5). Substitution of glutamine 54 with alanine had no effect. Substituting pairs of arginine with alanine residues at positions 52 and 53 or positions 55 and 56 reduced binding by >20-fold. Binding was restored to the wild-type level by substituting arginine residues 55 and 56 with lysine instead of alanine residues, whereas substituting arginine residues 52 and 53 with lysine residues restored binding to a level twofold lower than wild type. Replacing any combination of two arginine residues at positions 52, 53, 55, and 56 with alanine residues reduced binding by >20-fold.

Table 1. Dissociation constants of Tat 47-58 and variant peptides

47-58 ala52 ala53 ala54 ala55 ala56

ala52ala53 Iys52lys53 ala55ala56 Iys551ys56 ala52ala55 ala52ala56 ala53ala55 ala53ala56

38-58 62-48 scrambled-1 scrambled-2 SIV 76-91 Rev 34-50

YGRKKRRQRRRP YGRKKARQRRRP YGRKKRAQRRRP YGRKKRRARRRP YGRKKRRQARRP YGRKKRRQRARP

YGRKKAAQRRRP YGRKKKKQRRRP YGRKKRRQAARP YGRKKRRQKKRP YGRKKARQARRP YGRKKARQRARP YGRKKRAQARRP YGRRRRAQRARP

FITKALGISYGRKKRRQRRRP SGQPPRRRQRRKKRG YRKRRQRRGKRP YRKRGRQRRKRP YEKSHRRRRTPKKAKA TRQARRNRRRRWRERQR

K^ (X 1 0 - ' M )

6 10 12

5 12 12

> I 0 0 13

>100 4

>100 >100 >100 > I 0 0

6 3 4 3 6 2

Purified synthetic peptides were bound to wild-type TAR RNA (2 nM) at a minimum of five peptide concentrations. Binding constants were determined by calculating the concentration at which half the RNA was bound using the RNA gel shift assay. Experiments were repeated at least twice for each peptide, and the variation in binding constants between experiments was <20% in all cases.

GENES & DEVELOPMENT 203




Calnan et al.

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Figure 5. RNA binding and native gel of Tat 47-58 and variant peptides. [Top] RNA gel shift of each peptide at a 5 : 1 peptide/ TAR molar ratio (2 nM RNA). The extent of the gel shift is proportional to the peptide molecular weight. [Bottom) Native 20% polyacrylamide gel (pH 4.5) of peptides (2 |j,g each). A single band is observed for each peptide, and each migrates according to its molecular weight.

These results suggested that the overall charge density of Tat 47-58 is important but did not point to specific residues in the peptide as being essential for binding. To examine sequence specificity more closely, v e synthesized another peptide in w^hich the sequence of the basic region was reversed (synthesized from the carboxyl to amino terminus), and two in which the amino acid sequence was scrambled. Remarkably, all three peptides specifically bound TAR with a higher affinity than the wild-type peptide (Table 1; Fig. 5). It should be appreciated that it is not possible to change every residue in a single scrambled peptide because this region is so argin-ine-rich. For example, the scrambled-1 peptide contains a 6-amino acid homology to the wild-type peptide (KRRQRR). This sequence is not present in the scrambled-! peptide (Table 1).

We then asked whether an arginine-rich peptide from SIV Tat, which trans-activates the HIV-1 promoter, could bind to TAR and found that it bound with affinity and specificity equal to that of Tat 47-58 (Table 1; Fig. 5). A similar arginine-rich region from Rev, which can functionally substitute for the HIV-1 Tat basic region (Endo et al. 1989; Subramanian et al. 1990), also bound to TAR RNA with wild-type affinity and specificity (Table I; Fig. 5). Beyond the fact that the SIV Tat and Rev peptides are arginine-rich, they do not share significant sequence homology with the HIV-1 Tat basic region (Table I).

Figure 5 shows the binding of all peptides, at the same concentration, to compare relative affinities and migration. Each peptide-TAR complex resolved in the gel according to its relative peptide molecular weight, and the unbound peptides resolved by weight on native polyacrylamide gels (Fig. 5). It should be noted that Tat 38-58, which contains additional residues between the cys-teine-rich and basic regions of Tat, binds with an affinity equal to that of Tat 47-58. In addition, we measured the relative specificities of all peptides by comparing their binding to the 3-nucleotide bulge deletion and to wild-type TAR RNA. In all cases, binding specificity paral

leled the measured affinities (data not shown). Thus, it appears that most or all of the binding specificity for TAR resides within the basic region of Tat.

Tians-activation activity of basic region mutants

To determine whether RNA binding in vitro is relevant to trans-activation in vivo, we used the mammalian expression vector pSV2tat72 (Frankel and Pabo 1988) to construct a set of plasmids encoding mutant Tat proteins. Single alanine substitutions were introduced at arginine 53, glutamine 54, and arginine 55, and double alanine or double lysine substitutions were introduced at arginine 52/arginine 53 and arginine 55/arginine 56. We also made a mutant containing the scrambled-2 sequence. Plasmids were transfected into HeLa cells that contained an HIV-1 LTR chloramphenicol acetyl transferase (CAT) reporter (HL3TI cells; Felber and Pavlakis 1988), and trans-activation activity was measured. Single arginine to alanine substitutions at positions 53 and 55 reduced Tat activity somewhat (Fig. 6A), which correlated with a twofold decrease in peptide RNA binding (Table 1). The double arginine to alanine substitutions showed much weaker trans-activation activity, with substitution at positions 52 and 53 almost completely eliminating activity (Fig. 6A). These substitutions also reduced RNA binding markedly (Table 1). When arginine residues at positions 52/53 or 55/56 were replaced with lysine instead of alanine residues, trans-activation activity was restored (Fig. 6A), as was RNA binding (Table 1). Interestingly, substituting lysine residues at positions 52 and 53 did not quite restore trans-activation or RNA binding to wild-type levels, whereas substituting lysine residues at positions 55 and 56 completely restored both activities. The scrambled-2 protein showed wild-type trans-activation (Fig. 6A), which correlated with the wild-type affinity of the scrambled-2 peptide (Table 1). Substitution of glutamine 54 with alanine had no effect on trans-activation (Fig. 6A) or RNA binding (Table I).

To assess Tat activity quantitatively, each plasmid was transfected at several concentrations, and trans-activation activity was determined (Fig. 6B,C). Again, trans-activation paralleled RNA binding. Immunopre-cipitation of '^S-labeled extracts from COS-1-transfected cells showed that the level of expression of each protein was similar (data not shown). Trans-activation in the COS-1 cells (measured by cotransfecting Tat plasmids with an HIV-CAT reporter) was the same as in the HeLa cells (data not shown). These results demonstrate a strong correlation between in vitro RNA binding by the arginine-rich Tat peptides and trans-activation activity of the full-length protein in vivo.

Circular dichroism of Tat 47-58 and TAR RNA

As a preliminary examination of the structure of the basic region of Tat, we measured circular dichroism (CD) spectra of the Tat 47-58 peptide. The CD spectrum of the peptide (Fig. 7A) clearly indicates a random coil structure (for basis spectra, see Creighton 1984). We also






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Figure 6. Tra/js-activation by Tat basic region mutants. [A] CAT assay of HeLa cells (containing an HIV-LTR CAT reporter) transfected with 25 ng of each Tat plasmid indicated, adjusted to 1 [ig of total DNA with nonspecific plasmid DNA. Acetylated (ac) and unacetylated (cm) [''*C|chloramphenicol was separated by thin layer chromatography. pSV2tat72 encodes wild-type Tat (residues 1-72) expressed from the SV40 early promoter (Frankel and Pabo 1988). {B,C) HeLa cells were transfected with 1-500 ng of each plasmid and adjusted to 1 jjig of total DNA, and CAT activity was quantitated as described (Frankel et al. 1989). In A, some points are beyond the linear range of the CAT assay; however, for quantition {B and C), assays were repeated with an appropriate amount of extract. [B] (•) Wild type, K^ 6 nM; (•) scrambled-2, K^ 3 nM; (D) ala54, K^ 5 HM; (O) ala53, K^ 12 nM; (A) ala55, K^ 12 nM. (C) (•) Wild type, K^ 6 nM; (A) Iys55lys56, K^ 4 nM; (a) Iys52lys53, K^ 13 nM; (A) ala55ala56, K^ >100 nM; (•) ala52ala53, K^ >100 nM. (Dissociation constants are from Table 1.)

measured spectra of TAR RNA and of the peptide-TAR complex at a 1 : 1 stoichiometry (Fig. 7B). The spectrum of TAR alone is similar to that of other RNAs (Aboul-ela et al. 1988; Daly et al. 1990). The TAR spectrum was subtracted from the spectrum of the complex to give a difference spectrum (Fig. 7C) that contains contributions both from the peptide and from conformational changes in TAR that might have occurred upon peptide binding. The difference spectrum has a minimum near 260 nm, which is almost certainly due to the RNA, as there is no known peptide conformation that contributes to the el-lipticity at this wavelength. There is a second minimum near 216 nm, which probably consists of contributions from both the peptide and a change in RNA conformation. It is not possible to determine how much of the signal is due to peptide and how much to RNA, so we cannot accurately calculate a peptide secondary structure. However, it seems likely that the peptide contributes significantly to the signal, because most peptide conformations show a CD signal between 215 and 225 nm and would cause the observed red shift of the 216 nm minimum (compared to the 212 nm minimum in TAR alone). Furthermore, the 216 nm minimum is much larger than might be expected from changes in RNA alone (compared to the 260 nm minimum).

To assess the specificity of these changes, we measured the CD spectra of Tat 47-58 bound to TAR containing a deletion of the 3-nucleotide bulge. The difference spectrum of the complex was similar to that seen with wild-type TAR RNA, although the intensities of the minima were reduced by —50% (data not shown). We assume that this reduction was not due to incomplete peptide binding because the RNA and peptide concentrations were at least one order of magnitude above the

dissociation constant. Tat 47-58/tRNA complexes were also prepared but were found to be almost completely insoluble. Interestingly, the Tat 47-58/wild-type TAR complexes also became insoluble when the peptide-RNA ratio was >1 : I, suggesting that nonspecific binding occurs at higher stoichiometrics. These solubility properties are similar to those seen with Rev-Rev responsive element (RRE) and Rev/tRNA complexes (Daly et al. 1990). Consistent with nonspecific binding is that protamine/TAR complexes are insoluble. From the CD and solubility data we can tentatively conclude that Tat 47-58 is unstructured by itself, that it becomes partially or fully structured when bound to TAR, and that changes in the RNA conformation occur upon binding. Because some of these properties are also seen with the bulge deletion RNA, at least some changes probably result from nonspecific interaction. More detailed studies using nuclear magnetic resonance (NMR) and x-ray crystallography will be needed to fully determine the nature of these changes.

Discussion

We have shown that a 12-amino-acid arginine-rich peptide from the HIV-1 Tat protein, residues 47-58, binds specifically to TAR RNA but that the precise amino acid sequence necessary for specific RNA recognition is surprisingly flexible; a peptide synthesized in the opposite orientation, from the carboxyl to amino terminus, and peptides with scrambled sequences bind to TAR with affinity equal to the wild-type peptide. The overall charge appears to be essential for RNA binding because replacing single arginine with alanine residues reduces binding by twofold, whereas substituting pairs of argin-





Calnan et al.

o> S' -20000-

^ -30000

-40000

MMMlM*M«i««^a

220 240 260

X (nm)

•O 50000

0

B

V o

240 260

g" -30000-

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200 220 240 260 280 300

X (nm)

Figure 7. Circular dichroism of Tat 47-58 and TAR RNA. [A] Spectrum of Tat 47-58 alone. [B] Spectra of TAR RNA alone (•) and of the Tat 47-58/TAR RNA complex at a 1 : 1 stoichiom-etry (O). (C) Difference spectrum of the spectra shown in B. All values of mean molar ellipticity were normalized to the peptide concentration (3 JJLM) to allow direct comparison of the spectra.

ine with alanine residues reduces binding by >20-fold. When pairs of arginine residues are replaced with lysine instead of alanine residues, RNA binding is restored to near wild-type levels. Not every position in the sequence is equally important because substitution of arginine residues at positions 52 and 53 has a slightly greater effect than substitution of arginines at positions 55 and 56. The specific sequence requirements are subtle, however, because the sequence of the basic region can be scrambled and still give wild-type binding.

Our results show a strong correlation between peptide RNA-binding affinities and trans-activation activity,

supporting other studies suggesting that Tat binding to TAR is important for Tat function (Dingwall et al. 1989; Muller et al. 1990; Roy et al. 1990a; Weeks et al. 1990). This is supported further by the demonstration that RNA binding is important for Tat-mediated trans-activation of transcription in vitro (Marciniak et al. 1990a). Southgate et al. (1990) have shown indirectly that RNA binding is important for Tat function by making a Tat-Rev fusion protein and replacing TAR with the RRE to provide an RNA-binding site for the fusion protein. Only the fusion protein, and not Tat or Rev alone, could trans-activate. Similar results were observed when TAR was replaced with an MS2 coat protein-binding site or an API-binding site (Berkhout et al. 1990; Selby and Peter-lin 1990). These experiments also demonstrate that although Tat binds directly to RNA, the specific Tat-TAR interaction is not essential for trans-activation.

Our results and those of Weeks et al. (1990) suggest that the arginine-rich region of Tat is an independent RNA-binding domain and that Tat contains independent RNA-binding and trans-activation domains. These domains appear to be structurally distinct because proteases readily cleave Tat just amino-terminal to the basic region (Frankel et al. 1988a). Arginine-rich motifs similar to Tat are present in a number of RNA-binding proteins, including antiterminators, Gag proteins, ribosomal proteins, and HIV Rev (Lazinski et al. 1989). Our results are consistent with the proposal that these are specific RNA-binding domains (Lazinski et al. 1989) and suggest that although these domains have significantly different primary amino acid sequences, they may recognize similar RNA structures. This is supported by the findings that an arginine-rich peptide from the HIV Rev protein binds specifically to TAR (Table 1; Fig. 5) and can functionally substitute for the basic region of Tat (Endo et al. 1989; Subramanian et al. 1990), and that the sequence of the Tat basic region can be completely rearranged and still bind and function normally. The similarity of arginine-rich RNA recognition between proteins is further underscored by circular dichroism experiments, suggesting that both the Tat basic region and Rev cause similar conformational changes in the RNA structure upon binding (Fig. 7; Daly et al. 1990). Such changes in RNA structure might influence the binding of cellular proteins to other sites on the RNA.

The basic region of Tat has been shown to be a nuclear localization signal and also targets Tat to the nucleolus (Dang and Lee 1989; Endo et al. 1989; Hauber et al. 1989; Ruben et al. 1989; Siomi et al. 1990; Subramanian et al. 1990). Nucleolar localization correlates well with trans-activation activity. A reasonable interpretation is that nucleolar localization is a consequence of RNA binding; expression of the high levels of Tat needed for immunofluorescence may allow binding to nonspecific RNAs containing TAR-like bulges. Such bulges might be present in ribosomal RNAs that are highly structured (Stem et al. 1989) and are synthesized in the nucleolus. Interestingly, several ribosomal proteins contain arginine-rich regions similar to Tat, and these regions have been proposed to mediate specific RNA binding (Lazin-





RNA lecognition by peptides

ski et al. 1989). This interpretation is consistent with the behavior of a trans-dominant Tat mutant that localizes to the nucleus but not to the nucleolus (Pearson et al. 1990). This protein is truncated at glutamine 54 and therefore is unlikely to bind RNA but presumably can still interact with cellular factors or other Tat molecules in the nucleus to produce the trans-dominant pheno-type. Furthermore, our ala55/ala56 mutant is more active than our ala52/ala53 mutant (Fig. 6A,C), consistent with the idea that basic residues at positions 52 and 53 are involved in nuclear localization in addition to RNA binding.

How do arginine-rich regions interact specifically with RNA? The observations that Tat 47-58 is unstructured when not bound to RNA and that the sequence requirements for RNA binding are highly flexible suggest that the RNA may provide much of the information for recognition. Indeed, mutagenesis experiments show that many mutations abolish TAR function (Hauber and CuUen 1988; Jakobovits et al. 1988; Feng and Holland 1988; Garcia et al. 1989; Selby et al. 1989; Berkhout and Jeang 1989; Roy et al. 1990c), whereas here we show that the basic region of Tat can be completely rearranged and still retain its function. It seems likely that the TAR bulge is a highly structured element that interacts specifically with arginine residues. Other RNA hairpins and bulges are known to form very stable, ordered tertiary structures (Bhattacharyya et al. 1990; Cheong et al. 1990; Puglisi et al. 1990), and in the few well-studied examples of RNA-protein recognition, RNA structure is clearly an important component of the interaction (Romaniuk et al. 1987; Rouid et al. 1989; Stem et al. 1989). "Specific" contacts between Tat and TAR are likely to result from ionic interactions between arginine amino groups and RNA phosphates arranged in a specific tertiary conformation rather than from base-specific hydrogen bonds or other interactions such as those seen in DNA-binding proteins (Jordan and Pabo 1988). The ionic nature of the interaction is supported by arginine to lysine substitutions at positions 52, 53, 55, and 56, which still show specific RNA binding and trans-activation. The only nonbasic amino acid in this region, glutamine 54, seemed a good candidate for a base-specific contact because it is known that glutamine can form specific hydrogen bonds with bases in the major groove of DNA (Jordan and Pabo 1988); however, substituting alanine for this glutamine did not affect binding or trans-activation.

The sequence flexibility shown by these arginine-rich domains is reminiscent of acidic trans-activating domains of transcription factors (Ptashne 1988). Trans-activating domains have been proposed to be unstructured, "negative noodles" (Sigler 1988), which become structured upon interaction with other molecules, presumably components of the transcription apparatus. Our circular dichroism results suggest that the Tat peptide is unstructured when not bound to RNA and becomes partially or fully structured upon binding, suggesting that arginine-rich RNA-binding domains may interact with RNA in an analogous manner. "Induced" structure is also seen in the DNA-binding domains of leucine zipper

proteins. Several recent studies have shown that the basic DNA-binding domain adopts an a-helical structure upon specific DNA binding (O'Neil et al. 1990; Talanian et al. 1990; Weiss 1990). Thus, it appears that interaction of protein domains with other proteins, RNA, or DNA can all induce conformational changes required for specific recognition.

Materials and methods

Peptide synthesis, purification, and analysis

Syntheses were performed using Fmoc chemistry on a Milli-Gen/Biosearch model 9600 peptide synthesizer with a peptide amide linker-norleucine-4-methylbenzyhydrylamine (Pal-Nle-MBHAj polystyrene resin (MilliGen/Biosearch, 0.5 gram). The benzotriazolyloxytris (dimethylamino)phosphonium hexa-fluorophosphate/l-hydroxybenzotriazole (BOP/HOBt) and N-methylmorpholine/DMF coupling method was used with coupling times of 1-4 hr. Protecting groups were 2,2,5,7,8-penta-methyl chroman-6-sulfonyl (Arg), t-butyloxycarbonyl (Lys), trityl (Gin, Asn, His), t-butyl (Ser, Thr, Tyr), and t-butyl ester (Glu). All peptides were synthesized as their carboxy-terminal amides. After syntheses were completed, protecting groups were removed and the peptide chains were cleaved from the resin with trifluoroacetic acid/thioanisole/ethanedithiol/anis-ole [90 : 5 : 3 : 2 (vol/vol)] for 2 hr. The mixtures were filtered into cold anhydrous diethyl ether, and the precipitated peptides were filtered, washed with additional ether, and dried.

Amino acid composition was determined by hydrolysis in 6 M HCl containing 0.5% phenol at 110°C and analysis on an LKB 4151 Alpha Plus analyzer. Peptides were purified on a C4 reverse-phase HPLC column (Vydac) using an acetonitrile gradient of 0.2%/min in 0.1% trifluoroacetic acid. Peptide absorption spectra were recorded, and concentrations of most peptides were determined by tyrosine absorbance at 278 nm (e = 1420 M ' cm" '; Creighton 1984). The concentration of the Rev peptide was determined by trytophan absorbance at 278 nm (e = 5600 M " 'cm~ '; Creighton 1984) and the concentration of Tat 62—48 by peptide absorbance at 229 nm using Tat 47-58 as a standard. Purity and concentrations were confirmed by native gel electrophoresis (20% polyacrylamide in 30 mM sodium acetate at pH 4.5), and peptides were visualized by Coomassie blue staining. The mass of several peptides was confirmed by fast atom bombardment (FAB) mass spectrometry (University of California, Berkeley) as described previously (Frankel et al. 1989).

RNA synthesis and purification

TAR RNAs were transcribed in vitro by T7 RNA polymerase from synthetic oligonucleotide templates containing a 17-base double-stranded T7 promoter and single-stranded TAR sequences coding for sense TAR RNAs. All RNAs contained GG at their 5' end, which increases the efficiency of transcription (Milligan and Uhlenbeck 1989) and CC at the 3' end to base-pair with the Gs. Wild-type TAR RNA contained sequences -I-18 to -1-44 of the HIV LTR TAR site (see Fig. 1). Mutant TARs also contained this sequence but with substitutions or deletions as highlighted in Figure 3. All RNAs were purified on 10% poly-acrylamide/8 M urea gels, eluted from gels in 0.5 M ammonium acetate, 10 mM magnesium acetate, 1 mM EDTA, and 0.1% SDS, extracted twice with phenol, and ethanol-precipitated. Purified RNA was resuspended in sterile deionized water. The concen-

GENES &, DEVELOPMENT 207




Calnan et al.

trations of radiolabeled RNAs were determined from the specific activity of [^^P]CTP incorporated into the transcripts. Unlabeled RNA was quantitated by spectrophotometry.

RNA-binding gel mobility shift assays

Peptide and RNA were incubated together for 10 min on ice in lO-jil binding reactions containing 10 mM Tris-HCl (pH 7.5), 70 mM NaCl, 0.2 mM EDTA, and 5% glycerol. To determine binding constants, 2 nM radiolabeled wild-type TAR RNA was titrated with peptide. At least five concentrations of a given peptide were used to determine the binding constant of the peptide. Peptide-RNA complexes were resolved on 10% polyacryl-amide, 0.5 x TBE gels that had been prerun for 1 hr and allowed to cool to 4°C. Gels were electrophoresed at 200 V for 3 hr at 4°C, dried, and autoradiographed. A beta scanner was used to quantitate peptide-bound RNA and free RNA.

Construction of mutant plasmids, CAT assays, and imm unoprecipitation

Oligonucleotide cassettes containing the basic region mutants were synthesized and cloned into the tat gene of pSV2tat72 (Frankel and Pabo 1988). Mutations were confirmed by dideox-ynucleotide sequencing (Sequenase; U.S. Biochemicals). Various amounts of plasmid were transfected into HeLa cells or COS-1 cells (50% confluent in 25-mm wells) by lipofection (Feigner et al. 1987), cells were harvested 48 hr after transfection, and CAT activity was assayed and quantitated as described (Frankel et al. 1989). In all cases, plasmid concentrations were adjusted to 1 .g of total DNA with nonspecific pBR322 DNA. Cells were grown in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum and penicillin/streptomycin. For immunoprecipitations, COS-1 cells (50% confluent in 35-mm dishes) were transfected with the various plasmids for 48 hr, cells were incubated with serum-free medium containing [''^S]cysteine for 4 hr, and Tat was immunoprecipitated with a rabbit polyclonal antibody (Aldovini et al. 1986) and analyzed as described (Cullen 1987).

Circular dichroism

Circular dichroism spectra were measured using an Aviv model 60DS spectropolarimeter, equipped with a Hewlett-Packard Peltier temperature controller. Spectra were recorded from 300 to 200 nm and averaged over five scans, with a 10 sec averaging time at each wavelength. Samples were prepared in 10 mM potassium phosphate buffer (pH 7.0) and 70 mM potassium fluoride. Concentrations of peptide and TAR RNA were determined by absorption spectra. Samples were prepared in a 1-cm path-length cylindrical cuvette, and all spectra were recorded at 5°C. Mean molar residue ellipticity was calculated using a molecular mass for Tat 47-58 of 1657 daltons, and all spectra, including the TAR RNA, were normalized using these values to allow direct calculation of the difference spectrum.

Acknowledgments

We thank David Mann for help with CAT assays, Chris Siebel and Don Rio for T7 RNA polymerase, and Don Rio, Peter Kim, Carl Pabo, Stephen Fiarrison, Phillip Sharp, Alan Sachs, Douglas Vollrath, Mark Feinberg, and Robert Talanian for interesting discussions and helpful comments. This work was supported by the Lucille P. Markey Charitable Trust and by National Institutes of Fiealth National Institute of Allergy and Infectious Diseases grant AI29135 (A.D.F.).

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.

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