Three-nucleotide Bulge Research Articles

HIV-1 Tat Peptide Binding to TAR RNA by Electrospray Ionization Mass Spectrometry

Electrospray ionization mass spectrometry (ESI-MS) has been used to study the noncovalent complexes formed from the interaction between HIV-1 Tat peptide and Tat protein with TAR RNA. Both positive ion and negative ion ESI mass spectra showed a maximum stoichiometry of 3:1 between Tat peptide and TAR RNA. However, the higher order complexes only occurred at high relative concentrations of Tat peptide. The 1:1 Tat peptide-TAR RNA complex is believed to involve only specific interactions, whereas the higher order complexes involve nonspecific interactions. Relative binding affinities between Tat peptide and TAR RNA and its various mutants (TAR missing the three-nucleotide bulge, TAR with a poly(ethylene glycol) linker in the bulge region, and TAR with a poly(ethylene glycol) linker in the loop region) can be differentiated by competitive binding experiments and ESI-MS measurements. The gas phase mass spectrometry experiments are consistent with solution phase studies, as they show that mutations in the bulge region reduce TAR RNA affinity to Tat peptide.

Crystallization studies of the human immunodeficiency virus (HIV-1) Tat protein and its trans-activation response element (TAR) RNA.

Small single crystals are reported of a complex between a small peptide fragment of the HIV-1 Tat protein and a fragment of the RNA to which it binds. Tat is responsible for enhancing the level of expression of the human immunodeficiency virus type 1 (HIV-1) and is a logical target for AIDS therapy. Tat may function to increase the level of transcription initiation or to prevent premature termination of transcripts. In vitro, Tat binds through its basic domain (two Lys and six Arg in nine residues) to a three-nucleotide bulge of a stem-loop RNA structure called TAR. Complex formation between Tat and TAR is necessary for Tat activity. Peptides which contain the basic region of Tat also bind to TAR RNA. We have carried out crystallization experiments on a 27-nucleotide fragment of TAR RNA and on complexes between two Tat peptides and TAR.

Role of RNA structure in arginine recognition of TAR RNA.

The human immunodeficiency virus Tat protein binds specifically to an RNA stem-loop structure (TAR) that contains two helical stem regions separated by a three-nucleotide bulge. A single arginine within the basic region of Tat mediates specific binding to TAR, and arginine as the free amino acid also binds specifically to TAR. We have previously proposed a model in which interaction of the arginine guanidinium group with guanosine-26 (G26) and with a pair of phosphates is stabilized by formation of a base triple between U23 in the bulge and A27.U38 in the upper helix. Here we show by NMR spectroscopy that formation of the base triple is critical for arginine binding to TAR. Mutants of TAR that cannot form the base triple or that remove the guanine contact do not bind arginine specifically. These mutants also showed reduced transactivation by Tat. A triple mutant designed to form an isomorphous base triple between C23 and G27.C38 binds arginine and adopts the same conformation as wild-type TAR. These results demonstrate the importance of RNA structure for arginine binding and further demonstrate the direct correspondence between arginine and Tat binding.

Rotational symmetry in ribonucleotide strand requirements for binding of HIV-1 Tat protein to TAR RNA.

Transactivation of human immunodeficiency virus (HIV) gene expression requires binding of the viral Tat protein to a RNA hairpin-loop structure (TAR) which contains a two or three-nucleotide bulge. Tat binds in the vicinity of the bulge and the two adjacent duplex stems, recognising both specific sequence and structural features of TAR. Binding is mediated by an arginine-rich domain, placing Tat in the family of arginine-rich RNA binding proteins that includes other transactivators, virus capsid proteins and ribosome binding proteins. In order to determine what features of TAR allow Tat to bind efficiently to RNA but not DNA forms, we examined Tat binding to a series of RNA-DNA hybrids. We found that only one specific strand in each duplex stem region needs to be RNA, implying that interaction between Tat and a given stem may be solely or predominantly with one of the two strands. However, the essential strand is not the same one for each stem, suggesting a switch in the bound strand on opposing sides of the bulge.

Circular dichroism studies suggest that TAR RNA changes conformation upon specific binding of arginine or guanidine.

Short basic peptides from the HIV Tat protein bind specifically to a bulge region in TAR RNA, with a single arginine residue providing the only sequence-specific contact. The free amino acid arginine also binds specifically to TAR. Previous circular dichroism (CD) experiments suggested that peptide binding induces a conformational change in TAR. Here we confirm this observation using single arginine-containing peptides and show that arginine or guanidine binding also induces a conformational change in TAR. A peptide containing a single arginine within a stretch of histidines (CYHHHRHHHHHA) shows pH-dependent binding and a corresponding change in TAR conformation, as detected by a decrease in the CD signal at 265 nm. Arginine and guanidine, which bind to TAR with apparent Kd's of approximately 1.5 mM, induce similar CD changes. In contrast, lysine, which does not bind specifically to TAR, has no effect. Mutants of TAR that abolish specific binding (a U-->C substitution in the three-nucleotide bulge, a deletion of the bulge, or an A-U to U-A base pair change above the bulge) show no change in the CD signal upon binding of peptides, arginine, or guanidine. The results suggest that binding of a single guanidinium group to a specific site in TAR induces a change in RNA conformation.

Conformation of the TAR RNA-arginine complex by NMR spectroscopy.

The messenger RNAs of human immunodeficiency virus-1 (HIV-1) have an RNA hairpin structure, TAR, at their 5' ends that contains a six-nucleotide loop and a three-nucleotide bulge. The conformations of TAR RNA and of TAR with an arginine analog specifically bound at the binding site for the viral protein, Tat, were characterized by nuclear magnetic resonance (NMR) spectroscopy. Upon arginine binding, the bulge changes conformation, and essential nucleotides for binding, U23 and A27.U38, form a base-triple interaction that stabilizes arginine hydrogen bonding to G26 and phosphates. Specificity in the arginine-TAR interaction appears to be derived largely from the structure of the RNA.

Specific binding of arginine to TAR RNA.

A single arginine residue within the basic region of the human immunodeficiency virus Tat protein mediates specific binding of Tat peptides to a three-nucleotide bulge in TAR RNA. It has been proposed that arginine recognizes TAR by forming a network of hydrogen bonds with two structurally distinct phosphates, an interaction termed the "arginine fork." Here it is shown that L-arginine blocks the Tat peptide/TAR interaction, whereas L-lysine and analogs of arginine that remove specific hydrogen bond donors do not. Experiments using an L-arginine affinity column demonstrate that arginine and the Tat peptides bind to the same site in TAR. Modification of two phosphates located at the junction of the double-stranded stem and bulge and modification of two adenine N7 groups in base-paired regions of TAR interfere with specific arginine binding. The results emphasize the importance of RNA structure in RNA-protein recognition and provide methods to identify arginine-binding sites in RNAs.

Refolded HIV-1 tat protein protects both bulge and loop nucleotides in TAR RNA from ribonucleolytic cleavage

Substantial evidence indicates that HIV-1 trans-activation by tat protein is mediated through the TAR RNA element. This RNA forms a stem-loop structure containing a three-nucleotide bulge and a six-nucleotide loop. Previous mutagenic analysis of TAR indicates that the bulge residues and a 4 bp segment of the stem constitute, in part, the tat binding site. However, there appears to be no sequence-specific contribution of the six-base loop. We have employed a ribonuclease protection technique to explore the interaction of tat with single-stranded regions of TAR. The results indicate that tat interacts with both the bulge and loop regions of TAR. Treatment of TAR RNA with RNase A results in cleavage at U23 and U31, located in the bulge and loop regions, respectively. High concentrations (approximately 2 microM) of Escherichia coli derived tat protein, prepared by standard procedures, gave complete protection of TAR RNA from RNase A cleavage. However, under these conditions, truncated TAR derivatives in which no stem-loop structure is expected to form were also protected, indicating nonspecific binding. In order to obtain a tat preparation with enhanced specificity toward TAR RNA, methods were developed for refolding the recombinant protein. This treatment enhanced the affinity of tat for TAR by approximately 30-fold [Kd(apparent) less than 25 nM] and markedly increased its specificity for the TAR. Again, tat protected TAR RNA from RNase A cleavage at both U23 and U31. Protection was also observed with RNase T1 which cleaves TAR RNA at three G residues in the six-base loop.(ABSTRACT TRUNCATED AT 250 WORDS)