Conformation of the TAR RNA-Arginine Complex by NMR Spectroscopy

Abstract

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.

A conserved pseudouridine modification in eukaryotic U2 snRNA induces a change in branch-site architecture

“…NOEs involving protons in the bulge region of the cBP duplex, particularly those involving H2 protons, were very different from those of the unmodified sequence, pointing out that modification of uridine r pseudouridine in this sequence results in a marked conformational change in the bulged region (Tables 2 and 3)+ Compelling evidence that A24 was extruded from the helix came from NOE cross peaks in this region (Fig+ 3) and the complete lack of any NOEs involving A24 H2, and an i r i ϩ 2 NOE between the flanking bases+ Although the absence of NOEs could be explained by conformational averaging due to motion of the base on the NMR time scale, we do not favor this explanation because the H8 resonance line width of A24 is relatively narrow, and the A24 H2 resonance is only slightly broad+ In addition to NOE data supporting our assignment of A24 H2 and A23 H2 resonances, evidence from biochemical studies suggest that A24 is extrahelical+ The functional groups on the branch-site adenosine (corresponding to A24 in our constructs) that are recognized, and therefore accessible during spliceosome assembly (Query et al+, 1996), are consistent with our structural evidence of the extrahelical A24+ Other sequences in which NOEs between residues flanking bulged bases have been used to define their extrahelical position include the TAR-argininamide complex (in which an i r i ϩ 4 NOE was observed; Puglisi et al+, 1992) and the splice donor site of the SL1 RNA sequence from Caenorhabditis elegans (Greenbaum et al+, 1995(Greenbaum et al+, , 1996+ In addition to adenosine H2 NOE patterns (Table 2), other NOE data support the premise that the branchsite adenosine adopts an extrahelical position in cBP, besides the difference in adenosine H2 NOE patterns (Table 3)+ Base-base NOEs involving aromatic protons of A23, A24, and C25 present in cBP spectra, but not in uBP spectra (Table 3), suggest that these protons either are nearer in the modified construct, or that base motion is more restricted in the more thermally stable cBP duplex+ There is no H5 in a pseudouridine (Fig+ 1D), limiting the number of nonexchangeable NOEs between G5 and c6 (with respect to the number observed for equivalent bases in uBP)+ All resonance shifts of 0+07 ppm or greater occur only in the bulged-base region of the branch-site duplex, that is, between U4-G8 and A23-C25 (Table 1)+ A general trend in the bulge region differences between the two constructs is the small downfield shifting of A23H8, A24H8, C25H6, A23H2, A24H2, and A24H19 on the intron strand upon incorporation of the pseudouridine, suggesting a more exposed environment for these protons+ The upfield shift of A7H2 suggests a more protected environment for that proton, corroborated by the fact that we observe an NOE between U22 NH1 and A7H2 in exchangeable proton spectra of cBP, and not uBP+ The upfield shifts for the H19 of c6 are typical for a pseudouridine (Davis, 1995); therefore, no structural conclusions can be drawn from the widely different shifts for this proton between the two constructs+ The relative weakness of the sequential NOE between A23H8 and U22 H19, compared with its counterpart in uBP spectra, and the absence of an NOE between A7H2 and A23 H19 suggests a tilting of A23 toward C25 in cBP (schematically shown in Fig+ 3)+…”

A novel RNA motif that binds efficiently and specifically to the Tat protein of HIV and inhibits the trans‐activation by Tat of transcription in vitro and in vivo

“…The peaks were assigned to U6 and U22 on the basis of sequential imino±imino cross-peaks in the NOESY spectrum (Katahira et al, unpublished data). These results indicate that U:A:U base triples are formed when RNA Tat binds to CQ, supporting previous proposals (Puglisli et al 1992).…”

HIV-1 TAR RNA Spontaneously Undergoes Relevant Apo-to-Holo Conformational Transitions in Molecular Dynamics and Constrained Geometrical Simulations

“…In this regard, one of the best investigated RNA structures is the HIV-1 transactivation response element (TAR). [2][3][4][5][6] TAR RNA is a 59-nucleotide hairpin structure located at the 5′-end of all pre-mRNA in a HIV-1 transcript. Binding of the regulatory Tat protein to a bulge region within the TAR RNA structure is an essential step for HIV-1 viral replication.…”

“…7 In the search for therapeutic compounds, a promising strategy is to develop small molecules that either inhibit the Tat-TAR interaction or prevent the conformational change to the bound structure. [8][9][10] Currently, six different ligand-bound conformations are structurally known, comprising a conformation bound to a modification of the cognate Tat protein 2,11 and five conformations bound to small molecules that inhibit the TAR-Tat interaction ( Figure 1). 9,[12][13][14] The flexible nature of TAR RNA calls for docking approaches that consider target conformational changes, including backbone motions, when it comes to structurebased drug design.…”