Evidence for a base triple in the free HIV-1 TAR RNA

Abstract

We propose the existence of a novel base triple in the HIV-1 TAR hairpin. This triple is supported by covariation of loop residue 31 with residue 22, which is part of an unusual base pair with U40 below the 3-nucleotide bulge. A set of mutants was constructed to test the involvement of bases A22, U31, and U40 in a triple interaction. RNA structure probing, trans-activation assays, and structure modeling are consistent with the existence of this base triple in a bent conformation of the free TAR element. However, disruption of the base triple does not affect binding of a Tat-derived peptide. We therefore compared the structure of free and Tat-bound TAR RNA by footprinting and site-specific cross-linking analyses. These studies indicate that the Tat arginine-rich motif, in addition to its known binding site at the bulge, is in close contact with U31 in the TAR loop. Because binding of Tat to TAR is known to coincide with the formation of a base triple with residues U23, A27, and U38, we hypothesize that Tat binding and the associated straightening of TAR triggers the disruption of the (A22-U40)U31 triple.

First Snapshots of the HIV-1 RNA Structure in Infected Cells and in Virions

“…In TAR, the predicted bulges were highly reactive, whereas in the loop, the only highly reactive nucleotide was A34 (Fig. 3), which is in keeping with in vitro chemical probing experiments performed in the absence of proteins (5,35). Nucleotide C23 located in the Tat binding site was strongly modified (Fig.…”

“…3). The in vitro binding of a Tat-derived peptide to this three-nucleotide bulge strongly protected this residue against Me 2 SO 4 modification (35). Thus, our results suggested that Tat and the P-TEFb complex (positive transcription elongation factor complex b) that binds the TAR apical loop during transcription trans-activation (36) do not remain associated with the genomic RNA.…”

Insights into the mechanisms of RNA secondary structure destabilization by the HIV-1 nucleocapsid protein

“…Isothermal calorimetry titrations showed that TAR contained one high affinity site (110 nM), possibly with a second site of intermediate affinity (Heng et al 2012). Kanevsky et al (2005) showed that the apical loop G32 and G33 residue constitute a binding site for NC, this is probably not the case for G34, which has been shown to be involved in a transient cross-loop base pair (Huthoff et al 2004;Lee et al 2014). This finding was supported by the results of biophysical studies on the dynamic properties of the TAR apical loop demonstrating that G32 was highly dynamic (Dethoff et al 2008), constituting a favorable binding site for NC.…”

Thermodynamic examination of 1- to 5-nt purine bulge loops in RNA and DNA constructs

“…In a previous study, we had examined pyrimidine-rich trinucleotide bulge loop in HIV-1 TAR RNA, which gave a DG W 37 loop parameter of 5.94 kcal/ mol for CCC and 7.17 kcal/mol for UCU bulge loop in 1 M KCl in (Carter-O'Connell et al 2008). We are further examining the helical constructs for the trinucleotide bulge loops, as base triple formation is implicated for HIV-1 TAR RNA (Puglisi et al 1992;Huthoff et al 2004). It is possible that structures formed by the trinucleotide bulge loops allow for additional interactions between the bulge nucleotides and the stem leading to a more complex thermodynamic profile.…”

“…For small bulge loops, the unpaired nucleotides can be "flipped out" to allow a continual helical structure to form or can stack within the helix to bend the RNA (Turner 1992;Hermann and Patel 2000). Additional interactions between the bulge nucleotides and helical stems have been seen (Huthoff et al 2004). Bending of the RNA due to adenine or uracil bulge loops has been examined using gel electrophoresis (Bhattacharyya et al 1990), fluorescence resonance energy transfer (Gohlke et al 1994), and transient electric birefringence (Zacharias and Hagerman 1995a,b), among other techniques.…”