The interaction of the HIV-1 transactivator protein Tat with its transactivation response (TAR) RNA is an essential step in viral replication and therefore an attractive target for developing antivirals with new mechanisms of action. Numerous compounds that bind to the 3-nt bulge responsible for binding Tat have been identified in the past, but none of these molecules had sufficient potency to warrant pharmaceutical development. We have discovered conformationally-constrained cyclic peptide mimetics of Tat that are specific nM inhibitors of the Tat-TAR interaction by using a structure-based approach. The lead peptides are nearly as active as the antiviral drug nevirapine against a variety of clinical isolates in human lymphocytes. The NMR structure of a peptide-RNA complex reveals that these molecules interfere with the recruitment to TAR of both Tat and the essential cellular cofactor transcription elongation factor-b (P-TEFb) by binding simultaneously at the RNA bulge and apical loop, forming an unusually deep pocket. This structure illustrates additional principles in RNA recognition: RNA-binding molecules can achieve specificity by interacting simultaneously with multiple secondary structure elements and by inducing the formation of deep binding pockets in their targets. It also provides insight into the P-TEFb binding site and a rational basis for optimizing the promising antiviral activity observed for these cyclic peptides.NMR ͉ transcription elongation factor-b ͉ antiviral ͉ Tat-TAR interaction ͉ RNA recognition T ranscription of the HIV-1 RNA in infected cells is strongly activated by the complex between the viral Tat protein and its cognate transactivation response (TAR) RNA, a 59-nt RNA found at the 5Ј end of all nascent viral transcripts (Fig. 1A). Tat and its cellular cofactor, the transcription elongation factor-b (P-TEFb), are recruited to the elongating RNA polymerase II (RNAP II) through interactions with TAR and are required for reactivation of the integrated proviral genome in latently infected cells (1). The cooperative binding of Tat and P-TEFb to TAR activates the CDK9 kinase of P-TEFb that phosphorylates RNAP II and the repressive NELF factors, leading to greatly enhanced RNAP II processivity (1-3).The Tat-TAR complex is an attractive target for developing new antivirals because the interaction between Tat and TAR is unique to the virus, whereas P-TEFb is used widely for transcription of most host genes. Furthermore, TAR is extremely conserved among viral isolates and P-TEFb plays a key role in promoting infectivity through the Tat-TAR complex and in the emergence from latency. These considerations have led to the synthesis and evaluation of numerous small-molecule and peptidic inhibitors of the Tat-TAR interaction during the last 15 years (4-7). However, none of these molecules had sufficient potency or selectivity to progress into preclinical studies and, indeed, inhibiting the Tat-TAR interaction has proven challenging. First, there is little precedent for the pharmacological disruption...

Phosphorylation of the C-terminal domain of RNA polymerase II controls the co-transcriptional assembly of RNA processing and transcription factors. Recruitment relies on conserved CTD-interacting domains that recognize different CTD phosphoisoforms during the transcription cycle, but the molecular basis for their specificity remains unclear. We show that the CTD-interacting domains of two transcription termination factors, Rtt103 and Pcf11, achieve high affinity and specificity both by specifically recognizing the phosphorylated CTD and by cooperatively binding to neighboring CTD repeats. Single amino acid mutations at the protein-protein interface abolish cooperativity and affect recruitment at the 3′-end processing site in vivo. We suggest that this cooperativity provides a signal-response mechanism to ensure that its action is confined only to proper polyadenylation sites where Serine 2 phosphorylation density is highest.

We report that the cationic porphyrin TmPyP4, which is known mainly as a DNA G-quadruplex stabilizer, unfolds an unusually stable all purine RNA G-quadruplex (M3Q) that is located in the 5′-UTR of MT3-MMP mRNA. When the interaction between TmPyP4 and M3Q was monitored by UV spectroscopy a 22-nm bathochromic shift and 75% hypochromicity of the porphin major Soret band was observed indicating direct binding of the two molecules. TmPyP4 disrupts folded M3Q in a concentration-dependent fashion as was observed by circular dichroism (CD), 1D 1H NMR and native gel electrophoresis. Additionally, when TmPyP4 is present during the folding process it inhibits the M3Q RNA from adopting a G-quadruplex structure. Using a dual reporter gene construct that contained the M3Q sequence alone or the entire 5′-UTR of MT3-MMP mRNA, we report here that TmPyP4 can relieve the inhibitory effect of the M3Q G-quadruplex. However, the same concentrations of TmPyP4 failed to affect translation of a mutated construct. Thus, TmPyP4 has the ability to unfold an RNA G-quadruplex of extreme stability and modulate activity of a reporter gene presumably via the disruption of the G-quadruplex.