Recognition of RNA by the p53 tumor suppressor protein in the yeast three-hybrid system.

The p53 tumor suppressor protein is a homotetrameric transcription factor whose gene is mutated in nearly half of all human cancers. In an unrelated screen of RNA/protein interactions using the yeast three-hybrid system, we inadvertently detected p53 interactions with several different RNAs. A literature review revealed previous reports of both sequence-specific and -non-specific interactions between p53 and RNA. Using yeast three-hybrid selections to identify preferred RNA partners for p53, we failed to identify primary RNA sequences or obvious secondary structures required for p53 binding. The cationic p53 C-terminus was shown to be required for RNA binding in yeast. We show that while p53 strongly discriminates between certain RNAs in the yeast three-hybrid assay, the same RNAs are bound equally by p53 in vitro. We further show that the p53 RNA-binding preferences in yeast are mirrored almost exactly by a recombinant tetrameric form of the HIV-1 nucleocapsid (NC) protein thought to be a sequence-nonspecific RNA-binding protein. However, the possibility of specific RNA binding by p53 could not be ruled out because p53 and HIV-1 NC displayed certain differences in RNA-binding preference. We conclude that (1) p53 binds RNA in vivo, (2) RNA binding by p53 is largely sequence-nonspecific in the yeast nucleus, (3) some structure-specific RNA binding by p53 cannot be ruled out, and (4) caution is required when interpreting results of RNA screens in the yeast three-hybrid system because sequence-dependent differences in RNA folding and display can masquerade as sequence-dependent differences in protein recognition.

Crystal structure of NF-kappaB (p50)2 complexed to a high-affinity RNA aptamer.

We have recently identified an RNA aptamer for the transcription factor NF-kappaB p50 homodimer [(p50)2], which exhibits little sequence resemblance to the consensus DNA target for (p50)2, but binds (p50)2 with an affinity similar to that of the optimal DNA target. We describe here the 2.45-A resolution x-ray crystal structure of the p50 RHR/RNA aptamer complex. The structure reveals that two RNA molecules bind independent of each other to the p50 N-terminal Ig-like domains. The RNA secondary structure is comprised of a stem and a stem-loop separated by an internal loop folded into a kinked helix because of the cross-strand stacking of three internal loop guanines. These guanines, placed at the edge of the 3' helix, together with the major groove of the irregular 3' helix, form the binding surface for p50. Each p50 monomer uses the same surface to recognize the distorted RNA major groove as observed in the kappaB DNA/p50 RHR complex, resulting in strikingly similar interfaces. The structure reveals how the aptamer specifically selects p50 and discriminates against p65. We also discuss the physiological implications of RNA binding by (p50)2.

Yeast genetic selections to optimize RNA decoys for transcription factor NF-kappa B.

In vitro-selected RNA aptamers are potential inhibitors of disease-related proteins. Our laboratory previously isolated an RNA aptamer that binds with high affinity to human transcription factor NF-kappaB. This RNA aptamer competitively inhibits DNA binding by NF-kappaB in vitro and is recognized by its target protein in vivo in a yeast three-hybrid system. In the present study, yeast genetic selections were used to optimize the RNA aptamer for binding to NF-kappaB in the eukaryotic nucleus. Selection for improved binding to NF-kappaB from RNA libraries encoding (i) degenerate aptamer variants and (ii) sequences present at round 8 of 14 total rounds of in vitro selection yielded RNA aptamers with dramatically improved in vivo activity. Furthermore, we show that an in vivo-optimized RNA aptamer exhibits specific "decoy" activity, inhibiting transcriptional activation by its NF-kappaB target protein in a yeast one-hybrid assay. This decoy activity is enhanced by the expression of a bivalent aptamer. The combination of in vitro and in vivo genetic selections was crucial for obtaining RNA aptamers with in vivo decoy activity.

Having it both ways: transcription factors that bind DNA and RNA.

Multifunctional proteins challenge the conventional 'one protein-one function' paradigm. Here we note apparent multifunctional proteins with nucleic acid partners, tabulating eight examples. We then focus on eight additional cases of transcription factors that bind double-stranded DNA with sequence specificity, but that also appear to lead alternative lives as RNA-binding proteins. Exemplified by the prototypic Xenopus TFIIIA protein, and more recently by mammalian p53, this list of transcription factors includes WT-1, TRA-1, bicoid, the bacterial sigma(70) subunit, STAT1 and TLS/FUS. The existence of transcription factors that bind both DNA and RNA provides an interesting puzzle. Little is known concerning the biological roles of these alternative protein-nucleic acid interactions, and even less is known concerning the structural basis for dual nucleic acid specificity. We discuss how these natural examples have motivated us to identify artificial RNA sequences that competitively inhibit a DNA-binding transcription factor not known to have a natural RNA partner. The identification of such RNAs raises the possibility that RNA binding by DNA-binding proteins is more common than currently appreciated.

Binding stoichiometry of an RNA aptamer and its transcription factor target.

RNA molecules serve informational, structural, and catalytic roles in cells. RNA also offers an interesting raw material for the design or genetic selection of modifiers of gene expression. We have been interested in the possibility that natural and/or artificial RNA ligands might be identified for DNA-binding proteins. With these concepts in mind, our laboratory previously isolated a 31-nucleotide RNA aptamer that specifically binds to human transcription factor NF-kappaB. This RNA aptamer (alpha-p50) competitively inhibits DNA binding by NF-kappaB in vitro. The aptamer may target the DNA-binding groove formed by the junction of the two monomers of NF-kappaB, perhaps mimicking kappaB duplex DNA. This model predicts a binding stoichiometry of one RNA aptamer per NF-kappaB dimer. To test this hypothesis, two complementary biophysical methods were utilized. Both analytical ultracentrifugation and microelectrospray mass spectrometry suggest that 1 mol of alpha-p50 RNA binds per mole of NF-kappaB p50 homodimer. Such a result is consistent with the observed ability of the RNA aptamer to block the access of transcription factor NF-kappaB to its binding site on DNA and highlights the question of how an RNA stem-loop structurally mimics a DNA duplex. This work also demonstrates the successful application of mass spectrometry to characterize noncovalent RNA/protein interactions.