Identification of RNAs that bind to a specific protein using the yeast three-hybrid system.

We have adapted the yeast three-hybrid system to identify RNA ligands for an RNA-binding protein. In this assay system, a protein-RNA interaction is detected by the reconstitution of a transcriptional activator using two hybrid proteins and a hybrid RNA. The RNA molecule is tethered to the promoter of a reporter gene by binding to a hybrid protein consisting of the bacteriophage MS2 coat protein fused to the DNA-binding protein LexA; the RNA-binding domain to be analyzed is fused to the transcriptional activation domain of the yeast Gal4 protein; and the bifunctional RNA consists of binding sites for the coat protein and for the other RNA-binding domain. We built an RNA library such that short fragments of genomic DNA from yeast were transcribed in yeast together with binding sites for the coat protein. We screened this hybrid RNA library for RNAs that bound to the yeast Snp1 protein, a homolog of the human U1-70K protein. The screen yielded as the strongest positive the fragment of U1 RNA that contains loop I, which is known to bind to Snp1 in U1 snRNP. We also identified four other RNA ligands that produced weaker three-hybrid signals, suggesting lower affinities for Snp1 as compared to U1 RNA. In addition, this search also yielded a set of RNA sequences that can activate transcription on their own when bound to a promoter through a protein interaction.

A three-hybrid system to detect RNA-protein interactions in vivo.

RNA-protein interactions are pivotal in fundamental cellular processes such as translation, mRNA processing, early development, and infection by RNA viruses. However, in spite of the central importance of these interactions, few approaches are available to analyze them rapidly in vivo. We describe a yeast genetic method to detect and analyze RNA-protein interactions in which the binding of a bifunctional RNA to each of two hybrid proteins activates transcription of a reporter gene in vivo. We demonstrate that this three-hybrid system enables the rapid, phenotypic detection of specific RNA-protein interactions. As examples, we use the binding of the iron regulatory protein 1 (IRP1) to the iron response element (IRE), and of HIV trans-activator protein (Tat) to the HIV trans-activation response element (TAR) RNA sequence. The three-hybrid assay we describe relies only on the physical properties of the RNA and protein, and not on their natural biological activities; as a result, it may have broad application in the identification of RNA-binding proteins and RNAs, as well as in the detailed analysis of their interactions.

Strand and face: the topography of interactions between the SV40 origin of replication and T-antigen during the initiation of replication.

The mechanism by which a replicator (origin of replication) becomes denatured during the initiation of replication is not understood for any prokaryotic or eukaryotic system. To address this question, we chemically probed the molecular contacts on the SV40 origin of replication (ori) that are used by the SV40 large T-antigen and a single-stranded DNA-binding protein (SSB) during ori denaturation. Prior to the actual denaturation step, the T-antigen double hexamer bound ori utilizing sugar-phosphate contacts that were located on opposite strands in each flanking domain of ori. Each set of flanking phosphate contacts were also located on approximately opposite faces of the ori duplex. While the phosphate contacts had a 2-fold symmetry with respect to the ori center, T-antigen contacts with nucleotide bases were polar with critical interactions detected in only one of the two flanking domains. During origin denaturation catalyzed by T-antigen and a SSB, numerous new contacts to flanking phosphates were observed on the strand not initially bound by T-antigen, suggesting movement of each T-antigen hexamer outward from ori. These data suggest that T-antigen initially binds ori in a manner that facilitates transfer of each T-antigen hexamer to opposite strands during the initiation of SV40 replication.

Strand-specific recognition of a synthetic DNA replication fork by the SV40 large tumor antigen.

The mechanism by which DNA helicases unwind DNA was tested; an "unwinding complex" between the SV40 large tumor antigen (T antigen) and a DNA molecule designed to resemble a replication fork was probed. In an adenosine triphosphate (ATP)-dependent reaction, T antigen quantitatively recognized this synthetic replication fork and bound the DNA primarily as a hexamer. The T antigen bound only one of the two strands at the fork, an asymmetric interaction consistent with the 3'----5' directionality of the DNA helicase activity of T antigen. Binding to chemically modified DNA substrates indicated that the DNA helicase recognized the DNA primarily through the sugar-phosphate backbone. Ethylation of six top strand phosphates at the junction of single-stranded and double-stranded DNA inhibited the DNA helicase activity of T antigen. Neither a 3' single-stranded end on the DNA substrate nor ATP hydrolysis was required for T antigen to bind the replication fork. These data suggest that T antigen can directly bind the replication fork through recognition of a fork-specific structure.

Recognition of model DNA replication forks by the SV40 large tumor antigen.

The ability of the SV40 large tumor antigen (T antigen), a DNA helicase, to bind to model DNA replication forks was tested. DNA fork molecules were constructed either from two partially complementary oligonucleotides or from a single oligonucleotide able to form a 'panhandle' structure. T antigen specifically recognized the two-strand fork in a reaction dependent on the presence of ATP, dATP, or non-hydrolyzable analogs of ATP. T antigen asymmetrically bound the two-strand fork, protecting from nuclease cleavage a fork-proximal region on only one of the two strands. The asymmetric binding is consistent with the 3'-->5' directionality of the DNA helicase activity of T antigen. An analogous region on the one-strand fork was also bound by T antigen, suggesting that T antigen does not require a free single-stranded end to load onto the fork. Use of chemically modified DNA substrates indicated that T antigen binding to the fork utilized important contacts with the DNA sugar-phosphate backbone.