A one-hybrid system for detecting RNA-protein interactions.
BACKGROUND
mRNA translation, stability, and localization are controlled by regulatory proteins that bind to specific RNA motifs. Since biochemical isolation of such proteins has often proven to be difficult, a genetic system for studying RNA-protein interactions would be of great utility in the identification of novel RNA binding proteins and in understanding how these proteins recognize particular RNA sequences. The bacteriophage lambda gene product N protein is a sequence specific RNA binding protein that when bound to its target sequence allows RNA polymerase to ignore transcription termination signals. The fact that the binding of N protein to RNA is directly coupled to gene expression suggests that N protein could be used to develop a general system for studying RNA-protein interactions.


RESULTS
Our results show that fusion of the RNA binding protein, R17, to N causes antitermination in a beta-galactosidase reporter construct that has the R17 binding site substituted for the normal N target sequence. This system can both detect low affinity interactions as well as discriminate between binding events with equilibrium dissociation constants from 10(-5) to 10(-8)M. The differences in antitermination activity with various mutant binding sites can be reliably detected by colony colour on X-Gal plates as well as by liquid culture assay.


CONCLUSIONS
We have demonstrated that N protein can cause antitermination through a heterologous RNA-protein interaction and that the system is capable of detecting RNA-protein interactions of differing affinities. This approach may also be useful in screening libraries for proteins that bind to novel RNA regulatory elements. Our results are also consistent with a model of N protein function in which binding to the nascent transcript increases the effective concentration of N in the vicinity of RNA polymerase leading to antitermination.
Wilhelm
J E
JE
Department of Pharmacology, University of California, San Francisco 94143, USA.
Vale
R D
RD
D002213:Capsid / Q000235:genetics / Q000378:metabolism*; D036022:Capsid Proteins; D017930:Genes, Reporter / Q000235:genetics; D009875:Operator Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013728:Terminator Regions, Genetic; D014158:Transcription, Genetic / Q000235:genetics; D054334:Viral Regulatory and Accessory Proteins / Q000235:genetics / Q000378:metabolism*; D001616:beta-Galactosidase / Q000235:genetics
D036022:Capsid Proteins; C059417:N protein, Bacteriophage lambda; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D054334:Viral Regulatory and Accessory Proteins; D001616:beta-Galactosidase