Abstract

 

A one-hybrid system for detecting RNA-protein interactions.

Wilhelm, James E. and Vale, Ronald D. 1996. Genes to Cells 1:317-23.

ABSTRACT:

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 b-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 determined 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.


Research: RNA Transport


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