A system based on specific protein-RNA interactions for analysis of target protein-protein interactions in vitro: successful selection of membrane-bound Bak-Bcl-xL proteins in vitro.
Ribosome display systems are very effective and powerful tools for in vitro screening of transcribed mRNAs that encode proteins (or peptides) with specific (known or unknown) functions. We have modified such a system by exploiting the interaction between a tandemly fused MS2 coat-protein (MSp) dimer and the RNA sequence of the corresponding specific binding motif, C-variant (or Cv). We placed the MSp dimer at the N-terminus of a nascent protein and the Cv binding motif was attached to the 5' end of the protein's mRNA. This configuration enhanced the stability of the ribosome-mRNA complex. We demonstrate here that this improved ribosome display system provides an effective method for identifying the gene for a protein that binds to a protein of interest. We visualized the formation of polysome complexes in this advanced polysome display by atomic force microscopy (AFM) and found that the AFM images of polysomes in our system were different from those observed in the case of conventional ribosome display systems. Our results suggest that our technology might usefully complement yeast two-hybrid assays.
Sawata
Shinya Y
SY
Gene Function Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Central 4, 1-1-1 Higashi, Tsukuba Science City 305-8562, Japan.
Suyama
Eigo
E
Taira
Kazunari
K
D036022:Capsid Proteins / Q000235:genetics; D002474:Cell-Free System / Q000378:metabolism; D004247:DNA / Q000235:genetics; D005978:Glutathione / Q000737:chemistry; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D018625:Microscopy, Atomic Force; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011132:Polyribosomes / Q000648:ultrastructure; D014176:Protein Biosynthesis; D025941:Protein Interaction Mapping / Q000379:methods*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012270:Ribosomes / Q000378:metabolism*; D012685:Sepharose / Q000737:chemistry; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D014158:Transcription, Genetic; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051020:bcl-X Protein
C494772:BAK1 protein, human; C494811:BCL2L1 protein, human; D036022:Capsid Proteins; D008565:Membrane Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051020:bcl-X Protein; D004247:DNA; D012685:Sepharose; D013762:Tetrahydrofolate Dehydrogenase; D005978:Glutathione