A number of small RNA molecules that are high affinity ligands for the 46-kDa form of human 2′-5′ oligoadenylate synthetase have been identified by the SELEX method. Surface plasmon resonance analysis indicates that these RNAs bind to the enzyme with dissociation constants in the nanomolar range. Competition experiments indicate that the binding site for the small RNAs on the 2′-5′ oligoadenylate synthetase molecule at least partially overlaps that for the synthetic double-stranded RNA, poly(I)·poly(C). Several of the RNAs function as potent activators of 2′-5′ oligoadenylate synthetasein vitro, although there is no correlation between binding affinity and ability to activate. The RNA aptamers having the strongest activation potential appear to have few base-paired regions. This suggests that 2′-5′ oligoadenylate synthetase, which has previously been believed to be activated only by double-stranded RNA, can also be activated by RNA ligands with little secondary structure. Since 2′-5′ oligoadenylate synthetase possesses no homology to other known RNA-binding proteins, the development of small specific ligands by SELEX should facilitate studies of RNA-protein interactions and may reveal novel features of the structure-function relationships involving this enzyme.

Translation termination in eukaryotes is governed by termination codons in mRNA and two release factors, eRF1 and eRF3. In this work, human eRF1 and eRF3 have been produced in insect cells using a recombinant baculovirus expression system for the corresponding human cDNAs. Purification of eRF1 has led to a homogeneous 50‐kDa protein active in promoting ribosome‐dependent and termination‐codon‐dependent hydrolysis of formylmethionyl‐tRNA.esMet.rb.eif.rb. Purification of eRF3 yielded a full‐length protein and shorter polypeptides. Microsequencing of the N‐terminus of the shortest form detected a site of proteolytic cleavage between Arg91 and Gly92, probably due to exposed region(s) hypersensitive to proteolysis. The mixture of full‐length and truncated forms of eRF3 as well as bacterially expressed eRF3 lacking 138 N‐terminal amino acids (eRF3Cp) are active as an eRF1‐dependent and ribosome‐dependent GTPase and in stimulating the GTP‐dependent release activity of eRF1. Complex formation between eRF1 and eRF3Cp was demonstrated by affinity and gel‐filtration chromatographies and by native‐gel electrophoresis. An abnormal electrophoretic mobility observed for eRF1 as compared with the complex points to a significant conformational change of either eRF1 or both factors in the complex. Co‐expression of both factors in baculovirus‐infected insect cells and a yeast two‐hybrid assay were applied to monitor complex formation in vivo. In yeast cells, both eRF1 and eRF3 are either in a monomeric or in a heterodimeric but not in a homodimeric state.