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. 1997 Mar 18;94(6):2410–2414. doi: 10.1073/pnas.94.6.2410

Figure 1.

Figure 1

The human α-complex protects a 42-nt segment of the 3′UTR that is sufficient for α-complex assembly. (A) The α-complex was assembled in vitro and resolved on a nondenaturing polyacrylamide gel. Lane 1, [32P]α-3′UTR digested with RNaseT1; lane 2, [32P]α-3′UTR incubated with S100 extract from human erythroleukemia cells (K562) before RNaseT1 digestion; lane 3, [32P]α-3′UTR incubated with S100 in the presence of poly(C) competitor before RNaseT1 digestion. The position of the α-complex is indicated. The structure of the more slowly migrating alternative complex formed in the presence of the poly(C) competitor, in which the 39-kDa αCP is replaced by a 48-kDa poly(CU)-binding protein, is detailed elsewhere (17). (B) A gel slice containing the α-complex was excised from the gel shown in A, lane 2, the RNA was eluted, electrophoresed on 6% denaturing gel, and autoradiographed. Lane 1, full-length undigested [32P]α-3′UTR; lane 2, RNA protected by the α-complex (42-nt protected region; PR). (C) The 3′ end of the 32P-labeled protected fragment was mapped by site-directed RNaseH cleavage. The protected region mRNA was cleaved at a site specified by a hybridized antisense oligonucleotide, and the span of the resulting fragment were determined. Based on this data the termini of the 42-bp protected region were determined. Lane 1, protected region eluted from B, lane 2; lane 2, RNA segment eluted from B and subjected to site-directed RNase H cleavage. The size marker (nt) is indicated to the left. The diagram depicts the position of the antisense oligonucleotide that targeted the RNase H cleavage and indicates the sizes of the observed bands. (D) The RNA protected region isolated from an excised gel slice (B, lane 2) was incubated with S100 extract and was electrophoresed as in A. Lane 1, the eluted [32P]PR RNA digested with RNase T1 in the absence of S100; lane 2, [32P]PR incubated in S100 before digestion with RNase T1; lane 3, poly(C) competitor added to the S100 extract before incubation with the [32P]PR and RNase digestion.