C

SLBP A87Ni72C

Figure 6 In vitro translation of SLBP and determination of the RNA binding domain. (A) Translation of human SLBP and SLBP deletions in the reticulocyte lysate. The cloned DNAs were transcribed by T7 RNA polymerase, and the RNA was translated in the reticulocyte lysate for 90 min in the presence of 35S-methionine. The products were analyzed by electrophoresis on 12% polyacrylamide gels and detected by autoradiography. Lane 1: full-length SLBP; lanes 2—5, SLBP truncated 13, 52, 72, or 90 amino acids from the C terminus; lane 6; SLBP with the first 87 and the last 13 amino acids removed. (B) Binding of the stem—loop by the C-terminal deletions of SLBP. Fifteen femtomoles of radiolabeled stem-loop (lane 1) was incubated with 5 fig of the polyribosomal extract (lane 2) or with 12.5 /ill of reticulocyte lysate programmed with RNA encoding the full-length (lane 3) or C-terminal deletion mutants (lanes 4—6). The mutants used are indicated above each lane. Lane 7 is an incubation of 12.5 fil of the unprogrammed reticulocyte lysate with the probe. The arrow indicates the complex with full-length SLBP, and "NS" indicates a nonspecific complex formed in the reticulocyte lysate. (C) A 110-amino acid RNA binding fragment of SLBP specifically binds the stem-loop. In lane 1 the probe was incubated with a nuclear extract. In eiiro-translated full-length SLBP (lanes 2—5) and the 110-amino acid fragment A87NA72C (lanes 6—9) were incubated with 15 fmol of labeled 30-nt RNA containing the stem-loop. The complexes were resolved by electrophoresis on a 10% polyacrylamide gel. In lanes 3 and 7, a 100-fold excess of the 30-nt RNA was included, in lanes 4 and 8 a 100-fold excess of an RNA with the stem sequence reversed was included, and in lanes 5 and 9 a 100-fold excess of a nonspecific RNA (AAAUACCUACUUCAUACAA) was included. The arrows indicate the specific complexes formed. The unbound probe has been run off the gel to allow good resolution of the complex with the 110-amino acid protein.

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