Various derivatives of the internal ribosomal entry site (IRES) of encephalomyocarditis virus (EMCV) RNA have been used to analyze by UV-cross-linking its interaction with mRNA binding proteins from ascites carcinoma Krebs-2 cells. A doublet of proteins with Mr 58 and 60 kD bound to two regions of the IRES. One site is centered at nt 420-421 of EMCV RNA whereas the other is located between nt 315-377. Both sites form hairpin structures, the loops of which contain UCUUU motif, conserved among cardio- and aphthoviruses. The interaction of p58 and p60 with IRES is affected by the integrity of the stem-loop structure proximal to the start AUG codon (nts 680-787), although, under similar conditions, cross-linking of these proteins to this region was not detected. Deletions in the main recognition site of p58 strongly reduce the initiation activity of the IRES in vitro. However, elimination of p58 (p60) binding by these mutations does not completely abolish the ability of the IRES to direct polypeptide synthesis starting from the authentic AUG codon. The IRES can be assembled in vitro from two covalently unlinked transcripts, one containing the target site for p58 and the other encompassing the remaining part of the IRES fused to a reporter gene, resulting in considerable restoration of its activity. Implications of these findings for the mechanism of initiation resulting from internal entry of ribosomes are discussed.

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Direct contacts between 16-S RNA and split proteins S2, S3, S 5 , S14 and S21 inside the 30-S subunit of Eschevichia coli ribosomes were evidenced by the formation of ultraviolet-induced (A = 254 nni) RNA-protein cross-links. 30-S subunits were reassembled from core particles and a mixture of split proteins containing in each case a single '251-labelled protein. All the proteins tested are cross-linked as a result of a single-hit process; proteins S3 and S21 were cross-linked at the highest rate.The essential step in the study of nucleoproteins is detection of interactions between components of the complex.The evidence for direct RNA-protein contacts in ribosomes of Eschevichia coli was presented for a number of proteins [l -31. They belong mostly to a group of proteins which do not readily dissociate from 16-S RNA upon incubation of 30-S subparticles at high concentrations of LiCl or CsCl and which form specific RNA . protein complexes (core proteins) [4,5]. At the same time, there was no evidence for the existence of direct RNA-protein contacts in the 30-S subparticle for a number of so-called split proteins that readily dissociate from the ribosome at high concentrations of LiCl or CsCl and do not form strong complexes with 16-S RNA.Only recently [6] Hochkeppel and Craven showed that some split proteins are able to bind independently to 16-S RNA prepared by acetic acid urea extraction. They presented some evidence that six of these proteins (S3, S5, S9, S11, S12 and SlS) bind site-specifically to such isolated 16-S RNA. But the stability and specificity of nucleoproteins are conditioned by cooperative interactions. Therefore the data, obtained for incomplete complexes, seem not to be very conclusive.For detection of contacts between a number of split proteins (S2, S3, S5, S14 and S21) and 16-S RNA in the complete 30-S subunit of E. coli ribosomes we have used ultraviolet-induced cross-links. This method is adequate for fixation of polynucleotideprotein contacts in intact nucleoproteins [7,8] including ribosomes [9-121. 30-S subunits were reconstituted from a mixture of core subparticles and split proteins, containing in each case a single "'Ilabelled protein component. The reconstituted subunits were irradiated with ultraviolet light (i = 253.7 nm, 2.5-25 x lo5 quanta per subparticle) and the formation of cross-links was monitored by analysis of cosedimentation of 16-S RNA and the labelled protein in the presence of sodium dodecyl sulphate and EDTA.It was shown that proteins S3 and S21 cross-linked well with 16-S RNA, whereas proteins S2, S5 and S14 cross-linked under the same conditions to a much lesser extent. MATERIALS AND METHODS30-S subunits of E. coli MRE 600 ribosomes were prepared by zonal centrifugation [I 31. Individual ribosomal proteins were isolated according to [14]. 100-300 pg of each isolated protein were labelled with 1-3 pCi of Na'251 (Amersham, 18 mCi/pg) by the method of Ball et al. [15] with a slight modification [16]. The purity of the labelled proteins was checked by gel electrophoresis...

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