The amino acid sequence of rat ribosomal protein S8 was deduced from the sequence of nucleotides in a recombinant cDNA and confirmed from the NH2- and carboxyl-terminal amino acid sequences of the protein. Ribosomal protein

The amino acid sequence of rat ribosomal protein S8 was deduced from the sequence of nucleotides in a recombinant cDNA and confirmed from the NH2and carboxyl-terminal amino acid sequences of the protein. Ribosomal protein S8 contains 207 amino acids (the Nr^-terminal methionine is removed after translation of the mRNA) and has a molecular weight of 23,928. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 7-9 copies of the S8 gene. Ribosomal protein S8 contains a possible internal repeat that has 12 or 13 residues, is basic, and occurs 5 times in the protein. INTRODUCTION It is assumed that a molecular account of the function of eukaryotic ribosomes will only follow from knowledge of the structure and that this, in turn, requires information on the chemistry of the constituent proteins and nucleic acids. This inventory of data is necessary also for the second order structural problem, namely, the analysis of the chemistry of the interactions of the proteins with the nucleic acids. Progress has been made in this endeavor, although, a good deal remains to be done (1). The covalent structure of the four species of RNA in rat ribosomes has been established (2-7). In addition, eighty-four proteins have been isolated from the particles (8) and the sequence of amino acids in several has been determined directly (9-13). The latter task is being expedited now by the application of recombinant DNA technology. Thus, the structure of a number of rat ribosomal proteins has been deduced from the sequence of nucleotides in recombinant cDNAs (14-21). We report here the structure of rat ribosomal protein S8 which we have inferred from the sequence of nucleotides in a recombinant cDNA and which we have confirmed by sequencing portions of the protein. Ribosomal protein S8 can be crosslinked to eukaryotic initiation factor eIF-3 (22, 23); furthermore, the protein has been located at the interface between ribosomal subunits (24). These observations imply that protein S8 forms part of the domain concerned with the binding of initiation factors to the 40S subunit at the start of the translation of mRNA. © IRL Press Limited, Oxford, England. 9 4 5 1 Nucleic Acids Research EXPERIMENTAL PROCEDURES Preparation of Recombinant cDNAs Encoding Rat Ribsomal Protein S8. The recombinant DNA procedures and the methods used to determine the sequence of nucleotides in the nucleic acid were either described or cited before (20). The strategy that was used to design a probe for the cDNA encoding rat ribosomal protein S8, based on the sequence of 6 amino acids near the NH2 terminus of the protein has been reported (20). The probe, a mixture of 32 different oligodeoxynucleotides, 17 nucleotides in length, was synthesized on a solid support by the methoxyphosphoramidite method using an Applied Biosystems, Model 380B, DNA synthesizer (25) and the oligonucleotides were purified by polyacrylamide gel electrophoresis. Rat ribosomal protein S8 cDNA was hybridized to restriction enzyme digests of genomic DNA (26). Comparison of the Amino Acid Sequences of Ribosomal Proteins. The computer program, RELATE (27), was used to assess possible evolutionary relationships between S8 and other ribosomal proteins. The scoring matrix was DayhofFs MDM '78 (27). RESULTS AND DISCUSSION The Sequence of Nucleotides in a Recombinant cDNA Encoding Rat Ribosomal Protein S8. A cDNA library of 30,000 independent transformants was constructed from poly(A)mRNA prepared from regenerating rat liver (20). A random selection of 14,000 colonies from the library was screened for clones that hybridized to an oligonucleotide probe that was synthesized to be complementary to the sequence of nucleotides predicted to be present in the portion of the mRNA that encoded 6 amino acids (-AspAsn-Trp-His-Lys-Arg-) near the NH2 terminus of rat ribosomal protein S8. Seven colonies gave a positive hybridization signal with the probe. The DNA from the plasmids of the 7 transformants was isolated, digested with restriction endonucleases, and analyzed by gel electrophoresis. These clones had inserts that ranged in length from 0.45 to 0.7 kilobases and Southern blot hybridization with the oligonucleotide probe confirmed that all of the inserts contained cDNA for S8. The anticipated length of the S8 coding sequence, calculated from the molecular weight of the protein, is 670 nucleotides. The clone with the largest insert, pS8-14, was selected and the nucleotide sequence of the cDNA was determined. The sequences of nucleotides from both strands of the DNA, and overlapping sequences for each restriction site, were obtained. The cDNA insert in pS8-14 contains 735 nucleotides and includes the 5' homopolymer linkers, the 5' noncoding sequence, and a single open reading frame (Fig. 1). In the other two reading frames the sequence is interrupted by many termination codons. The open reading frame of 627 nucleotides begins at an ATG codon at position