Molecular cloning and analysis of cDNA sequences for two ribosomal proteins from Artemia. The coordinate expression of genes for ribosomal proteins and elongation factor 1 during embryogenesis of Artemia.

The large subunit of eukaryotic ribosomes contains acidic phosphoproteins which are related to L7/L12 from Escherichia coli. In the brine shrimp Artemia these proteins are designated eL12 and eL12'. We have isolated cDNA clones for these proteins from a cDNA bank that was constructed by the use of size-fractionated poly(A)-rich RNA (8-10S fraction) from Artemia and a synthetic oligonucleotide as primer. Clones containing DNA sequences coding for eL12 and eL12 were characterized by hybrid-selected translation and DNA sequencing. The proteins eL12 and eL12' share an identical peptide of 22 amino acids at their carboxy termini whereas the remaining part of the protein shows little sequence homology. The nucleotide sequences show a different codon use for the amino acids in the common carboxy terminus, thereby excluding a common exon coding for this part of both proteins. Despite the differences in amino acid sequence in the major part of eL12 and eL12' the proteins have a considerable degree of homology on the basis of the distribution of hydrophobic and hydrophilic amino acids over the polypeptide chains, in agreement with a related folding and function of both proteins. Relative levels of mRNA coding for eL12, eL12' and elongation factor 1 alpha were determined during the development of Artemia from a dormant cyst to a nauplius. The data show a coordinate expression of the genes for EF-1 alpha and both ribosomal proteins, excluding a differential expression of the genes for these related ribosomal proteins during embryogenesis. Analysis of the gene copy number for eL12 and eL12' indicates the presence of a few genes for each protein.

Localization of Lys-51 of L7/L12 on 50S ribosomes from Escherichia coli.

Ribosomal protein L7/L12 from Escherichia coli was modified specifically at Lys-51 with 4-(6-formyl-3-azido-phenoxy)butyrimidate. Reconstitution of ribosomal cores, lacking L7/L12, with imidate-modified L7/L12 resulted in back formation of 50S particles which were fully active in elongation-factor-dependent processes. By use of the formylazidophenoxy moiety as hapten, the position of Lys-51 of L7/L12 on the 50S ribosome was determined by immune electron microscopy. The results show that an L7/L12 dimer is present in the L7/L12 stalk in such a way that Lys-51 is located at the far cytoplasmic end of the stalk. The experimental data are discussed in relation to a proposed model for the L7/L12 dimer.

Characterization of the region on protein L7/L12 involved in binding to ribosomal particles.

Tryptic digestion of reductively methylated protein L7/L12 yields a large tryptic fragment, which comprises amino acids 1-59. At the most, two molecules of this fragment can bind to a 50-S ribosomal particle, deprived of protein L7/L12. Besides, binding of each single 1-59 fragment competes with binding of one dimeric L7/L12 molecule. Molecular weight studies on the fragment reveal a monomeric structure. Digestion of the 1-59 fragment with carboxypeptidase Y leads to the formation of a 1-55 fragment. The binding characteristics of the latter fragment are similar to those of the 1-59 fragment. The results suggest that a monomeric stretch of L7/L12, comprising the first 55 amino acids, is sufficient for attaching L7/L12 to the ribosome.

Structural analysis of ribosomal protein L7/L12 by the heterobifunctional cross-linker 4-(6-formyl-3-azidophenoxy)butyrimidate.

The structure of the dimeric form of the protein L7/L12 from ribosomes from Escherichia coli was studied by using the heterobifunctional cross-linker 4-(6-formyl-3-azidophenoxy)butyrimidate. The imidate group of the cross-linker reacts very specifically with Lys-51 of L7/L12. Subsequent cross-linking of this modified L7/L12 by reductive alkylation of the aldehyde group of the cross-linker results in the formation of a covalent cross-link between both polypeptide chains of the L7/L12 dimer. This covalently cross-linked dimer is fully active in reconstitution of elongation factor G dependent GTP hydrolysis of 50S cores lacking L7/L12, suggesting a conformation of the cross-linked protein similar to the conformation of native L7/L12. Analysis of the tryptic peptides of cross-linked L7/L12 shows the points of attachment of the cross-linker to be Lys-51 in one polypeptide chain and Lys-29 in the other. On the basis of a combination of this result with published data, a structure for the N-terminal region of L7/L12 dimers is proposed. The important feature of this model is a shifted parallel alignment of both polypeptide chains resulting in one free N-terminal stretch for each L7/L12 dimer which attaches the protein to the ribosome via protein L10.