Elongation factor P (EFP) is a protein that stimulates the peptidyltransferase activity of fully assembled 70 S prokaryotic ribosomes and enhances the synthesis of certain dipeptides initiated by N-formylmethionine. This reaction appears conserved throughout species and is promoted in eukaryotic cells by a homologous protein, eIF5A.Here we ask whether the Escherichia coli gene encoding EFP is essential for cell viability. A kanamycin resistance (Kan R ) gene was inserted near the N-terminal end of the efp gene and was cloned into a plasmid, pMAK705, that has a temperature-sensitive origin of replication. After transformation into a recA ؉ E. coli strain, temperature-sensitive mutants were isolated, and their chromosomal DNA was sequenced. Mutants containing the efp-Kan R gene in the chromosome grew at 33°C only in the presence of the wild-type copy of the efp gene in the pMAK705 plasmid and were unable to grow at 44°C. Incorporation of various isotopes in vivo suggests that translation is impaired in the efp mutant at 44°C. At 44°C, mutant cells are severely defective in peptide-bond formation. We conclude that the efp gene is essential for cell viability and is required for protein synthesis.The most important catalytic function of the ribosome is the synthesis of peptide bonds. A variety of approaches have been used to deduce the components that comprise this catalytic center. The results of in vitro reconstitution studies, photochemical cross-linking of substrates, and mutagenesis of conditionally lethal or antibiotic-resistant phenotypes have implicated domain V of the 23 S rRNA as well as proteins L2, L3, and L4 as the minimum components of this active center (1-6).A surprising finding is that the in vitro reconstituted peptidyltransferase cannot condense all aminoacyl-tRNA template combinations (7). This anomaly is reflected in the fact there is a subsite on domain V of 23 S rRNA that is specific for hydrophobic amino acids (2). In retrospect, it has been known for more than two decades that puromycin, which is one of the most common substrate analogues used to study this reaction, has a special three-dimensional structure (a U shape) that favors peptide-bond synthesis (8). Substitution of the aromatic residue of puromycin by that of other amino acids distorts this structure and drastically impairs peptide-bond synthesis (9). This specificity is reflected in the 50 S catalyzed "fragment" reaction that has been used to deduce the components of the peptidyltransferase catalytic center.Reconstitution studies as well as photoaffinity labeling experiments indicate that several proteins of the 50 S particle enhance peptide-bond synthesis. The assembled peptidyltransferase in the 70 S ribosome catalyzes peptide bonds at a higher rate than does the peptidyltransferase of the 50 S subunit, but does not efficiently condense nonaromatic amino acids (7, 10). In addressing this issue, we asked whether proteins that stimulate reconstitution of translation from homogeneous translation factors enhance the condensation of ...

The structure of the chicken link protein gene has been determined from a series of genomic clones that cover the entire coding region as well as the complete 3'-untranslated region and a small portion of the 5'-untranslated region. The gene is >80 kilobase pairs long and is present in a single copy in the chicken genome. The link protein gene contains at least five exons with four encoding the entire protein. We have reported (3) the isolation of cDNA clones encoding the entire chicken cartilage link protein (LP) and deduced the amino acid sequence from the nucleotide sequence. A comparison between the complete amino acid sequence of rat LP (4) and chicken LP (3) shows a very high degree of homology. The analysis of the amino acid sequence of LPs reveals that the protein can be divided into several domains. The N-terminal half of LP has homology with the repeating units of the human free secretory component, the 'y chain of the T-cell receptor, and three types of immunoglobulin variable regions (5). The C-terminal half contains tandemly repeated sequences that may be the hyaluronic acid binding regions of LP (3,4,6).Here we report on the isolation of chicken LP genomic clones covering the entire protein coding region as well as the complete 3'-untranslated region of the mRNA. The structure of the gene is compared to the domain structure of the protein. MATERIALS AND METHODSConstruction and Screening of the Chicken Genomic Library. High molecular weight DNA, isolated from 9-day chicken embryos, was partially digested with Sau3AI and inserted into the BamHI site of XEMBL3 vector DNA as described by Frischauf et al. (7). Ligation mixtures were packaged in vitro, and 0.5-1 x 106 recombinant phages were amplified in Q359 host cells.The library was screened according to the method of Benton and Davis (8) using '2P-labeled LP cDNA probes (3). Duplicate filters were hybridized as described by Maniatis et al. (9). Phage DNA was isolated (10), and restriction maps were established based on the results of single and double digests of phage DNA. Restriction fragments were subcloned in pUC8/pUC9 (11) or in M13mp8/Ml3mp9 (12). These genomic fragments as well as different LP cDNA fragments were used as probes in Southern hybridizations (13) to confirm the restriction map.DNA fragments were labeled with 32P by random oligonucleotide priming (14). Hybridization and washing conditions of Southern blots of restriction digests of genomic DNA were carried out according to Jeffreys and Flavell (15).Electron Microscopy. Separated complementary DNA strands of genomic clones were isolated from agarose gels as described (16). Double heteroduplexes were prepared by incubating the complementary DNA strands of appropriate phages with 3-5 ,ug of chicken sternal total RNA in 80% (vol/vol) formamide and 0.1 M Hepes (pH 8.3), 0.4 M NaCl, 0.01 M EDTA (17) in a final volume of 25 ,ul at a DNA concentration of 5 uzg/ml. Incubation was done for 15 hr at 45°C. Excess RNA was removed by chromatography on a Sephacryl S-1000 column (2 x 45 mm...

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