Two families of small peptides that bind to the human thrombopoietin receptor and compete with the binding of the natural ligand thrombopoietin (TPO) were identified from recombinant peptide libraries. The sequences of these peptides were not found in the primary sequence of TPO. Screening libraries of variants of one of these families under affinity-selective conditions yielded a 14-amino acid peptide (Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala) with high affinity (dissociation constant approximately 2 nanomolar) that stimulates the proliferation of a TPO-responsive Ba/F3 cell line with a median effective concentration (EC50) of 400 nanomolar. Dimerization of this peptide by a carboxyl-terminal linkage to a lysine branch produced a compound with an EC50 of 100 picomolar, which was equipotent to the 332-amino acid natural cytokine in cell-based assays. The peptide dimer also stimulated the in vitro proliferation and maturation of megakaryocytes from human bone marrow cells and promoted an increase in platelet count when administered to normal mice.

We have used an in vitro protein synthesis system to construct a very large library of peptides displayed on polysomes. A pool of DNA sequences encoding 1012 random decapeptides was incubated in an Escherichia coil S30 coupled transcription/translation system. Polysomes were isolated and screened by afflnity selection of the nascent peptides on an immobilized monoclonal antibody specific for the peptide dynorphin B. The mRNA from the enriched pool of polysomes was recovered, copied into cDNA, and amplified by the polymerase chain reaction (PCR) to produce template for the next round of in vitro synthesis and selection. A portion of the amplified template from each round was cloned into a filamentous phagemid vector to determine the specificity of peptide binding by phage ELISA and to sequence the DNA. After four rounds of affinity selection, the majority of clones encoded peptides that bound specifically to the antibody and contained a consensus sequence that is similar to the known epitope for the antibody. Synthetic peptides corresponding to several of these sequences have binding affinities ranging from 7 to 140 nM. The in vitro system described here has the potential to screen peptide libraries that are three to six orders of magnitude larger than current biological peptide display systems.Peptide libraries derived from the cloning and expression of random-sequence oligonucleotides provide a rich source of ligands. These libraries, which often contain >108 recombinants, are characterized by the physical linkage of each peptide to its encoding DNA. This feature permits the affinity purification of peptides and associated DNA on an immobilized receptor and the DNA is sequenced to identify the peptide. This general approach provides a powerful tool for discovering ligands.A widely used version of this strategy is the display of peptides on the outer surface of filamentous phage particles. The peptide sequences are encoded by random oligonucleotides inserted into the 5' region of the genes encoding the capsid proteins pIII or pVIII (1-3). The peptides are expressed on the phage fused to the N terminus of the coat proteins. Several rounds of screening and amplification result in the enrichment of phage expressing peptides that bind to the receptor. Another system employs peptides fused to the C terminus of the lac repressor Lacd (4). The repressor protein physically links the peptides to the plasmid encoding them by binding to the lac operator sequences on the plasmid. The peptide-LacI-plasmid complexes are screened in a manner analogous to the phage system. These library approaches to ligand discovery often rely on the generation of huge numbers of peptides with the expectation that those of the appropriate structure will be rare. The size of a cell-based library is limited by the number of recombinants recovered from the transformation step. To create a peptide display system that avoids this limitation and is capable of screening much larger libraries, we developed the in vitro polysome system describe...

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A mutant of Saccharomyces cerevisiae defective in the S-adenosylmethionine (AdoMet)-dependent methyltransferase step of diphthamide biosynthesis was selected by intracellular expression of the F2 fragment of diphtheria toxin (DT) and shown to belong to complementation group DPHS. The DPH5 gene was cloned, sequenced, and found to encode a 300-residue protein with sequence similarity to bacterial AdoMet:uroporphyrinogen Il methyltransferases, enzymes involved in cobalamin (vitamin B12) biosynthesis. Both DPH5 and AdoMet:uroporphyrinogen Ill methyltransferases lack sequence motifs commonly found in other methyltransferases and may represent a new family of AdoMet:methyltransferases. The DPH5 protein was produced in Escherichia coli and shown to be active in methylation of elongation factor 2 partially purified from the dphS mutant. A null mutation of the chromosomal DPHS gene did not affect cell viability, in agreement with other studies indicating that diphthamide is not required for cell survival. The dph5 null mutant survived expression of three enzymically attenuated DT fragments but was killed by expression of fuly active DT fragment A. Consistent with these results, elongation factor 2 from the dph5 null mutant was found to have weak ADP-ribosyl acceptor activity, which was detectable only in the presence of high concentrations of fragment A.

The spc operon of Escherichia coli encodes 10 ribosomal proteins in the order L14, L24, L5, S14, S8, L6, L18, S5, L30, and L15. This operon is feedback regulated by S8, which binds near the translation start site of L5 and inhibits translation of L5 directly and that of the distal genes indirectly. We constructed plasmids carrying a major portion of the spc operon genes under lac transcriptional control. The plasmids carried a point mutation in the S8 target site which abolished regulation and resulted in overproduction of plasmidencoded ribosomal proteins upon induction. We showed that alteration of the AUG start codon of L5 to UAG decreased the synthesis rates of plasmid-encoded distal proteins, as well as L5, by approximately 20-fold, with a much smaller (if any) effect on mRNA synthesis rates, indicating coupling of the distal cistrons' translation with the translation of L5. This conclusion was also supported by experiments in which S8 was overproduced in trans. In this case, there was a threefold reduction in the synthesis rates of chromosome-encoded L5 and the distal spc operon proteins, but no decrease in the mRNA synthesis rate. These observations also suggest that transcription from ribosomal protein promoters may be special, perhaps able to overcome transcription termination signals. We also analyzed the state of ribosomal protein mRNA after overproduction of S8 in these experiments and found that repression of ribosomal protein synthesis was accompanied by stimulation of processing (and degradation) of spc operon mRNA. The possible role of mRNA degradation in tightening the regulation is discussed.It is known that the synthesis of many of the ribosomal proteins (r-proteins) in Escherichia (0li is coordinately and stoichiometrically balanced with the assembly of mature ribosomes and that' a posttranscriptional feedback mechanism is largely responsible for this regulation (for reviews, see references 17, 21,'and 28). Key r-proteins have been identified which, when synthesized in excess of the need for ribosome assembly, act as translational feedback inhibitors on their polycistronic mRNA to control the synthesis of all r-proteins within their regulatory unit.How does a single repressor r-protein inhibit the synthesis of'more than one r-protein? This question was first studied with the Lii operon, which encodes the genes for Lii and LI and is regulated by Li. It was demonstrated that Li interacts with mRNA at a single target site near the translation start site of Lii and inhibits translation of both Lii and Li (2,43). The inhibition of Lii translation by Li takes place directly and that of Li translation takes place only indirectly as a result of inhibition of Lii synthesis. It was shown that the synthesis of Li does not take place unless the preceding Lii cistron is translated, that is, LI synthesis is translationally coupled to Lii synthesis, and this is the basis of coregulation of the two genes by the single translational repressor LI (2,35,43). Recent experiments have also demonstrated the presen...