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Publisher Summary This chapter describes the methods that are most useful for the study of the biogenesis of the major outer membrane proteins of Escherichia coli K12: lipoprotein, the matrix proteins, and ompA protein. The pro-ompA protein accumulated in the nonfluid state can be completely processed to ompA protein during a 2-min chase. During the chase, aliquots are removed from the cultures at various time intervals, and membranes are isolated. Bacterial membrane proteins are more resistant to puromycin and kasugamycin, but more sensitive to tetracycline and sparsomycin than are soluble proteins. An important approach to the study of the biosynthesis and assembly of bacterial membrane proteins is the development of RNA-dependent cell-free translation systems. Although, bacterial membrane proteins are translated in eukaryotic protein-synthesizing systems, the use of a bacterial system is not as restrictive and can reveal more details about in vivo biosynthetic mechanisms.

Publisher Summary This chapter discusses the chemical and biological aspects of sparsomycin, an antibiotic from streptomyces. Sparsomycin is a strong inhibitor of protein biosynthesis, its site of interaction being the large ribosomal subunit, which prevents peptide transfer by interfering with the peptidyl transferase centre. Valuable information on the structure of this peptidyl transferase center and of the ribosomes as a whole has been obtained from studies using sparsomycin. A decision on whether sparsomycin—or its most promising analog—shows sufficiently auspicious activity to warrant further clinical development as an anticancer drug is required. This antibiotic has achieved prominence as a tool to study the protein biosynthesis machinery. A comprehensive review of sparsomycin, its chemistry, biological activity, and potential as an anticancer drug is timely. The compound has attracted much attention since its discovery, not only because of its biological activity but also for its unique structural characteristics and synthetic challenge.

Mainly protein L16 and to a lesser extent L2, L15, L27 of the bacterial ribosome and proteins L21/23, L26, L27, L28, L29 and L36 of rat liver ribosomes have been postulated as being involved in the ribosomal peptidyl transferase center. However no single protein has been found individually responsible from this activity and when bacterial ribosomes are treated with proteinase K, all these proteins that might be involved in peptidyl transferase activity are lost at faster rate than the activity itself. Furthermore other studies suggest that there is no identity between any of the individual proteins of the bacterial and eukaryotic ribosomes that were postulated as being involved in the peptidyl transferase activity, although there are a number of antibiotics (sparsomycin, blasticidin S, gougerotin, amicetin and anthelmycin) which are equally active in blocking peptidyl transferase activity by bacterial and eukaryotic ribosomes. Furthermore the treatment with a number of chemical reagents that modify the proteins have very little or no effect on the peptidyl transferase of the bacterial ribosome. On the other hand there is increasing evidence supporting the relevant role of the ribosomal RNA in the peptidyl transferase center. Thus the replacement of Mg2+ by Ba++ induces the breakage of the ribosomal RNA with a concomitant decrease in the peptidyl transferase activity. Furthermore ribosome treatment with a number of reagents that chemically modify the ribosomal RNA also affect drastically the peptidyl transferase activity. Therefore we propose that antibiotics which specifically inhibit peptide bond formation by bacterial and eukaryotic ribosomes (sparsomycin, gougerotin, blasticidin S, amicetin and anthelmycin) might recognize some moiety common in the RNA of the larger ribosomal subunit.

Publisher Summary The chapter describes the action of antibiotics on chain-initiating and on chain-elongating ribosomes. Procedures for preparing endogenous polysomes, and polysomes on viral RNA, that lack initiation factors (IF) are described. The ribosomes in these preparations are active in chain elongation, but they do not reinitiate with added phage RNA after runoff unless IF is added. The chapter also describes the application of such IF-free polysomes, compared with an initiation-dependent system, in analyzing the action of ribosomal inhibitors. There is increasing evidence that certain antibiotics selectively bind and exert their inhibitory actions on free or initiating ribosomes; elongating ribosomes carrying ligands, that is, nascent peptides and mRNA, bind differently or not at all to the antibiotics. By comparing the effects of antibiotics on peptide synthesis by elongating ribosomes finishing their incomplete nascent chains with the effects on initiating ribosomes starting with mRNA recognition, this selective action may be recognized. The specific effect can then be further analyzed. The actions of antibiotics on chain-initiating and on chain-elongating ribosomes are classified into 4 types: sparsomycin type, streptomycin type, aurintricarboxylate type, and erythromycin type. Comparison of the effects of antibiotics on chain-initiating and chain-elongating systems indicates whether an antibiotic selectively inhibits initiating ribosomes, at concentrations that do not inhibit elongating ribosomes.

Publisher Summary This chapter describes an assay that measures the reaction of polylysyl-tRNA with puromycin (Assay I) or with lysyl-tRNA (Assay II) in the presence of purified ribosomes and polyadenylic acid. The ability of the ribosomes to catalyze the puromycin reaction can be destroyed by prolonged storage at -15°, or by maintaining them at 0° at high dilution. Washed ribosomes chromatographed on DEAE-cellulose, or heated at 50° for 5 minutes retain the ability to carry out the puromycin reaction. The two assays show different template specificities. Triadenosine diphosphate supports the puromycin reaction; hexaadenosine pentaphosphate are required for the lysine addition reaction. Puromycin does not react with lysyl-tRNA. Chloramphenicol and its analogs, amicetin, blasticidin, sparsomycin, and gougerotin, inhibit the puromycin reaction, while chlortetracycline inhibits the lysine addition reaction, but not the puromycin reaction. This is consistent with the hypothesis that this antibiotic prevents the binding of acylated tRNA to the ribosomal A site.