Microcin C is a ribosome-synthesized heptapeptide that contains a modified adenosine monophosphate covalently attached to the C-terminal aspartate. Microcin C is a potent inhibitor of bacterial cell growth. Based on the in vivo kinetics of inhibition of macromolecular synthesis, Microcin C targets translation, through a mechanism that remained undefined. Here, we show that Microcin C is a subject of specific degradation inside the sensitive cell. The product of degradation, a modified aspartyl-adenylate containing an N-acylphosphoramidate linkage, strongly inhibits translation by blocking the function of aspartyl-tRNA synthetase.Microcins are a class of small (Ͻ10 kDa) ribosomally synthesized peptide antibiotics produced by Enterobacteriaceae (1). Whereas some microcins are active as unmodified peptides (2), others are produced as polypeptide precursors that are heavily modified by dedicated maturation enzymes (3). Interest is attached to such post-translationally modified microcins due to their highly unusual structures and the fact that they target important cellular processes that are attractive targets for antibacterial drug development.Genes responsible for microcin production are usually plasmidborne. Plasmids encoding microcin structural and maturation genes also encode determinants of immunity specific to the microcin produced. Based on cross-immunity, post-translationally modified microcins can be subdivided into the B, C, and J types. Microcin B (MccB) 4 is a 43-residue peptide with 8 thiazole and oxazole rings that are synthesized by the McbBCD maturation enzyme complex from multiple serine and cysteine residues present in the MccB precursor (4). MccB is a potent inhibitor of DNA gyrase; it traps the enzyme at the stage of DNA strand passage (5). Microcin J, a 21-amino acid peptide, contains an unusual lactam bond between its N-terminal glycine and the ␦-carboxyl group of an internal glutamate; it assumes a highly unusual threaded-lasso structure (6 -8). MccJ inhibits bacterial RNA polymerase by occluding a narrow channel that is used to traffic transcription substrates, NTPs, to the catalytic center of the enzyme (9, 10).The structure of the subject of this study, Microcin C (McC) is shown in Fig. 1A. McC is a heptapeptide containing a modified adenosine monophosphate covalently attached to its C terminus through an N-acylphosphoramidate linkage (11, 12). The phosphoramidate group of the nucleotide part of McC is additionally modified by a propylamine group. Additionally, in mature McC, the peptide moiety, which is encoded by the mccA gene, is modified and the C-terminal asparagine residue specified by mccA is converted to an aspartate (18, 19), through an unknown mechanism. In vivo, McC appears to target translation (12). Guijarro et al. (12) also reported that large concentrations of McC, as well as of synthetic peptide of the same sequence but without the nucleotide modification, mildly inhibit translation in vitro. They therefore concluded that the peptide part of McC is responsible for transl...

In vitro synthesis of firefly luciferase and its folding into an enzymatically active conformation were studied in a wheat germ cell‐free translation system. A novel method is described by which the enzymatic activity of newly synthesized luciferase can be monitored continuously in the cell‐free system while this protein is being translated from its mRNA. It is shown that ribosome‐bound polypeptide chains have no detectable enzymatic activity, but that this activity appears within a few seconds after luciferase has been released from the ribosome. In contrast, the renaturation of denatured luciferase under identical conditions occurs with a half‐time of 14 min. These results support the cotranslational folding hypothesis which states that the nascent peptides start to attain their native tertiary structure during protein synthesis on the ribosome.

We have recently isolated and characterized a novel protein associated with Escherichia coli ribosomes and named protein Y (pY).Here we show that the ribosomes from bacterial cells growing at a normal physiological temperature contain no pY, whereas a temperature downshift results in the appearance of the protein in ribosomes. The protein also appears in the ribosomes of those cells that reached the stationary phase of growth at a physiological temperature. Our experiments with cell-free translation systems demonstrate that the protein inhibits translation at the elongation stage by blocking the binding of aminoacyl-tRNA to the ribosomal A site. The function of the protein in adaptation of cells to environmental stress is discussed. INTRODUCTIONThe involvement of ribosomes in low temperature adaptation of bacteria was implied a long time ago . It was reported that the ribosomal fraction of the bacterial cell is responsible for the arrest of translation at low temperatures . Some antibiotics specifically affecting bacterial ribosomes were shown to mimic cold shock or heat shock response, and so ribosomes were claimed to be sensors of cold and heat shock in bacteria . More recently, a ribosome-binding factor, RbfA, was discovered that proved to be a cold shock protein . RbfA was shown to bind with 30S ribosomal subunit and suppress a cold-sensitive mutation in 16S ribosomal RNA . Another cold shock protein, CsdA, was also characterized as a ribosome-associated protein, and its capacity to unwind doublestranded RNA was observed . The major cold shock protein of Escherichia coli, CspA (Goldstein et al., 1990), was reported to be an RNA-binding protein and qualified as an RNA chaperone . A number of other cold shock proteins were discovered in bacteria . In no case, however, were the mechanisms and effects of cold shock protein on protein synthesis and cell growth clarified. RESULTS AND DISCUSSIONThe presence of the recently discovered ribosome-associated protein pY in ribosomes was analysed depending on the conditions of the E. coli culture growth. To our surprise, the protein was not found in the ribosomal fraction of cells grown at the temperature for physiological activity in E. coli (37°C). The protein appeared in the ribosomal fraction when the cultivated cells were chilled rapidly to 15°C followed by incubation at the same temperature, or cooled gradually to 4°C. Figure 1 presents the area of the two-dimensional electrophoresis slab where pY migrates among ribosomal proteins. It can be seen that the proteins extracted from ribosomes of cells grown under normal physiological conditions contain very little, if any, pY ( Figure 1A), whereas a significant amount of the protein is + Corresponding author. Tel/Fax: +7 095 924 0493; E-mail: spirin@vega.protres.ru D.E. Agafonov, V.A. Kolb & A.S. Spirin scientific reports detected in the ribosomal fraction from cells subjected to a temperature downshift prior to harvesting ( Figure 1B). The appearance pY in some of the preparations of ribosomes from cells grown at ...

Surface labeling of Escherichia coli ribosomes with the use of the tritium bombardment technique has revealed a minor unidentified ribosome-bound protein (spot Y) that is hidden in the 70S ribosome and becomes highly labeled on dissociation of the 70S ribosome into subunits. In the present work, the N-terminal sequence of the protein Y was determined and its gene was identified as yfia, an ORF located upstream the phe operon of E. coli. This 12.7-kDa protein was isolated and characterized. An affinity of the purified protein Y for the 30S subunit, but not for the 50S ribosomal subunit, was shown. The protein proved to be exposed on the surface of the 30S subunit. The attachment of the 50S subunit resulted in hiding the protein Y, thus suggesting the protein location at the subunit interface in the 70S ribosome. The protein was shown to stabilize ribosomes against dissociation. The possible role of the protein Y as ribosome association factor in translation is discussed.ribosomal proteins ͉ ribosome dissociation ͉ ribosomal surface ͉ tritium labeling T he hot tritium bombardment technique is based on replacement of hydrogen by tritium in covalent bonds of thin surface layer in macromolecules (1). The technique appears to be the most direct approach for studies of protein topography on surfaces of biological structures. Protein exposure on the surfaces of viruses (1-3), membranes (4), and ribosomes (5-9) has been determined by the use of this technique.Studying proteins of the ribosome surface with this method has lead to the finding that unidentified minor component of the ribosome corresponding to spot Y on the ribosomal protein map becomes highly exposed on dissociation of the 70S ribosome into subunits (5, 7, 9). The reassociation of ribosomal subunits by increasing Mg 2ϩ concentration resulted in reshielding of the protein Y, suggesting its location at the ribosomal subunit interface (9). In this study, the protein was identified and isolated. Its interface location was confirmed by experiments on hot tritium bombardment. The protein was shown to support the 70S ribosome in associated state at low concentration of Mg 2ϩ . Materials and MethodsMaterials. Buffer reagents were obtained from Sigma, acrylamide and methylenebisacrilamide were from Fluka, urea was from Bio-Rad, DNase I was from Serva, Butyl-Toyopearl 650S from Toyo Soda (Tokyo), and DEAE-Sepharose Fast Flow from Pharmacia. Sucrose, acetone, hydrogen peroxide, and acetic acid were from ReaKhim (Moscow, Russia).Preparation of 70S Ribosomes and Total Ribosomal Protein. Ribosomes were prepared from Escherichia coli MRE-600 cells according to Staehelin et al. (10) with modifications described in ref. 9. Salt-washed ribosomes used in binding assay, as well as ribosomal subunits, were prepared as described by Gavrilova et al. (11) with substitution of pelleting by ultracentrifugation for precipitation with (NH 4 ) 2 SO 4 at the final stage of the procedure.The standard procedure of acetic acid extraction and precipitation with acetone (12) was used to p...