Daisuke Nakada

Formation of ribosomes by a "relaxed" mutant of Escherichia coli.

A preferential synthesis of ribosomal protein was observed in a methionine-requiring “relaxed” mutant of Escherichia coli during recovery from methionine starvation. The ribosomal RNA-eontaining “relaxed” particles, which had accumulated during methionine starvation, rapidly combined with the ribosomal protein, subsequently produced upon restoration of methionine, to form ribosomes. When 5-fluorouracil was present during methionine starvation, “relaxed” particles containing the analogue in their RNA were produced but these particles failed to form ribosomes after removal of the analogue and addition of methionine: preferential synthesis of ribosomal protein did not occur during recovery in this case. On the other hand, normal “relaxed” particles formed ribosomes in the presence of 5-fluorouracil when methionine was restored to methionine-starved cells: a preferential synthesis of ribosomal protein also took place in the presence of the analogue. A cell-free preparation containing “relaxed” particles appeared to produce a substantial amount of ribosomal protein in vitro: a partial conversion of “relaxed” particles to 30 s and 50 s particles was observed in this system. A similar preparation consisting of “relaxed” particles which contained 5-fluorouracil failed to do so. Since the ribosomal RNA in “relaxed” particles showed template activity for in vitro amino acid incorporation, it is proposed that the ribosomal RNA of “relaxed” particles directs the synthesis of ribosomal protein. “relaxed” particles, free of normal ribosomes, incorporated amino acids in the presence of an energy-generating system, transfer RNA and the necessary enzymes, but in the apparent absence of messenger RNA.

Fate of the ribosomal RNA produced by a “relaxed” mutant of Escherichia coli

The fate of the RNA produced during methionine starvation by a strain of Escherichia coli with “relaxed” control of RNA synthesis has been investigated. This RNA consists of amino acid transfer RNA (4 s), messenger RNA and ribosomal RNA (16 s and 23 s); the ribosomal RNA forms part of a particle (18 s to 25 s). The RNA of the “relaxed” particles is not stable: it disappears slowly (half-life approximately 20 minutes) when RNA synthesis is inhibited by 2,4-dinitrophenol. When methionine is restored to cells that have accumulated the “relaxed” particles, the RNA of these particles is rapidly incorporated intact into newly formed ribosomes. This incorporation also occurs when cells that have accumulated the “relaxed” particles in a glucose medium free of methionine are transferred to a succinate medium containing methionine: the “shift-down” prevents synthesis of new RNA, but permits the synthesis of the protein required for the conversion of the particles to complete ribosomes.

Translational control of bacteriophage MS2 RNA cistrons by MS2 coat protein: Polyacrylamide gel electrophoretic analysis of proteins synthesized in vitro☆

Abstract MS2-specific proteins synthesized in a cell-free, protein-synthesizing system under the direction of MS2 RNA were analyzed by polyacrylamide gel electrophoresis in a buffer containing sodium dodecyl sulfate. In addition to coat protein, which accounted for half or more of the total [14C]phenylalanine-labeled proteins synthesized, a few non-coat proteins were resolved. When a ribonucleoprotein complex (termed complex I), formed in vitro by interaction of MS2 RNA with several molecules of MS2 coat protein, was used instead of MS2 RNA to direct in vitro protein synthesis, the synthesis of some non-coat proteins was greatly reduced, whereas the synthesis of coat protein was almost unaffected. These results support our previous proposition that complex I is involved in the mechanism for the shut-off of synthesis of non-coat proteins in MS2-infected cells. In the cell-free, protein-synthesizing system, coat protein appeared to be the first protein synthesized. This is consistent with the notion that the coat protein cistron in MS2 RNA is located close to the initiation point of translation.

Relaxed synthesis of ribosomal RNA by a stringent strain of Escherichia coli.

A stringent, leucine-requiring strain of Escherichia coli produced ribosomal RNA in the absence of leucine when the bacteria were starved of magnesium prior to leucine starvation. The ribosomal RNA produced under these conditions appeared in abnormal particles containing pre-existing ribosomal protein, which was released from ribosomes due to the degradation of the ribosomal RNA during the previous magnesium starvation. The participation of pre-existing ribosomal protein in the subsequent formation of the ribosomal RNA-containing particle suggests a possible role of the protein in the regulation of ribosomal RNA synthesis.

Ribosome formation by puromycin-treated Bacillus subtilis

Abstract Puromycin causes an unbalanced synthesis of RNA in Bacillus subtilis , while net increase in protein of the cell is prevented. Under these conditions, an accumulation of abnormal particles (between 18 S and 25 S) containing newly formed ribosomal RNA (16 S and 23 S) occurs. Experiments on the fate of the RNA contained in these particles have provided the following information. 1. 1. The ribosomal RNA contained in the abnormal particles is unstable, as indicated by its degradation to acid-soluble material when synthesis of RNA de novo is blocked by actinomycin D. 2. 2. When puromycin is removed from the culture, most of the pre-formed ribosomal RNA is incorporated into ribosomes by combining with ribosomal protein synthesized preferentially by the recovering cells. 3. 3. The preferential synthesis of ribosomal protein during recovery from puromycin treatment can also occur in the presence of actinomycin D, resulting in incorporation of some of the pre-formed RNA directly into ribosomes. An accumulation of the information for ribosomal protein during puromycin treatment is indicated.

Proteins of ribosomes formed from “relaxed particles”

During starvation for methionine, a methionine-requiring, relaxed mutant of Escherichia coli accumulates subribosomal relaxed particles. The proteins of the particles are mostly derived from pre-existing proteins of the cell. These proteins are electrophoretically identical with some of the normal ribosomal proteins. When methionine is restored to the starved culture, the relaxed particles combine with preferentially synthesized new ribosomal proteins to form ribosomes. These ribosomes, separated from the pre-existing ribosomes by the density-transfer technique, contain both old and new ribosomal proteins. In acrylamide gel electrophoresis, both old and new ribosomal proteins labeled with different radioisotopes appear in most, if not all, of the ribosomal protein components although they are different in quantity.

Functional activity of ribosomes formed in vivo from “relaxed particles”

Abstract The ribosomes formed in vivo from “relaxed particles” appear to have normal functional activity. This was indicated by the following: (a) a ribosome preparation in which about 23.5 % of the ribosomes were formed from “relaxed particles” showed a poly (U)-dependent polyphenylalanine-synthesizing activity about equal to that of normal ribosomes; (b) ribosomal “cores” obtained by CsCl-gradient centrifugation of the ribosomes formed from “relaxed particles” synthesized polyphenylalanine when combined with normal ribosomal proteins; and (c) ribosomes formed from “relaxed particles” participated in the formation of functionally active polysomes.