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Biomedical subjects

T Nakamoto

Publications and source records attributed to T Nakamoto.

At least 217 records · Page 12Linked to original sources
PubMed1978

On the accessibility and selection of the initiator site of mRNA in protein synthesis.

The specificity of the cell-free system of Escherichia coli for mRNA was examined, and the "accessibility" of some natural and synthetic RNAs to the ribosomes was determined by measurement of AcPhe-tRNA and fMet-tRNA binding, AcPhe-puromycin and fMet-puromycin formation, and polypeptide synthesis. The E. coli system effectively initiates the translation of various synthetic RNAs with AcPhe-tRNA or fMet-tRNA under conditions optimal for the translation of viral RNA. Poly(A,G,U) is accessible to the ribosomes according to all of the above criteria. Poly(A,C,G,U), 23 S rRNA, R17 RNA, and MS2 RNA, on the other hand, show limited accessibility when tested for initiator tRNA binding, or for AcPhe-puromycin and fMet-puromycin formation. MS2 and R17 RNA, but not poly(A,C,G,U) and 23 S rRNA, show accessibility when measured by polypeptide synthesis. The results suggest that, except at initiator sites of natural mRNA, an RNA containing about equal amounts of all four bases is inaccessible to E. coli ribosomes for polypeptide synthesis. Rate constants obtained for fMet-tRNA binding with MS2 RNA, poly(A,G,U), and poly(C,G,U) indicate that the ribosomes do not have any special affinity for the viral RNA. Thus, the selection of the initiator site in protein synthesis may be critically determined more by the accessibility of the initiator codon than by ribosomal recognition of the site.

Base Sequence↗
PubMed1978

Translational specificity of Bacillus stearothermophilus ribosomes.

The translational specificity of Bacillus stearothermophilus ribosomes was studied by determining the effectiveness of various synthetic RNAs as templates at 37 degrees and at higher temperatures. The effectiveness of poly(G,U) was maximal at a G:U ratio of 1:3; it declined with lower G content because of reduced ribosomal affinity for the RNA and, with higher G, because of interference by secondary structure. The effectiveness of poly(A,C,G,U) also declined when secondary structure was increased by increasing (G+C) content. Escherichia coli ribosomes exhibited a similar specificity for poly-(G,U), but had a lower sensitivity to interference by RNA secondary structure. In both bacterial species, sensitivity to secondary structure was determined by the 30S ribosomal subunit.

Base Sequence↗
PubMed1977

Stimulation, by two Escherichia coli supernatant proteins, of the initiation of polypeptide synthesis.

Two protein factors (A and B) have been partially purified from Escherichia coli supernatant which, in combination, are more effective than 0.5 M NH4Cl in stimulating ribosomes for AcPhe-tRNA and fMet-tRNA binding, for the puromycin reaction, and for incorporating acetylphenylalanine from AcPhe-tRNA into polypeptide. The factors appear to differ from the initiation factors, the elongation factor EF-T, and ribosomal proteins. Some uncertainty exists as to whether factor B is different from EF-G. To maximize the effect of the factors in initiator tRNA binding, we preincubated the ribosomes with the factors and carried out the binding assay for a short period at 15 degrees C. Maximal stimulation of binding occurred after about a 2-min preincubation at 37 degrees C. Longer preincubation times were required at 15 degrees C, and only slight stimulation was observed after preincubation at 0 degrees C. The extent of stimulation by the factors was not affected when the NH4Cl concentration was increased from 40 to 500 mM in the preincubation. The presence of both the 30S and 50S ribosomal subunits is required for the enhancement of AcPhe-tRNA binding. Polyphenylalanine synthesis carried out without AcPhe-tRNA is inhibited by the factors. It is suggested that the factors may act by inducing a structural rearrangement of the ribosomes.

Bacterial Proteins↗
PubMed1977

Effect of protein-energy malnutrition on the growth of mandible and long bone in newborn male and female rats.

Several parameters were measured in the growth centers of mandible and long bone of rat pups suckled on dams fed 25% or 6% protein diets. No sex-related differences were found in any parameter either during normal growth or in response to malnutrition. While the total contents of protein, DNA, RNA and calcium were reduced in both tissues, mandible was less affected than long bone, although it appeared to be affected earlier. Moreover, malnutrition reduced the number of cells in the mandible without changing cell size or calcium content. In contrast, long bone appeared to be affected later than mandible, and to be modified differently. In this case, not only cell number, but also cell size and cellular content of calcium were reduced. The results can be interpreted to mean that the critical periods of growth for mandible and long bone are different. Moreover, some suggestion of differences in relative sensitivity to malnutrition of these two hard tissues was also observed.

Age Factors↗
PubMed1975

Kinetic evidence for the obligatory formation of a 30S initiation complex in polyphenylalanine synthesis initiated with N-acetylphenylalanyl-5RNA.

The problem of whether the initiation of bacterial protein synthesis involves the obligatory formation of a 30S initiation complex intermediate was examined in a model system with N-acetylphenylalanyl-tRNA as initiator 5RNA nad poly(uridylic acid) as mRNA. The time courses of the formation of the 30S and 70S initiation complex with Escherichia coli ribosomes were measured simultaneously by stopping the reaction with dextran sulfate and differentiating the N-acetylphenylalanyl-tRNA bound to 30S ribosomal subunits from that bound to 70S ribosomes with RNase I, which hydrolyzes N-acetylphenylalanyl-tRNA bound to 30S subunits but not that bound to 70S ribosomes. A maximum in the 30S complex concentration was observed within the first 10-15 sec of the reaction, whereas 70S complex formed formed more slowly with a slight initial time lag. When an analog computer was programmed with rate constants determined separately for the formation of the 30S initiation complex from preformed 30S complex, kinetic curves very similar to the empirical curves were obtained for the entire time course of the reaction. The results show clearly that formation of the 70S complex obeys the kinetic laws for consecutive reactions, and the 30S complex is, therefore, an obligatory intermediate in the initiation of polyphenylalanine synthesis in the model system.

Binding Sites↗
PubMed1974

Requirement of initiation factor 3 in the initiation of polypeptide synthesis with N-acetylphenylalanyl-tRNA.

Initiation factor 3 is required, along with initiation factors 1 and 2, for the incorporation of N-acetylphenylalanine into polypeptides and the formation of N-acetylphenylalanylpuromycin. Initiation factor 3 also strongly stimulates the binding of N-acetylphenylalanyl transfer RNA to isolated 30S ribosomal subunits. Phosphocellulose fractions of initiation factor 3 were found to catalyze N-acetylphenylalanine incorporation differentially with different synthetic messenger RNAs not containing any codons for N-formylmethionine. The results suggest that ribosomes recognize the initiator codon only through the initiator transfer RNA.

Binding Sites↗