Protein synthesis in E. coli

Early studies by Terawaki and Greenberg on the antibiotic activity of carzinophilin established that it inhibited DNA synthesis but not RNA or protein synthesis in E. coli strain Bo and in Bacillus subtilis [134]. They also found that exposure to carzinophilin removed the transforming capacity of B. subtilis DNA [135]. They... 

TABLE 27-5 Components Required for the Five Major Stages of Protein Synthesis in E. coli... 

The bulk of the cellular RNA is ribosomal RNA. Although seven genes exist in E. coli for rRNA, they all lead to essentially the same three ribosomal RNA molecules (see table 28.1) which differ substantially in size. The three rRNAs are always found in a complex with proteins in a functional component known as the ribosome. The ribosome is the site where mRNA and tRNAs meet to engage in protein synthesis. In E. coli, ribosomes are referred to as 70S particles, a measure of their rate of sedimentation and hence their size (S refers to Svedberg units, which are defined in chapter 6). A 70S ribosome consists of two dissociable subunits A 50S subunit and a 30S subunit. Each of these contains both RNA and protein. The 50S subunit contains 23S and 5S rRNAs. The 30S subunit contains a single 16S rRNA (fig. 28.5). Eukaryotic ribosomes are similar in structure, although they are somewhat larger (80S) and con-... 

Nirenberg, M. W., and J. H. Mattaei, The dependence of cell-free protein synthesis in E. coli upon naturally occurring or synthetic polyribonucleotides. Proc. Natl. Acad. Sci. 

The possible role of guanosine 3 -pyrophosphate-5 -triphosphate (30) in protein synthesis in E. coli has been examined. Although it can substitute for GTP reactions catalysed by initiation factor (IF)2 and elongation factor... 

The third approach used repeating ribonucleotide polymers containing known repeating sequences (Fig. 26.4C). When these were used as templates for in vitro protein synthesis, it was found that each ribonucleotide polymer could specify as many as three different repeating polypeptide products. Of the 64 possible codons, 61 were found to specify amino acids and 3 were later defined as termination codons. Figure 26.5 shows the genetic code for protein synthesis in E. coli, which is for the most part applicable to mRNA translation in mammalian cells. Note that methionine and tryptophan are only specified by single codons, whereas almost all the other amino acids have from two to four codons (except leucine and serine which are encoded by six codons). Methionine is the first amino acid in essentially all proteins however, methionine residues are also found within the polypeptide sequence. The amino-terminal methionine is called the initiator methionine. 

A number of workers have shown that novobiocin inhibits protein synthesis in bacteria. For example, it inhibits /S-galactosidase synthesis in both Staph, aureus and E. coli [48, 64]. Although M-protein synthesis in a Group A streptococcus is not inhibited by novobiocin [70], the inhibition of protein synthesis in Strept. faecium has been attributed to the inhibition of tRNA synthesis [23]. Novobiocin inhibits protein synthesis in Staph, aureus [26], but as the inhibition of other macro-molecules also occurs to a similar extent, this effect may not be the primary action of the antibiotic. The antibiotic also inhibits protein synthesis in E. coli [33, 71] but this inhibition appears much later than the observed inhibition of deoxyribonucleic acid (DNA) synthesis. 

Figure 2.4. Effect of novobiocin on (a) growth, as recorded by changes in optical density, (b) DNA synthesis, (c) RNA synthesis, and (d) protein synthesis in E.coli in nutrient broth at 37°C. Novobiocin concentrations (ng/ml) 0, 0—0 20, — 100, — 500, A—A. (From Morris and Russell [72], by courtesy of Microbios.)...

Provided the synthesis of virus-specific RNA is dependent on the prior production of a virus-specific polymerase, RNA synthesis should be inhibited or abolished by the presence of inhibitors of protein synthesis. Protein synthesis in E, coli was more than 90% inhibited by either puromycin or chloramphenicol, and the fate of single-stranded RNA in E, coli investigated. The rate of conversion of viral RNA into double-stranded RNA was unaffected by chloramphenicol but was reduced to about half in the presence of puromycin. These results indicate that an RNA polymerase is already present in E, coli. 

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