MECHANISM OF ACTION OF STREPTOMYOCIN IN E. COLI: INTERAPTION OF THE RIBOSOME CYCLE AT THE INITATION OF PROTEIN SYNTHESIS.
By L. Luzzatto
There are several kind of antibiotics with a different antimicrobial mechanism which can be used against gram positive and negative bacteria. There are also many kinds of classifications can be used in order to classify the antibiotics. Some of these antibiotics have a broad-spectrum bacterial effect whereas others have narrow- spectrum bacterial effect. Some of them have bactericidal (they kill the bacteria) impact and others have bacteriostatic (they inhibit the bacterial growth or replication) impact. Some antibiotics can be both bactericidal and bacteriostatical.
Different antibiotics have different modes of action and target sites within bacterial cells. There are five basic mechanisms of antibiotic action against bacterial cells;
- Inhibition of cell wall synthesis
- Inhibition of protein synthesis (translation)
- Alteration of cell membranes
- Inhibition of nucleic acid synthesis
- Antimetabolite activity
Inhibition of cell wall synthesis is the most common mechanism of antibiotics. Second largest class antibiotics are showing their antimicrobial effect by inhibiting the translation mechanism in the cell. In the paper of Luzzatto and colleagues, they proved the antimicrobial mechanism of streptomycin on Escherichia coli by the paper published on 1968.
There are a couple of ways of antibiotics inhibiting translation in the cell. These are;
- tRNA mimicry
- Inhibitors of peptide-bone formation
- Inhibitors of binding of tRNA to the A site
- Inhibitors of translocation
- Binding to 23S RNA
- Binding to the 30S ribosome
Streptomycin belongs to aminoglycoside class antibiotic and it is known as a protein synthesis inhibitor.
Back at 1964, in a study called “Streptomycin, Suppression and the Code” conducted by Julian Davies and Walter Gilbert, they were aware of the protein synthesis inhibiting the power of streptomycin, however, the mechanism was unknown. Previous studies of this study had suggested that streptomycin blocked the protein synthesis by strongly binding to nucleic acids. Julian Davies had concluded that streptomycin had done some alterations in the coding properties. Moreover provided the evidence that the ribosomes control the accuracy of the reading and may have a role in suppression. After that study, in 1968 Luzzatto lightened the unknown mechanism of streptomycin inhibiting the translation by this research.
In the paper of Luzzatto, they found that a certain concentration (lethal concentration) of streptomycin cause the accumulation of 70S ribosomes in E. coli cells. These ribosomes are incapable of doing translation in the cell. These 70S ribosomal units that accumulated were called “streptomycin monosomes”. Moreover, they found that these units consist of a complex of 30S and 50S subunits, tRNA, mRNA, and streptomycin. They observed that the 70S subunits which consist streptomycin had abnormal initiation complexes that cannot elongate; therefore they accumulate. So, they conclude that streptomycin molecules somehow “freeze” the protein initiation in E. coli.
According to their findings; Streptomycin blocks bacterial protein synthesis at initiation. After intact bacteria are exposed to streptomycin, polysomes become rapidly depleted and 70S particles. “The streptomycin monomers” build up. Although the formation of initiation complex is not affected, the complex formed in the presence of streptomycin cannot synthesize protein and remains fixed in the position. It is proposed that ribosome beyond the initiation stage are able to continue their movement and detachment so that a 70S ribosomal complex of mRNA and 50S and 30S units with bound streptomycin results. In effect, the initiation complex is frozen.
In their experiment, they used Escherichia coli mutant sud 24 and grow it in fragile form to be able to lyse and analyze during the translations. To trace the RNA during translations; they used radioactively labelled RNAs. They measured the speed of the molecules by using sucrose gradient analysis to determine the size distribution.
They used a streptomycin resistant derivative (N21) and susceptible AB301 strain, labelled ala-transfer tRNA, and labeled F-met tRNA, natural mRNA (f-met dependent), synthetic mRNA (poly AUG, f-met independent) and also they used a phage protein called R17.
Their results were;
- Polyribosome metabolism in cultures treated with streptomycin
At the moment they add streptomycin the ribosome was the 30S and the 50S form as well as bound to mRNA. After 20 and 40 min intervals; they released (a) a decrease in large polyribosomes and in free 30S and 50S particles. (b) Accumulation of 70S monomers. (fig.1)
- Streptomycin blocks the function of natural mRNA.
The protein synthesis directed by the phage R17 was observed alone with streptomycin and streptomycin was observed alone without the phage R17. Their finding had shown that streptomycin blocked the function of the natural mRNA (fig. 2)
- Streptomycin blocks normal initiation of protein synthesis
Streptomycin blocked the RNA synthesis directed by natural mRNA but not with the synthetic mRNA. The natural mRNA initiates the protein synthesis by using f-met however the synthetic mRNA doesn’t require the f-met to start the translation. Thereby, they released that the streptomycin was blocking somehow the f-met boundary on the 30S ribosomal subunit to and block the F-met to come and attached in order to initiate the translation process. However, the synthetic RNA experiment did not block by adding the streptomycin. The amount of S35 f-met-tRNA was reduced by streptomycin and H3-ala-tRNA bound to ribosomes.
They realized that the initiated protein synthesis with 70S ribosomes was not blocked and continue when streptomycin added but only new translation processes did not initiate. So, by using the synthetic mRNA and natural and also by using a radioactive label with the knowledge of f-met requirement for the translation they were able to understand this mode of action of streptomycin was at the initiation right after ribosomal subunits association.