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Abstract:
Bacteriophage R17 and its RNA were found to contain significant amounts of spermidine but not of putrescine. When isolated at 0.01 M KCl, up to 1,000 molecules of spermidine were associated with the virion. The phage RNA isolated with phenol plus sodium lauryl sulfate contained approximately 70 to 90 molecules of spermidine. The association appeared to be ionic because the bound spermidine could be dissociated by KCl, MgCl2, or both. Effects of polyamines on in vitro translation were studied using both poly(U) and phage R17-RNA as mRNA. Addition of spermidine to the system at suboptimal concentrations of Mg2+ resulted in marked stimulations of the rate of protein synthesis. Putrescine alone had no effect but stimulated the incorporation in the presence of suboptimal concentrations of spermidine plus Mg2+. The isolated amino acid-incorporating system contained suboptimal soluble and bound polyamines. A comparison of incorporation was made in this system using R17-RNA with and without bound spermidine. No effects of these bound cations were detected on the rate or extent of incorporation of valine. The ratio of incorporation of histidine (present in non-coat proteins) to valine (total protein) revealed little difference as a functions of cation in the system or a function of the spermidine present in R17-RNA.
Insights
Bacteriophage R17 virions and RNA contain spermidine, a polyamine that enhances in vitro protein synthesis. This spermidine binding is ionic and affects translation rates, particularly under suboptimal magnesium conditions.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- Bacteriophages are viruses that infect bacteria and have been instrumental in understanding molecular biology.
- Polyamines, such as spermidine and putrescine, are small organic molecules with biological significance in nucleic acid stabilization and gene expression.
- The interaction between viral components and polyamines is crucial for viral structure and function.
Purpose of the Study:
- To investigate the presence and binding characteristics of polyamines, specifically spermidine and putrescine, in bacteriophage R17.
- To elucidate the role of spermidine in the in vitro translation process using phage R17 RNA as a messenger RNA (mRNA).
- To determine the impact of polyamines on protein synthesis rates and amino acid incorporation.
Main Methods:
- Isolation of bacteriophage R17 and its RNA.
- Quantification and characterization of polyamine content (spermidine, putrescine) within the virion and isolated RNA.
- Ionic dissociation experiments using varying concentrations of KCl and MgCl2 to study polyamine binding.
- In vitro translation assays using poly(U) and R17 RNA as mRNA, with varying concentrations of spermidine, putrescine, and Mg2+.
- Analysis of amino acid incorporation rates (histidine and valine) in isolated systems.
Main Results:
- Bacteriophage R17 virions and RNA were found to contain significant amounts of spermidine, but not putrescine.
- Spermidine associated with the virion is ionically bound and can be dissociated by salts (KCl, MgCl2).
- Spermidine significantly stimulated in vitro protein synthesis rates when Mg2+ concentrations were suboptimal.
- Putrescine showed no effect alone but enhanced incorporation in the presence of suboptimal spermidine and Mg2+.
- Bound spermidine on R17 RNA did not affect the rate or extent of valine incorporation in isolated systems.
Conclusions:
- Spermidine is a key polyamine associated with bacteriophage R17, playing a role in its structure and function.
- Spermidine enhances in vitro translation, particularly under conditions of limited magnesium, suggesting a role in stabilizing mRNA or the translation machinery.
- The ionic nature of spermidine binding and its stimulatory effect on protein synthesis highlight the importance of polyamines in viral gene expression.