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In vitro translation studies of the cytoplasmic nonpolysomal particles containing messenger RNA
Abstract:
We analyzed the translational capacity of different kinds of free cytoplasmic messenger ribonucleoprotein complexes (free mRNP) in a Hela cell cell free system. Native free mRNP are not translated although free mRNP washed with 0.5 M KC1 can direct polypeptide synthesis. Furthermore, the 0.5 M KC1 wash possesses a factor which inhibits the translation of 0.5 M KC1 washed free mRNP as well as globin mRNA naked mRNA from plasmocytoma, or Hela cells. We also demonstrated that native free mRNP are able to form a complex with ribosomal subunits in the presence of initiation factors. This indicates that inhibition of translation by the 0.5 M KC1 wash occurs either at some point after initiation complex formation or at the elongation step.
Insights
Native messenger ribonucleoprotein complexes (mRNP) are not translated. A 0.5 M KCl wash removes an inhibitory factor, restoring translational capacity and revealing translation occurs post-initiation or during elongation.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Cytoplasmic messenger ribonucleoprotein complexes (mRNP) are essential for gene expression.
- The translational control mechanisms of free mRNP are not fully understood.
Purpose of the Study:
- To investigate the translational capacity of different free mRNP populations.
- To identify factors regulating mRNP translation.
Main Methods:
- Utilized a Hela cell-free system to analyze mRNP translational activity.
- Fractionated mRNP using 0.5 M KCl washes.
- Assessed polypeptide synthesis and complex formation with ribosomal subunits.
Main Results:
- Native free mRNP showed no translational activity.
- 0.5 M KCl-washed free mRNP regained the ability to direct polypeptide synthesis.
- The 0.5 M KCl wash contained an inhibitory factor affecting translation of various mRNAs.
- Native free mRNP formed initiation complexes with ribosomal subunits.
Conclusions:
- Translation inhibition by the 0.5 M KCl wash occurs after initiation complex formation or during elongation.
- A regulatory factor in native free mRNP inhibits translation, which can be removed by salt washing.