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Regulation of protein synthesis in mitotic HeLa cells
Abstract:
Mitotic HeLa cells (M cells) synthesize protein at about 25% of the rate of S phase cells. This decrease in protein synthesis is due to a reduction in the rate of initiation. However, extracts prepared from M cells are almost as active in protein synthesis as S cell extracts. Both cell extracts are quite active in in vitro initiation of protein synthesis. Moreover, two steps in initiation, binding of Met-tRNAf to 40S ribosomal subunits and binding of mRNA to ribosomes, show similar activity in both extracts. The difference in protein synthesizing activity observed in vivo is largely eliminated in the preparation of cell-free systems. The ribosomes of M cells contain small mol wt RNA, which inhibits protein synthesis in vitro. This RNA, which has possibly a nuclear origin, may be a cause of the reduction in the rate of protein synthesis in M cells.
Insights
Mitotic HeLa cells show reduced protein synthesis due to decreased initiation. A small RNA molecule in mitotic cell ribosomes inhibits this process in vitro, potentially explaining the in vivo reduction.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Protein synthesis rates differ significantly between mitotic (M) and S phase HeLa cells.
- Mitotic cells exhibit approximately 25% of the protein synthesis rate observed in S phase cells.
- This reduction is primarily attributed to a decreased rate of translation initiation.
Purpose of the Study:
- To investigate the molecular mechanisms underlying the reduced protein synthesis in mitotic HeLa cells.
- To identify factors responsible for the diminished initiation rate during mitosis.
- To compare the protein synthesis and initiation activities of M cell and S cell extracts.
Main Methods:
- Preparation and analysis of cell-free extracts from mitotic (M) and S phase HeLa cells.
- In vitro assays to measure protein synthesis activity.
- Assessment of specific initiation steps: Met-tRNAf binding to 40S subunits and mRNA-ribosome binding.
- Analysis of ribosomal components from M cells for inhibitory factors.
Main Results:
- Cell-free extracts from M cells showed comparable protein synthesis activity to S cell extracts.
- In vitro initiation steps, including tRNA and mRNA binding, were similarly active in both M and S cell extracts.
- Ribosomes from M cells contained a low molecular weight RNA that inhibited in vitro protein synthesis.
Conclusions:
- The significant reduction in protein synthesis observed in vivo in mitotic HeLa cells is largely mitigated in cell-free systems.
- A small molecular weight RNA associated with M cell ribosomes acts as an inhibitor of protein synthesis in vitro.
- This inhibitory RNA, possibly of nuclear origin, is a likely cause for the decreased protein synthesis rate during mitosis.