Related Experiment Videos
Binding of wheat germ ribosomes to fragmented viral mRNA
Abstract:
The specificity of binding of wheat germ ribosomes to mRNA was greatly altered by cleavage of the message. Fragmentation of reovirus mRNA allowed wheat germ ribosomes to bind and protect a variety of internal sequences which were not accessible to ribosomes in the intact message. In experiments using the polycistronic mRNA from bacteriophage R17, wheat germ ribosomes bound preferentially at the beginning of the lysis peptide and synthetase cistrons, and at a third site which may be derived from the C-terminal region of the A protein cistron. This result is similar to that reported previously in a mammalian translational system (J.F. Atkins et al., Cell 18:246-256, 1979) except that, in the present study, limited cleavage of the phage RNA was necessary to activate these sites. More extensive fragmentation of R17 RNA permitted wheat germ ribosomes to bind and protect a great many additional sites. Thus, presence of an (exposed) 5'-terminus on an RNA molecule appears to be necessary and sufficient for attachment of eucaryotic ribosomes.
Insights
Wheat germ ribosomes bind to mRNA. Cleaving mRNA exposes internal sequences, allowing ribosome binding to new sites, indicating a necessary 5
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Ribosome binding to messenger RNA (mRNA) is a crucial step in protein synthesis.
- The specificity of ribosome-mRNA interaction dictates translation initiation.
- Eukaryotic ribosomes exhibit distinct binding characteristics compared to prokaryotic systems.
Purpose of the Study:
- To investigate how mRNA cleavage affects the binding specificity of wheat germ ribosomes.
- To identify internal mRNA sequences that become accessible to ribosomes upon fragmentation.
- To compare ribosome binding sites in wheat germ with those in mammalian systems.
Main Methods:
- Fragmentation of reovirus and bacteriophage R17 mRNA.
- Incubation of fragmented mRNA with wheat germ ribosomes.
- Analysis of ribosome-protected mRNA sequences.
Main Results:
- Cleavage of reovirus mRNA enabled wheat germ ribosomes to bind internal sequences.
- Wheat germ ribosomes preferentially bound to specific sites on fragmented bacteriophage R17 mRNA, including lysis peptide and synthetase cistrons.
- Limited mRNA cleavage was required to activate these binding sites, similar to mammalian systems.
- Extensive fragmentation led to binding at numerous additional sites.
Conclusions:
- The 5'-terminus of an RNA molecule is necessary and sufficient for eukaryotic ribosome attachment.
- mRNA integrity influences ribosome binding specificity.
- Wheat germ ribosomes can recognize and bind to internal mRNA sequences when accessible.