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Initiation of protein synthesis. Binding of messenger RNA
Abstract:
Complexes between 30 S ribosomal subunits and fMet-tRNA are formed during incubation of 30 S subunits with fMet-tRNA and all other components for initiation of protein synthesis, except R17 bacteriophage RNA. That these complexes serve as intermediates in the binding of messenger RNA is demonstrated directly by the finding that upon addition of R17 RNA, fMet-tRNA in preformed fMet-RNA-30 S complexes preferentially enters fMet-tRNA-30 S-R17 RNA complexes. On the other hand, incubation of 30 S ribosomal subunits with R17 RNA and all other components for initiation except fMet-tRNA does not yield 30 S-R17 RNA complexes that can act subsequently as functional intermediates in the binding of fMet-tRNA: formation of fMet-tRNA-30 S-R17 RNA complexes does not occur when fMet-tRNA is added and further binding of R17 RNA to 30 S subunits is prevented by specific inhibitors. These experiments lead to an unambiguous order of events in the sequence of initiation, in which binding of fMet-tRNA to the small ribosomal subunit must occur before messenger RNA can be bound and phased correctly. Complexes between fMet-tRNA and 60 S subunits are in rapid equilibrium with the free components, and have a half-life of less than 2 min at 37 degrees. This explains why such complexes are not detected in sucrose gradients, unless they are first fixed with glutaraldehyde. Attachment of R17 RNA, however, results in formation of an fMet-tRNA-30 S-R17 RNA complex that is stabilized greatly; fMet-tRNA in this complex exchanges only very slowly with free fMet-tRNA. Initiation factor IF-3 has two functions in initiation. The first is to direct the binding of messenger RNA to the 30 S-fMet-tRNA complex. This function is not needed when initiation complex formation occurs on ApUpG triplets, in which case the second function of IF-3 is detected, that of providing free 30 S subunits for initiation. The ability of IF-3 to bind directly to R17 RNA may be related to its requirement in messenger RNA recognition. However, since IF-3 exhibits a greater affinity for the 30 S subunit than for R17 RNA, it appears that the recognition function of IF-3 is expressed while IF-3 is associated with the 30 S subunit.
Insights
Messenger RNA (mRNA) binding to ribosomal subunits requires prior binding of initiator tRNA. This study clarifies the precise order of events in protein synthesis initiation, establishing a foundational sequence for molecular biology research.
Area of Science:
- Molecular Biology
- Protein Synthesis
- Ribosome Function
Background:
- Protein synthesis initiation is a complex process involving ribosomal subunits, initiator tRNA, and mRNA.
- The precise order of binding events for these components is crucial for accurate translation.
- Bacteriophage R17 RNA serves as a model mRNA for studying initiation complex formation.
Purpose of the Study:
- To determine the sequential order of binding between the 30S ribosomal subunit, fMet-tRNA, and R17 RNA during protein synthesis initiation.
- To elucidate the role of initiation factor IF-3 in mRNA recognition and 30S subunit availability.
Main Methods:
- Formation and analysis of pre-initiation complexes using 30S ribosomal subunits, fMet-tRNA, and R17 RNA.
- Utilizing specific inhibitors to block certain binding events and observe subsequent complex formation.
- Investigating the stability and exchange kinetics of formed complexes using techniques like sucrose gradient centrifugation.
Main Results:
- Preformed complexes of 30S subunits and fMet-tRNA directly bind R17 RNA, confirming fMet-tRNA binding precedes mRNA binding.
- Complexes lacking fMet-tRNA do not form functional intermediates for subsequent fMet-tRNA binding.
- Initiation factor IF-3 has dual roles: directing mRNA binding and providing free 30S subunits, with a higher affinity for the 30S subunit.
Conclusions:
- The binding of fMet-tRNA to the 30S ribosomal subunit is a mandatory prerequisite for messenger RNA binding and correct phasing during protein synthesis initiation.
- Initiation factor IF-3 plays a critical role in both mRNA recognition and ensuring the availability of 30S subunits for initiation.
- Understanding this ordered sequence is fundamental for comprehending the fidelity and regulation of gene expression.