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Ribosome initiation complex formation with the pseudoknotted alpha operon messenger RNA
G Spedding1, T C Gluick, D E Draper
1Department of Chemistry, Johns Hopkins University, Baltimore, MD 21218.
Journal of Molecular Biology
|February 5, 1993
Summary
The Escherichia coli alpha mRNA pseudoknot is stable and influences translation initiation. Its structural transition regulates the rate of ternary complex formation, impacting protein synthesis.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- The Escherichia coli alpha mRNA possesses a complex pseudoknot structure crucial for translational regulation.
- This pseudoknot serves as a binding site for ribosomal protein S4 and regulates ribosome binding.
Purpose of the Study:
- To determine the stability of the alpha mRNA pseudoknot during translational initiation.
- To elucidate the role of the pseudoknot structure in the kinetics of initiation complex formation.
Main Methods:
- Calorimetry and ultraviolet hyperchromicity were used to monitor RNA thermal denaturation.
- Reverse transcriptase primer extension assays ('toeprints') measured the kinetics of ternary complex formation.
Main Results:
- The alpha mRNA secondary structure exhibits a stability of -7.4 kcal/mol at 37°C.
- A transition around 38°C suggests pseudoknot rearrangement.
- Translational initiation occurs in fast and slow phases, dependent on mRNA conformation and temperature.
- The inactive mRNA conformation stabilizes the pseudoknot, hindering tRNA binding.
Conclusions:
- The pseudoknot structure is stable and influences translational initiation kinetics.
- An inactive-to-active transition of the pseudoknot is essential for tRNA binding and is rate-limiting at lower temperatures.
- mRNA structure impacts both ribosome binding affinity and kinetic steps in initiation complex formation.