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Puromycin reaction for the A site-bound peptidyl-tRNA
Y u Semenkov1, T Shapkina, V Makhno
1B.P. Konstantinov Petersburg Nuclear Physics Institute, Academy of Sciences, Saint Petersburg district, USSR.
Abstract:
AcPhe2-tRNA(Phe) synthesized in 70S ribosomes after consecutive binding of AcPhe-tRNA(Phe) at the P sites and EF-Tu-directed binding of Phe-tRNA(Phe) at the A sites is able to react quantitatively with puromycin in the absence of EF-G. A detailed study of the kinetics of the puromycin reaction, its comparison with that of spontaneous translocation, the use of antibiotic viomycin as an effective inhibitor of spontaneous translocation revealed that, besides spontaneous translocation, this peptidyl-tRNA could react with puromycin being located at the A site. This leads to the conclusion that the transpeptidation reaction per se triggers conformational changes in the ribosomal complex bringing the 3'-end of a newly synthesized peptidyl-tRNA nearer to the peptidyl-site of the peptidyltransferase center. This is detected functionally as the ability of such an A site bound peptidyl-tRNA to react with puromycin. This reaction is highly pronounced at elevated (25 degrees C) temperature but can be hardly detected at 0 degrees C.
Insights
This study reveals that peptidyl-tRNA can react with puromycin while bound to the ribosomal A site, not just after translocation. This occurs due to conformational changes triggered by the transpeptidation reaction itself.
Area of Science:
- Molecular Biology
- Biochemistry
- Ribosome Function
Background:
- Peptidyl transfer RNA (tRNA) plays a crucial role in protein synthesis.
- Understanding the precise positioning and reactivity of peptidyl-tRNA within the ribosome is essential for elucidating translation mechanisms.
Purpose of the Study:
- To investigate the reactivity of peptidyl-tRNA with puromycin at the ribosomal A site.
- To determine the role of transpeptidation and ribosomal conformational changes in this reactivity.
Main Methods:
- Kinetic analysis of the puromycin reaction with peptidyl-tRNA.
- Comparison with spontaneous translocation.
- Utilizing the antibiotic viomycin as an inhibitor of spontaneous translocation.
Main Results:
- AcPhe2-tRNA(Phe) at the P site and Phe-tRNA(Phe) at the A site react quantitatively with puromycin without EF-G.
- Peptidyl-tRNA at the A site can react with puromycin, independent of spontaneous translocation.
- This A site reactivity is enhanced at 25°C and minimal at 0°C.
Conclusions:
- The transpeptidation reaction induces ribosomal conformational changes that bring the peptidyl-tRNA's 3'-end closer to the peptidyltransferase center.
- This functional change allows A site-bound peptidyl-tRNA to react with puromycin.
- Temperature significantly influences the rate and extent of this ribosomal conformational change and subsequent reactivity.