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Structure and function of ribosomal RNA
H F Noller1, R Green, G Heilek
1Center for Molecular Biology of RNA, Sinsheimer Laboratories, University of California, Santa Cruz 95064, USA.
Biochemistry and Cell Biology = Biochimie Et Biologie Cellulaire
|November 1, 1995
Summary
A refined 30S ribosomal subunit model incorporates new experimental data to detail 16S rRNA folding and essential tRNA binding sites. This research clarifies the Watson-Crick base pair interaction crucial for peptidyl transferase activity.
Area of Science:
- Structural Biology
- Molecular Biology
- Biochemistry
Background:
- The 30S ribosomal subunit plays a critical role in protein synthesis.
- Understanding the precise folding of 16S ribosomal RNA (rRNA) and its interactions with transfer RNA (tRNA) is essential for elucidating translation mechanisms.
Purpose of the Study:
- To develop a refined model of 16S rRNA folding within the 30S ribosomal subunit.
- To identify nucleotides in 16S rRNA critical for P-site tRNA binding.
- To investigate the base-pairing interactions between tRNA and 23S rRNA, and their role in peptidyl transferase activity.
Main Methods:
- Hydroxyl radical footprinting of individual 30S ribosomal proteins.
- Localized hydroxyl radical cleavage using Fe2+ tethered to ribosomal proteins.
- Modification interference strategy to identify essential bases for tRNA binding.
- Hydroxyl radical probing of rRNA neighborhood near the tRNA acceptor end.
- In vitro genetics and chemical probing to demonstrate and test tRNA-23S rRNA base-pairing interactions.
- Chimeric in vitro reconstitution to assess peptidyl transferase activity.
Main Results:
- A refined 30S subunit model was generated using hydroxyl radical footprinting and localized cleavage data.
- Four essential bases (G926, 2mG966, G1338, G1401) in 16S rRNA were identified for P-site tRNA binding.
- Hydroxyl radical probing revealed rRNA neighborhood interactions with the tRNA acceptor end, particularly in 23S rRNA domains.
- A specific Watson-Crick base-pairing interaction between tRNA (C74) and 23S rRNA (2252) was demonstrated to be essential for tRNA-ribosome interaction.
- The peptidyl transferase reaction was shown to depend on this Watson-Crick base pair.
Conclusions:
- The study provides a more accurate model of 16S rRNA folding in the 30S subunit.
- Key nucleotides essential for P-site tRNA binding and their location within the 30S subunit model were identified.
- A direct base-pairing interaction between tRNA and 23S rRNA is crucial for the peptidyl transferase activity of the ribosome.