Ribosome or Ribozyme?

Welcome to Transformative Advances in Molecular Biology, a YouTube series prepared by graduate students in a journal colloquium at the University of Florida. Dr. Mark Settles and I decided that instead of the traditional discussion of contemporary science literature, that it'd be fun and informative to look backwards to review the seminal discoveries that seeded contemporary molecular biology. This series offers student presentations of milestone papers in this area.

The Mechanism of Translation, by Tufan Gökermek and Chip Hunter. Over the course of the past 60 years, the manner of how mRNA transcripts are translated into proteins has been gradually elucidated. As an mRNA molecule progresses through a ribosome, 3-base codons are recognized by certain tRNAs covalently attached to specific amino acids. As the homologous tRNAs move from the ribosome's aminoacyl site, or A site, to the peptidyl site, or P site, a peptide bond is formed between the amino acid on the donor tRNA and an elongating polypeptide chain, which eventually forms a protein. The mechanism of how the ribosome catalyzes this reaction, that of peptide bond formation, has been one of the primary focuses of researchers since the discovery of the central dogma. Here we will present the evidence that has led to our current understanding of the process of translation.

In this presentation, we discuss the mechanism of translation, addressing the question of whether the ribosome acts as an enzyme or ribozyme, a question that was debated for many years. We'll be going through a Science paper published by Nielsen et al. in 2000, which has shaped our current understanding of how the ribosome functions. It has been shown that The peptidyl transferase activity responsible for peptide bond formation is intrinsic to the large subunit of the ribosome. The ribosome consists of both proteins and RNA. In this figure, you see the protein components of large and sub small subunits of the ribosome in purple, and RNA components in pink. In bacteria, the large ribosomal subunit contains about 35 different proteins and two RNAs. By 1980. The list of components potentially responsible for the enzymatic activity of the ribosome had been reduced to a half-dozen proteins and the 23S rRNA of the large subunit. After the discovery of catalytic RNAs, it was proposed that 23S rRNA could be the sole catalytic unit.

In this diagram, you can see the secondary structure of the large ribosomal subunit. In 1984, Knoller and colleagues published results showed— that showed that bases U2619 and U2620 of the 23S rRNA are adjacent to the CCA end of P-site-bound transfer RNA, which is where the active site of the ribosome is expected to be. These nucleotides are part of a highly conserved internal loop in the center of domain 5 of the 23S rRNA. The hypothesis that this loop is intimately involved in the peptidyl transferase activity has been supported by mutations in this region and by deprotonation studies of large ribosomal subunits, which were able to remove all ribosomal proteins except for 2, proteins L2 and L3, while maintaining peptidyl transferase activity. Indeed, there was one report by Watanabe et al. that claimed success in eliciting peptidyl transferase activity from in vitro synthesized protein-free 23S rRNA.

In this presentation, we will show the evidence that answers these 3 questions. 1, which of the micromolecular components of the large ribosomal subunit contribute to its peptidyl transferase activity? And are they proteins or RNA components? 2, where is the active site located in the ribosome? And 3, what's the mechanism of translation?

Here you see the structure of the tRNA molecule. The anticodon loop recognizes the complementary mRNA coding sequence, and the 3' aminoacyl acceptor binds to the corresponding amino acid through the— through an ester bond. By knowing where tRNAs bind in ribosomes, we can predict the approximate location of the active site.

The study we will discuss now utilizes translational inhibitors that stably bind to the ribosome. The ARIS tetrahedral intermediate analog mimics a tRNA at the time of peptide bond formation. It contains the antibiotic molecule, pramycin. The RS reagent binds to the ribosome in an AP transition state, meaning that it is partially in both the A site and the P site at the same time. The N-aminoacylated mini-helix mimics the acceptor stem of an amino acid-bound tRNA and stably binds to the A site. By crystallizing ribosomes in the presence of these inhibitors and showing where they bind, the precise location of the active site can be determined. elucidated. Shown here is a difference electron density map between ribosomes bound and not bound by the ARIS reagent. As you can see, the ARIS reagent fits almost perfectly in the space difference between these 2 states.

Here is the crystal structure of the ribosome bound to puromycin. In this space-filling model, proteins are colored blue, ribosomal RNAs are orange and white, And the inhibitor is colored red. This structure provides strong evidence that the ribosome acts as a ribozyme, as the inhibitor is bound to an area of the large subunit composed entirely of rRNA.

Here you see the, you see the combined model of the CCA portion of the mini-helix bound to the A site and the ARIS region bound to the AP transition site. Watson-Crick base pairing occurs between C74 and C75 of fromycin and G2285 and G2284 of P-site. Watson-Crick base pairing happens between C75 of mini-helix and G2588 of A-loop. This indicates that these, these bases of the 23S rRNA are found at the ribosome's active site.

In this view, you can see that no ribosomal protein comes within 18 angstroms of the ribosome's active site, Which is much too far away to influence catalysis of peptide bond formation. The RNA that surrounds the substrate analogs is closely packed, much like the active site of a protein enzyme, and the nucleotides, colored red here, in contact with the inhibitor are 95% conserved in all 3 kingdoms of life. Indeed, these RNAs are so tightly packed around the active site that there is physically no way that some other protein is present when the peptidyl bond formation is catalyzed. Therefore, the ribosome must act as a ribozyme, with the ribosomal RNA itself forming the active site in which the peptide bond formation occurs.

As for the actual mechanism of how peptide bond formation is catalyzed, it must involve the RNA bases found at the active site.

Indeed, it is the adenine base of the nucleotide A2486 that serves as the proton acceptor allowing nucleophilic attack of the A site-bound tRNA's amino group on the carbonyl carbon that acylates the 3' hydroxyl of the tRNA in the P site. This reaction is analogous to the reverse of the acylation step in serine proteases during peptide hydrolysis.

Thus, acting like an enzyme by precisely positioning the substrates and providing the correct local environment for peptide bond formation, In an area solely made up of RNA, the ribosome acts as a ribozyme.

Thank you for watching, and please check out the other exciting topics in the series.