The Ribosome Is a Ribozyme

Until recently, proteins were thought to be the only molecules with catalytic ability. Then the self-splicing ability of the Tetrahymena snRNP showed that RNA can also catalyze reactions. In 2000, the complete structure of the large ribosomal subunit was determined by X-ray crystallography to 2.4-A (0.24-nm) resolution 

Many amino acids, such as citrulline and ornithine (which are found in the urea cycle), are not building blocks of proteins. Other nonstandard amino acids, such as hydroxyproline, are formed after translation by posttranslational modification. When discussing amino acids and translation, the magic number was always 20. Only 20 standard amino acids were put onto tRNA molecules for protein synthesis. In the late 1980s, another amino acid was found in proteins from eukaryotes and prokaryotes alike, including humans. It is selenocysteine, a cysteine residue in which the sulfur atom has been replaced by a selenium atom. 

1 Components Required for Each Step of Protein Synthesis in Escherichia coli I  

Amino acid activation Amino acids tRNAs Aminoacyl-tRNA synthetases ATP, Mg2+ 

Chain initiation fmet-tRNAf Initiation codon (AUG) of mRNA 30S ribosomal subunit 50S ribosomal subunit Initiation factors (IF-1, IF-2, and IF-3) GTP, Mg2+ 

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The ribosome is a ribozyme this is how Cech (2000) commented on the report by Nissen et al. (2000) in Science on the successful proof of ribozyme action in the formation of the peptide bond at the ribosome. It has been known for more than 30 years that in the living cell, the peptidyl transferase activity of the ribosome is responsible for the formation of the peptide bond. This process, which takes place at the large ribosome subunit, is the most important reaction of protein biosynthesis. The determination of the molecular mechanism required more than 20 years of intensive work in several research laboratories. The key components in the ribosomes of all life forms on Earth are almost the same. It thus seems justified to assume that protein synthesis in a (still unknown) common ancestor of all living systems was catalysed by a similarly structured unit. For example, in the case of the bacterium E. coli, the two subunits which form the ribosome consist of 3 rRNA strands and 57 polypeptides. Until the beginning of the 1980s it was considered certain that the formation of the peptide bond at the ribozyme could only be carried out by ri-bosomal proteins. However, doubts were expressed soon after the discovery of the ribozymes, and the possibility of the participation of ribozymes in peptide formation was discussed. 

Cech, T. R. The Ribosome Is a Ribozyme. Science 289, 878-879 (2000). [A classic paper describing the RNA-based catalysis of the ribosome.]... 

The atomic structure of this subunit and its complexes with substrate analogs revealed the enzymatic activity of the rRNA backbone. Thus, the ribosome is in fact a ribozyme P Nissen, J Hansen, N Ban, PB Moore, TA Steitz. Science 289 920-930, 2000. Atomic structure of the ribosome s small 30S subunit, resolved at 5 A WM Clemons Jr, JL May, BT Wimberly, JP McCutcheon, MS Capel, V Ramakrishnan. Nature 400 833-840, 1999. The 8-A crystal structure of the 70S ribosome reveals a double-helical RNA bridge between the 50S and the 30S subunit GM Culver, JH Cate, GZ Yusupova, MM Yusupov, HF Noller. Science 285 2133-2136, 1999. 

The peptidyl transferase centre of the ribosome is located in the 50S subunit, in a protein-free environment (there is no protein within 15 A of the active site), supporting biochemical evidence that the ribosomal RNA, rather than the ribosomal proteins, plays a key role in the catalysis of peptide bond formation. This confirms that the ribosome is the largest known RNA catalyst (ribozyme) and, to date, the only one with synthetic activity. Adjacent to the peptidyl transferase centre is the entrance to the protein exit tunnel, through which the growing polypeptide chain moves out of the ribosome. 

Fig. 11. Comparison of the peptidyl transfer reaction in the ribosome and in the selected peptidyltransferase ribozyme. The ribosome contains a binding site for the peptidyl-tRNA (P-site) and for the aminoacyl-tRNA (A-site). In the selected ribozyme the binding site for the AMP-Met-Bio substrate would be analogous to the P-site. The attacking a-amino group which is bound in the A-site in the ribosome is covalently attached to the 5 -end in the ribozyme. Catalytically active RNAs preferentially attach the biotin tag onto themselves and can thus be separated from the inactive ones...

The ribosome is both the site of protein synthesis and an active participant in the process. The eukaryotic ribosome is constructed from two subunits the smaller 40S subunit and the larger 60S subunit. Basically, the 40S subunit binds the mRNA and monitors the recognihon between the mRNA codon and tRNA anticodon. The 60S subunit has the binding sites for aminoacyl-tRNAs and catalyzes the formation of peptide bonds. Remarkably, the catalytic entity for peptide bond formahon in the 60S subunit is the RNA component, not the protein component. Therefore, the 60S subunit acts as a ribozyme. 

The ribosome is where the message carried by the mRNA is translated into the amino sequence of a protein. How it occurs is described in the next section. One of its most noteworthy aspects was discovered only recently. It was formerly believed that the RNA part of the ribosome was a structural component and the protein part was the catalyst for protein biosynthesis. Present thinking tilts toward reversing these two functions by ascribing the structural role to the protein and the catalytic one to rRNA. RNAs that catalyze biological processes are called ribozymes. Catalysis by RNA is an important element in origins of life theories as outlined in the accompanying boxed essay RNA World. ... 

Peptide bond formation the second step, peptide bond formation, is catalyzed by peptidyl transferase, part of the large ribosomal subunit. In this reaction the carboxyl end of the amino acid bound to the tRNA in the P site is uncoupled from the tRNA and becomes joined by a peptide bond to the amino group of the amino acid linked to the tRNA in the A site (Fig. 5). A protein with peptidyl transferase activity has never been isolated. The reason is now clear in E. coli at least, the peptidyl transferase activity is associated with part of the 23S rRNA in the large ribosomal subunit. In other words, peptidyl transferase is a ribozyme, a catalytic activity that resides in an RNA molecule (see also Topic G9). 

Translation is the process, where the RNA transcript of the DNA, the messenger RNA is translated as specified by the genetic code and new proteins are synthesized fiom aminoacids by the ribosome which is a ribozyme . 

X-ray crystallographic study of the SOS ribosomal subunit of H. marismortui has shown that macrolide antibiotics can act chiefly as inhibitors of peptidyltrans-ferase, that is, a ribozyme bearing a charge-relay system. It is likely that the poor water solubility of the macrolide antibiotic could give rise to drug deposition at a binding site near the peptidyltransferase, and that the extent of the solubility of the drug could determine bacteriostatic or bactericidal action. 

The ribosome is actually a ribozyme. There are no amino acids at the active site where the peptidyl transferase reaction occurs. Specific bases on the rRNA are believed to catalyze the reaction. 

A ribozyme is RNA that has catalytic activity without the intervention of protein at the active site. The catalytic portion of RNase P is a ribozyme. The self-splicing rRNA of Tetrahymena is the classic example, and it has recently been shown that the peptidyl transferase activity of the ribosome is actually a ribozyme. 

The authors next turn to the structure and composition of the ribosome, a molecular machine that coordinates charged tRNAs, mRNA, and proteins, leading to protein synthesis. The fact that the ribosome is now recognized to be a ribozyme, with the RNA components playing the major role in catalysis, is introduced. The experiments that showed the polarities of polypeptide formation and the translation of mRNA are presented next. Then initiation is described, and the roles of a specialized... 

EXAMPLE 5.6 An example of an RNA-dependent catalytic reaction that is arguably one of the most important in any cell is the RNase P process in tRNA it is present in all known forms of life. The reaction takes place on the large ribosomal subunit, hence the ribosome is an ancient ribozyme The mechanism of the reaction involves stabilization of a transition-state complex (see Prob. 5.3) by the formation of hydrogen bonds, like that shown below. 

The large (SOS) ribosomal subunit is where protein synthesis, catalyzed formation of peptide bonds, takes place. In light of the discoveries by Thomas Cech and Sidney Altman of ribozymes, RNA molecules that behave as enzymes, the question of catalysis of protein synthesis by RNA or proteins in the ribosome was a major one. The 3 OS ribosomal subunit is where transfer RNAs bind with ribosomal RNA codons. The 50S ribosomal subunit can be further separated into a 23S secondary subunit and a smaller 5S secondary subunit that are normally held together by protein molecules. The 23S subunit includes 3045 nucleotides and 31 proteins. There are six discrete RNA domains in the S23 unit particle. The 5S unit effectively adds a seventh domain. The proteins permeate the exterior of the RNA of S23, but are located over 18 A distant from the active site of catalytic protein bond formation. The structures of complexes of the S23 subunit with two inactive substrate molecules suggest mechanistic similarities to the enzyme chymotrypsin. The S23 structure indicates that an adenine base at the active site plays a role analogous to that of histidine-... 

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