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Brown TA. Genomes. 2nd edition. Oxford: Wiley-Liss; 2002.

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Genomes. 2nd edition.

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Box 11.1Peptidyl transferase is a ribozyme

A ribosome-associated protein that has the peptidyl transferase activity needed to synthesize peptide bonds during translation has never been isolated. The reason for this lack of success is now known: the enzyme activity is specified by part of the 23S rRNA.

When the base-paired structures of rRNAs (see Figure 11.11) were first determined in the early 1980s, the possibility that an RNA molecule could have enzymatic activity was unheard of, the breakthrough discoveries with regard to ribozymes not being made until the period 1982-86. Ribosomal RNAs were therefore initially assigned purely structural roles in the ribosome, their base-paired conformations being looked upon as scaffolds to which the important components of the ribosome - the proteins - were attached. Problems with this interpretation began to arise in the late 1980s when difficulties were encountered in identifying the protein or proteins responsible for the central catalytic activity of the ribosome - the formation of peptide bonds. By now the existence of ribozymes had been established and molecular biologists began to take seriously the possibility that rRNAs might have an enzymatic role in protein synthesis.

Locating the site of peptidyl transferase activity in the ribosome

Over the years, antibiotics and other inhibitors of protein synthesis have played an important role in studies of ribosome function. In 1995, a new inhibitor called CCdA-phosphate-puromycin was synthesized, this compound being an analog of the intermediate structure formed when two amino acids are joined by formation of a peptide bond during protein synthesis. CCdA-phosphate-puromycin binds tightly to the bacterial ribosome and, because of its structure, this binding site must be at precisely the position where peptide bonds are formed in the functioning ribosome. Would it be possible to use the inhibitor to find out where in the ribosome peptide bonds are made?

X-ray crystallography (Section 9.1.3) has revealed exactly where CCdA-phosphate-puromycin binds within the 50S subunit. Its position is deep down within the body of the subunit. The view shown here depicts the critical part of CCdA-phosphate-puromycin as a red dot, marking the position where the chemical reaction that creates a dipeptide must occur. This position is closely associated with the 23S rRNA of the large subunit (the rRNA is not shown in the figure) but is 18.4 Å away from the nearest protein, L3, and slightly more distant from L2, L4 and L10 (10 Å = 1 nm).

In atomic terms, 18–24 Å is a massive distance and it is inconceivable that any biochemical activity occurring at such a position could be catalyzed by one of the four proteins shown in the figure. The positioning of CCdA-phosphate-puromycin, and hence of the active site for peptide bond formation, provides convincing evidence that peptidyl transferase must be a ribozyme.

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Now that the evidence has finally been obtained, researchers are moving on to determine exactly how the rRNA backbone acts as a ribozyme in peptide bond formation. Attention was initially concentrated on an adenine nucleotide at position 2451 in the E. coli 23S rRNA, because this adenine has unusual charge properties compared with other nucleotides. The hypothesis was that an interaction between this adenine and a nearby guanine, at position 2447, is the key to protein synthesis. But this model has been thrown into disarray by mutational studies, which have shown that both A2451 and G2447 can be replaced by other nucleotides without a detectable effect on the ability of the ribosome to carry out peptide bond synthesis.

These results have prompted a re-evaluation of the roles of A2451 and G2447 in peptide bond formation, and attention is now turning to other nucleotides present in the parts of the 23S rRNA that are located in the vicinity of the active site. Much work still needs to be done, but the ribozymal basis for peptidyl transferase activity is gradually being tracked down.

References

  1. Nissen P, Hansen J, Ban N, Moore PB, Steitz TA. The structural basis of ribosome activity in peptide bond synthesis. Science. (2000);289:920–930. [PubMed: 10937990]
  2. Polacek N, Gaynor M, Yassin A, Mankin AS. Ribosomal peptidyl transferase can withstand mutations at the putative catalytic nucleotide. Nature. (2001);411:498–501. [PubMed: 11373685]

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