Lecture 21 - Lecture21...

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Lecture 21 Fig 8.48 23 rRNA has peptidyl transferase acting activity peptidyl transferase activity resides exclusively in the 23S rRNA. Both these state- ments are wrong! It is believed that the terminal A of the CAA at the 3’ end of the tRNA is the P site and the base that catalyzes peptide bond formation It was believed that peptidyl transferase would be catalyzed by ribosome protein, when they did the crystal structure of the ribosome they discovered that no side chain of any ribosomal protein’s amino acid was anywhere near where the peptide bond was formed. That eliminated any protein being involved in catalysis. This means that the ribosome is a ribozyme ribozymes - enzyme reaction catalyzed by RNA not by a protein ribosome is considered the largest known ribozyme After looking where the peptide is formed, researchers found this free nitrogen on a rRNA it was believed that it would be an acid base catalyzed reaction. The amino group attacks the carbonyl of the peptide bond, the tRNA in the P site. To make it nucleo- philic; they thought this free nitrogen would be the base, which would pull the hy- drogen off of the amino group making it more nucleophilic so it would attack the carbonyl. This was wrong because the pK of the base was incorrect to be receiving a hy- drogen. In fact, it would be deprotonized. The evidence showing catalysis in vitro turned out to be the group on an HPLC column. Many groups confirmed that the ring nitrogen was completely protonated, in fact peptide bond formation is not stimulated by higher pH. As an acid base catalyzed reaction, it would be expected that the more deprotonated the the base would be the faster the reac- tion would be, in fact it wasn’t dependent on pH at all. The final proof that the model was incorrect, was they mutated the base so that it wouldn’t happen as free nitrogen in the right place and there was only a modest reduction of rate of peptide bond formation. Whereas if the free nitrogen was involved in peptide bond formation then peptide bond formation would have been eliminated. What were scientists left with? there were no proteins that are left in catalysis and no other residues on the ri- bosomal RNA that could do the catalysis either. It was proposed that the mechanism was substrate catalyzed, which means that one of the substrates is catalyzing the reaction
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Have a tRNA in the A site and the amino acid that is going to be added to the poly- peptide chain. The tRNA in the P site (top left of the picture) and the terminal ribose on the terminal A. One of the reasons that this reactions seems plausible is that the CCA, the last three nucleotides at the ends of tRNAs, which are added after the tRNA is tran- scribed. They are added as a part of processing, which is a variant. There is no known organism that doesn’t have a CCA The ribose on the terminal A is called a76, this tRNA is only 76 residues long and this is the tRNA, the last A; the free 3’OH has the amino acid and the rest of the
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Lecture 21 - Lecture21...

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