Essential Features of the Peptidyl Transferase Center in theYeast Mitochondrial Ribosome

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Mason 9419340 The forrnation of peptide bonds in the peptidyl transferase center (PTC) of the large nbosomal subunit is the primary catalytic activity of all ribosomes. An important objective in the field of ribosome research is to understand the structure and catalytic mechanism of the PTC. The sequence and structure of the large subunit ribosomal RNA (LSU rRNA) in the PTC are very highly conserved in nature, and there is abundant evidence supporting the possibility that rRNA acts as a ribozyme in catalyzing the PT reaction. Current thinking favors a structural role for the r proteins in maintaining the conformation of active sites in the rRNA, although a catalytic role for r proteins has not been ruled out completely. Using the yeast mitochondrial ribosome as an experimental system, the current project has focused on the molecular genetic analysis of mitochondrial homologues of four bacterial PTC proteins (L2, L3, L16 and L27) and an rRNA ribose methyltransferase (Pet56p) that catalyzes the formation of 2' O methylguanosine at a universally conserved G nucleotide in the PTC of LSU rRNAs. The importance of modified nucleotides in rRNA is suggested by models of the E. coli ribosome in which the majority of the approximately 24 modified nucleotides are clustered around the mE~NA tRNA peptide complex in the functional center of the ribosome. Three of the modifications in the 23S rRNA of E. coli are ribose methylations at highly conserved nucleotides in the PTC of domain V. The retention of three modified nucleotides in the otherwise minimally modified mitochondrial LSU rRNA points to a fundamental role for these particular modifications in ribosome assembly or function or both. Indeed, pet56 mutants fail to assemble 54S ribosomal subunits. A largely molecular genetic approach will be used to study the functional requirement for Gm2251 in yeast mitochondrial ribosomes. A strategy based on functional complementation of E. coli mutants by yeast PET56 will be used to i dentify the gene for the bacterial homologue of Pet56p. These studies will provide new information about the structure, function and assembly ofthe principal catalytic center ofthe ribosome. %%% Linking amino acids through peptide bonds is the fundamental reaction of biological protein synthesis. This reaction takes place in the peptidyl transferase center (PTC) of the large ribosomal subunit and is thought to occur by the same catalytic mechanism in all cells. An important objective is to understand the respective roles ofthe ribosomal RNA (rRNA) and ribosomal proteins in the structure and catalytic activity of the PTC. Current thinking favors a structural role for the ribosomal proteins in maintaining the catalytic active sites in the rRNA, but there is only a rudimentary understanding of the underlying molecular architecture. There is a functional requirement for nucleotide modification in the PTC. Recent results have shown that the addition of a methyl (CH3) group to the sugar of a specific G nucleotide is an essential step in the formation of the PTC in yeast mitochondrial ribosomes. This G nucleotide is conserved in all ofthe known LSU rRNAs. The well studied E. coli ribosome contains ca. 24 modified nucleotides, all clustered around the the functional center of the ribosome. In contrast, the yeast mitochondrial ribosome has only three modified nucleotides, each identical to one of the modifications in the PTC of E. coli LSU rRNA. The presence of only three modified nucleotides in a functional LSU rRNA underscores the potential importance of these particular modifications. The major objective of this research is to understand why the addition of a single methyl group is so crucial in a huge macromolecular complex containing a 3,000 nucleotide long rRNA and approximately 40 ibosomal proteins. Overall, these studies will provide new information about the principal catalytic center ofthe ribosome. ***