Ribosomal peptidyl transferase can withstand mutations at the putative catalytic nucleotide.
Peptide bond formation is the principal reaction of protein synthesis. It takes place in the peptidyl transferase centre of the large (50S) ribosomal subunit. In the course of the reaction, the polypeptide is transferred from peptidyl transfer RNA to the alpha-amino group of amino acyl-tRNA. The crystallographic structure of the 50S subunit showed no proteins within 18 A from the active site, revealing peptidyl transferase as an RNA enzyme. Reported unique structural and biochemical features of the universally conserved adenine residue A2451 in 23S ribosomal RNA (Escherichia coli numbering) led to the proposal of a mechanism of rRNA catalysis that implicates this nucleotide as the principal catalytic residue. In vitro genetics allowed us to test the importance of A2451 for the overall rate of peptide bond formation. Here we report that large ribosomal subunits with mutated A2451 showed significant peptidyl transferase activity in several independent assays. Mutations at another nucleotide, G2447, which is essential to render catalytic properties to A2451 (refs 2, 3), also did not dramatically change the transpeptidation activity. As alterations of the putative catalytic residues do not severely affect the rate of peptidyl transfer the ribosome apparently promotes transpeptidation not through chemical catalysis, but by properly positioning the substrates of protein synthesis.
Polacek
N
N
Center for Pharmaceutical Biotechnology (MC 870), University of Illinois, Chicago, Illinois 60607, USA.
Gaynor
M
M
Yassin
A
A
Mankin
A S
AS
D000225:Adenine / Q000378:metabolism*; D001483:Base Sequence; D002384:Catalysis; D004926:Escherichia coli; D008969:Molecular Sequence Data; D009154:Mutation; D009711:Nucleotides / Q000378:metabolism; D010458:Peptidyl Transferases / Q000235:genetics / Q000378:metabolism*; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism*; D012338:RNA, Ribosomal, 23S / Q000235:genetics / Q000378:metabolism*; D012270:Ribosomes / Q000201:enzymology / Q000378:metabolism*; D013824:Thermus
D009711:Nucleotides; D016337:RNA, Catalytic; D012338:RNA, Ribosomal, 23S; D010458:Peptidyl Transferases; D000225:Adenine