title: Activities of the peptidyl transferase center of ribosomes lacking protein L27.
abstract: The ribosome is the molecular machine responsible for protein synthesis in all living organisms. Its catalytic core, the peptidyl transferase center (PTC), is built of rRNA, although several proteins reach close to the inner rRNA shell. In the Escherichia coli ribosome, the flexible N-terminal tail of the ribosomal protein L27 contacts the A- and P-site tRNA. Based on computer simulations of the PTC and on previous biochemical evidence, the N-terminal α-amino group of L27 was suggested to take part in the peptidyl-transfer reaction. However, the contribution of this group to catalysis has not been tested experimentally. Here we investigate the role of L27 in peptide-bond formation using fast kinetics approaches. We show that the rate of peptide-bond formation at physiological pH, both with aminoacyl-tRNA or with the substrate analog puromycin, is independent of the presence of L27; furthermore, translation of natural mRNAs is only marginally affected in the absence of L27. The pH dependence of the puromycin reaction is unaltered in the absence of L27, indicating that the N-terminal α-amine is not the ionizing group taking part in catalysis. Likewise, L27 is not required for the peptidyl-tRNA hydrolysis during termination. Thus, apart from the known effect on subunit association, which most likely explains the phenotype of the deletion strains, L27 does not appear to be a key player in the core mechanism of peptide-bond formation on the ribosome.
authors.affiliation: Department of Physical Biochemistry, Max Planck Institute for Biophysical Chemistry, 37077 Goettingen, Germany.
authors.forename: Cristina
authors.initials: C
authors.lastname: Maracci
authors.affiliation: Department of Physical Biochemistry, Max Planck Institute for Biophysical Chemistry, 37077 Goettingen, Germany.
authors.forename: Ingo
authors.initials: I
authors.lastname: Wohlgemuth
authors.affiliation: Department of Physical Biochemistry, Max Planck Institute for Biophysical Chemistry, 37077 Goettingen, Germany.
authors.forename: Marina V
authors.initials: MV
authors.lastname: Rodnina
mesh_terms: D057773:Cold Shock Proteins and Peptides / Q000096:biosynthesis / Q000737:chemistry; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000737:chemistry; D007700:Kinetics; D014176:Protein Biosynthesis; D012269:Ribosomal Proteins / Q000737:chemistry* / Q000502:physiology; D012270:Ribosomes / Q000737:chemistry* / Q000502:physiology
chemical_list: D057773:Cold Shock Proteins and Peptides; C498192:CspA protein, E coli; D029968:Escherichia coli Proteins; D012269:Ribosomal Proteins; C034661:ribosomal proteins L27
keywords: peptide-bond formation; ribosome; ribozyme; tRNA; translation