Ribosomal protein L27 participates in both 50 S subunit assembly and the peptidyl transferase reaction.
Protein L27 has been implicated as a constituent of the peptidyl transferase center of the Escherichia coli 50 S ribosomal subunit by a variety of experimental observations. To define better the functional role of this protein, we constructed a strain in which the rpmA gene, which encodes L27, was replaced by a kanamycin resistance marker. The deletion mutant grows five to six times slower than the wild-type parent and is both cold- and temperature-sensitive. This phenotype is reversed when L27 is expressed from a plasmid-borne copy of the rpmA gene. Analysis of ribosomes from the L27-lacking strain revealed deficiencies in both the assembly and activity of the 50 S ribosomal subunits. Although functional 50 S subunits are formed in the mutant, an assembly "bottleneck" was evidenced by the accumulation of a prominent 40 S precursor to the 50 S subunit which was deficient in proteins L16, L20, and L21, as well as L27. In addition, the peptidyl transferase activity of 70 S ribosomes containing mutant 50 S subunits was determined to be three to four times lower than for wild-type ribosomes. Ribosomes lacking L27 were found to be impaired in the enzymatic binding of Phe-tRNAPhe to the A site, although the interaction of N-acetyl-Phe-tRNAPhe with the P site was largely unperturbed. We therefore infer that L27 contributes to peptide bond formation by facilitating the proper placement of the acceptor end of the A-site tRNA at the peptidyl transferase center.
Wower
I K
IK
Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, Massachusetts 01003-4505, USA.
Wower
J
J
Zimmermann
R A
RA
D001665:Binding Sites / Q000235:genetics; D003432:Cross-Linking Reagents / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000378:metabolism*; D005819:Genetic Markers; D007612:Kanamycin / Q000494:pharmacology; D007700:Kinetics; D009154:Mutation / Q000235:genetics; D010444:Peptide Elongation Factor Tu / Q000378:metabolism; D010458:Peptidyl Transferases / Q000378:metabolism*; D010641:Phenotype; D012360:RNA, Transfer, Phe / Q000378:metabolism; D012269:Ribosomal Proteins / Q000502:physiology*; D012270:Ribosomes / Q000737:chemistry*
D003432:Cross-Linking Reagents; D005819:Genetic Markers; D012360:RNA, Transfer, Phe; D012269:Ribosomal Proteins; C034661:ribosomal proteins L27; D007612:Kanamycin; D010458:Peptidyl Transferases; D010444:Peptide Elongation Factor Tu