On the structural and functional importance of the highly conserved Glu56 of Thermus thermophilus L4 ribosomal protein.
The structural and functional importance of the highly conserved amino acid residue glutamic acid 56 (Glu56) of the ribosomal protein L4 from Thermus thermophilus (TthL4) has been investigated by replacing this residue by alanine or glutamine, and by incorporating the resulted mutants into Escherichia coli ribosomes. The catalytic properties of peptidyltransferase estimated for the mutants as well as for the wild-type TthL4 by the puromycin reaction, were quite different. The binding of tRNA to the P and A-site was affected. In addition, replacement of the native L4 protein by wild-type TthL4 or by TthL4-Ala56 mutant resulted in reduced capability of 50S subunits for association with 30S subunits. In contrast, neither the assembly of the 50S subunits nor the fixation of the tRNA 3'-end at the P or A-site was affected. These results are used to discuss critically the hypothesis that the delta-carboxyl group of the highly conserved Glu56 is essential for stabilizing a flexible loop of L4, which extended into the ribosome interior region, influences the mechanism of peptide bond formation. Mutations concerning the semi-conserved glycine 55 (Gly55) were investigated. Replacement of Gly55 by serine did not affect the measured functions. In contrast, replacement of Gly55 by alanine resulted in enhanced peptidyltransferase activity and increased tRNA affinity for the P and A-sites, indicating a possible implication of this amino acid in the local loop conformation of TthL4.
Leontiadou
Fotini
F
Laboratory of Biochemistry, School of Chemistry, Aristotle University of Thessaloniki, TK 54006 Thessaloniki, Greece.
Xaplanteri
Maria A
MA
Papadopoulos
Georgios
G
Gerassimou
Christina
C
Kalpaxis
Dimitrios L
DL
Choli-Papadopoulou
Theodora
T
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D018698:Glutamic Acid / Q000737:chemistry / Q000378:metabolism*; D005998:Glycine / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D012269:Ribosomal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D016415:Sequence Alignment; D012694:Serine / Q000737:chemistry; D016963:Thermus thermophilus / Q000737:chemistry* / Q000378:metabolism
D001426:Bacterial Proteins; D012269:Ribosomal Proteins; C056368:ribosomal protein L4; D018698:Glutamic Acid; D012694:Serine; D000409:Alanine; D005998:Glycine