Structural and functional domains of Escherichia coli ribosomal protein L7/L12. The hinge region is required for activity.
Variant forms of Escherichia coli ribosomal protein L7/L12 were constructed, overexpressed, and purified. These included proteins that deleted residues 35-52 (delta 35-52) and 42 to 52 (delta 42-52), others that contained single cysteine substitutions at residues 63 and 89, and combinations of the deletions and cysteine substitutions. Chemical modification of the introduced cysteine residues with [14C]iodoacetamide was used to radiolabel the protein variants in order to quantify their binding to the ribosome. Neither of the deletions in the hinge domain, delta 35-52 and delta 42-52, had any effect on L7/L12 dimer formation as detected by cross-linking by dimethyl suberimidate. Perpendicular urea gradient gel electrophoresis showed that both deletion variants retained a compact structural element attributable to the globular C-terminal domain. Reconstitution of core particles depleted of wild type L7/L12 with the deletion proteins showed that delta 42-52 bound normally in 4 copies per particle, whereas delta 35-52 bound in only 2.5 copies following isolation of the particles by high speed centrifugation or gel filtration. Ribosomes mixed with an excess of the deletion variants and assayed directly for polyphenylalanine synthesis were completely inactive. The results suggest that the flexibility conferred by the hinge is required for activity, perhaps by allowing the C-terminal domain to occupy a location near the base of the L7/L12 stalk.
Oleinikov
A V
AV
Department of Biological Chemistry, School of Medicine, University of California, Davis 95616.
Perroud
B
B
Wang
B
B
Traut
R R
RR
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003545:Cysteine; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005798:Genes, Bacterial; D014644:Genetic Variation; D007461:Iodoacetates / Q000378:metabolism; D019807:Iodoacetic Acid; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D012269:Ribosomal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism; D017384:Sequence Deletion
D001426:Bacterial Proteins; D007461:Iodoacetates; D011994:Recombinant Proteins; D012269:Ribosomal Proteins; C016700:ribosomal protein L7-L12; D003545:Cysteine; D019807:Iodoacetic Acid