Abstract
A monoclonal antibody specific for Escherichia coli ribosomal protein L5 was isolated from a cell line obtained from Dr. David Schlessinger. Its unique specificity for L5 was confirmed by one- and two-dimensional electrophoresis and immunoblotting. The antibody recognized L5 both in 50 S subunits and 70 S ribosomes. Both antibody and Fab fragments had similar effects on the ribosome functions tested. Antibody bound to 50 S subunits inhibited their reassociation with 30 S subunits at 10 mM Mg2+ but not 15 mM, the concentration present for in vitro protein synthesis. The 70 S couples were not dissociated by the antibody. The antibody caused inhibition of polyphenylalanine synthesis at molar ratios to 50 S or 70 S particles of 4:1. The major inhibitory effect was on the peptidyltransferase reaction. There was no effect on either elongation factor binding or the associated GTPase activities. The site of antibody binding to 50 S was determined by electron microscopy. Antibody was seen to bind beside the central protuberance or head of the particle, on the side away from the L7/L12 stalk, and on or near the region at which the 50 S subunit interacts with the 30 S subunit. This site of antibody binding is fully consistent with its biochemical effects.
Highlights
A monoclonal antibody specific for Escherichia coli ribosomal protein L5was isolated from a cellline obtained from Dr David Schlessinger
Ribosomalprotein L5 of the Escherichia coli large ribosomal subunit has been well described with respect to its stoichiometric binding to 5 S RNA along with proteins L18 and L25 [1, 2]
Protein-protein cross-links between L5 and L18 and L31 were found in purified 5 S RNP complexes [5];crosslinks with L2, L6, L25, L32 [6], and L7/L12 [7] were found in intact 50 S subunits [5]; and cross-links with S13 and S19 were found in 70 S ribosomes [8].Immune electron microscopy has shown the 3’ and 5’ ends of the 5 S RNA to be located in the central protuberance of the 50 S particle (91l),and by inference the 5 S RNA binding proteins arelocated in this neighborhood (this has been shown for L18 and L25 directly [12, 13])
Summary
A cell line producing antibodies to L5 was kindly provided by Dr D. One unit (40 Phe. 0, intact anti-15 antibody; 0, Fab fragments; 0, nonimmune pmol) of 50 S ribosomal subunits were preincubated with an equimouse IgG. Molar amount of nonimmune mouse IgG( A ) ,intact anti-15 antibody (B),or Fab fragments( C )and mixed with a 3-fold molar excess before adding 30 S subunits and completing the assay. Row D shows complexes in which the antibody lies close to the central protuberance and is sometimes partially obscured by it; if a stalk is visible, antiis consistent with the major binding site (area I, site A of Table I). Columns 1 and 2) in which antibody binding is seen neatrhe central protuberance but aftromthecentralprotuberance.Resultsarequantitated in or near the flat surface or face of the subunit.
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