Modification of the accessibility of ribosomal proteins after elongation factor 2 binding to rat liver ribosomes and during translocation.
Free- and EF-2-bound 80 S ribosomes, within the high-affinity complex with the non-hydrolysable GTP analog: guanylylmethylenediphosphonate (GuoPP(CH2)P), and the low-affinity complex with GDP, were treated with trypsin under conditions that modified neither their protein synthesis ability nor their sedimentation constant nor the bound EF-2 itself. Proteins extracted from trypsin-digested ribosomes were unambiguously identified using three different two-dimensional gel electrophoresis systems and 5 S RNA release was checked by submitting directly free- and EF-2-bound 80 S ribosomes, incubated with trypsin, to two-dimensional gel electrophoresis. Our results indicate that the binding of (EF-2)-GuoPP[CH2]P to 80 S ribosomes modified the behavior of a cluster of five proteins which were trypsin-resistant within free 80 S ribosomes and trypsin-sensitive within the high-affinity complex (proteins: L3, L10, L13a, L26, L27a). As for the binding of (EF-2)-GDP to 80 S ribosomes, it induced an intermediate conformational change of ribosomes, unshielding only protein L13a and L27a. Quantitative release of free intact 5 S RNA which occurred in the first case but not in the second one, should be related to the trypsinolysis of protein(s) L3 and/or L10 and/or L26. Results were discussed in relation to structural and functional data available on the ribosomal proteins we found to be modified by EF-2 binding.
Marzouki
A
A
Laboratoire de Biochimie Médicale, Université Claude Bernard, Villeurbanne, France.
Lavergne
J P
JP
Reboud
J P
JP
Reboud
A M
AM
D000818:Animals; D015180:Electrophoresis, Gel, Two-Dimensional; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D008099:Liver / Q000378:metabolism*; D008954:Models, Biological; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D012269:Ribosomal Proteins / Q000302:isolation & purification / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*; D014357:Trypsin
D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors; D010750:Phosphoproteins; D012269:Ribosomal Proteins; D006153:Guanosine Diphosphate; C004805:5'-guanylylmethylenebisphosphonate; D006160:Guanosine Triphosphate; D014357:Trypsin