Modification of histidine residues on proteins from the 50S subunit of the Escherichia coli ribosome. Effects on subunit assembly and peptidyl transferase centre activity.
L2, L3, L4, L16 and L20 are proteins of the 50S ribosomal subunit of Escherichia coli which are essential for the assembly and activity of the peptidyl transferase centre. These proteins have been modified with the histidine-specific reagent, diethylpyrocarbonate, while L17 and L18 were treated as controls. Each modified protein tested was able to participate in the reconstitution of a 50S particle when replacing its normal counterpart, although the particles assembled with modified L2 were heterogeneous. However, although they could support assembly, modified L16 and L20 were not themselves reconstituted stably, and modified L2 and L3 were found in less than stoichiometric amounts. Particles assembled in the presence of modified L16 retained significant peptidyl transferase activity (60-70% at 10 mM diethylpyrocarbonate) whereas those reconstituted with modified L2, L3, L4 or L20 had low activity (10-30% at 10 mM diethylpyrocarbonate). The particles assembled with the modified control protein, L17, retained 80% of their peptidyl transferase activity under the same conditions. The histidine residues within the essential proteins therefore contribute to ribosome structure and function in three significant ways; in the correct assembly of the ribosomal subunit (L2), for the stable assembly of the proteins within the ribosomal particle (L20 and L16 in particular), and directly or indirectly for the subsequent activity of the peptidyl transferase centre (L2, L3, L4 and L20). The essential nature of the unmodified histidines for assembly events precludes the use of the chemical-modification strategy to test the proposal that a histidine on one of the proteins might participate in the catalytic activity of the centre.
Sumpter
V G
VG
Department of Biochemistry, University of Otago, Dunedin, New Zealand.
Tate
W P
WP
Nowotny
P
P
Nierhaus
K H
KH
D002499:Centrifugation, Density Gradient; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006639:Histidine / Q000378:metabolism*; D010458:Peptidyl Transferases / Q000378:metabolism*; D012329:RNA, Bacterial / Q000235:genetics*; D012335:RNA, Ribosomal / Q000235:genetics*; D012269:Ribosomal Proteins / Q000378:metabolism*; D013329:Structure-Activity Relationship
D012329:RNA, Bacterial; D012335:RNA, Ribosomal; D012269:Ribosomal Proteins; D006639:Histidine; D010458:Peptidyl Transferases; D004047:Diethyl Pyrocarbonate