Histidine 229 in protein L2 is apparently essential for 50S peptidyl transferase activity.
It has recently been suggested that peptidyl transferase activity is primarily a property of ribosomal RNA and that ribosomal proteins may act only as scaffolding. On the other hand, evidence from both photoaffinity labeling studies and reconstitution studies suggest that protein L2 may be functionally important for peptidyl transferase. In the work reported here, we reconstitute 50S subunits in which the H229Q variant of L2 replaces L2, with all other ribosomal components remaining unchanged, and determine the catalytic and structural properties of the reconstituted subunits. We observe that mutation of the highly conserved His 229 to Gin results in a complete loss of peptidyl transferase activity in the reconstituted 50S subunit. This is strong evidence for the direct involvement of L2 in ribosomal peptidyl transferase activity. Control experiments show that, though lacking peptidyl transferase activity, 50S subunits reconstituted with H229Q-L2 appear to be identical with 50S subunits reconstituted with wild-type L2 with respect to protein composition and 70S formation in the presence of added 30S subunits. Furthermore, as shown by chemical footprinting analysis, H229Q-L2 appears to bind 23S RNA in the same manner as wild-type L2. Thus, the effect of H229 mutation appears to be confined to an effect on peptidyl transferase activity, providing the most direct evidence for protein involvement in this function to date.
Cooperman
B S
BS
Department of Chemistry, University of Pennsylvania, Philadelphia 19104-6323, USA.
Wooten
T
T
Romero
D P
DP
Traut
R R
RR
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification; D006639:Histidine / Q000737:chemistry*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010458:Peptidyl Transferases / Q000378:metabolism*; D012335:RNA, Ribosomal / Q000235:genetics; D012269:Ribosomal Proteins / Q000737:chemistry*
D001426:Bacterial Proteins; D012335:RNA, Ribosomal; D012269:Ribosomal Proteins; D006639:Histidine; D010458:Peptidyl Transferases