Characterization of functionally active subribosomal particles from Thermus aquaticus.
Peptidyl transferase activity of Thermus aquaticus ribosomes is resistant to the removal of a significant number of ribosomal proteins by protease digestion, SDS, and phenol extraction. To define the upper limit for the number of macromolecular components required for peptidyl transferase, particles obtained by extraction of T. aquaticus large ribosomal subunits were isolated and their RNA and protein composition was characterized. Active subribosomal particles contained both 23S and 5S rRNA associated with notable amounts of eight ribosomal proteins. N-terminal sequencing of the proteins identified them as L2, L3, L13, L15, L17, L18, L21, and L22. Ribosomal protein L4, which previously was thought to be essential for the reconstitution of particles active in peptide bond formation, was not found. These findings, together with the results of previous reconstitution experiments, reduce the number of possible essential macromolecular components of the peptidyl transferase center to 23S rRNA and ribosomal proteins L2 and L3. Complete removal of ribosomal proteins from T. aquaticus rRNA resulted in loss of tertiary folding of the particles and inactivation of peptidyl transferase. The accessibility of proteins in active subribosomal particles to proteinase hydrolysis was increased significantly after RNase treatment. These results and the observation that 50S ribosomal subunits exhibited much higher resistance to SDS extraction than 30S subunits are compatible with a proposed structural organization of the 50S subunit involving an RNA "cage" surrounding a core of a subset of ribosomal proteins.
Khaitovich
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P
Center for Pharmaceutical Biotechnology, m/c 870, University of Illinois, 900 South Ashland Avenue, Chicago, IL 60607, USA.
Mankin
A S
AS
Green
R
R
Lancaster
L
L
Noller
H F
HF
D010458:Peptidyl Transferases / Q000378:metabolism*; D012338:RNA, Ribosomal, 23S / Q000302:isolation & purification; D012341:RNA, Ribosomal, 5S / Q000302:isolation & purification; D012269:Ribosomal Proteins / Q000302:isolation & purification; D012270:Ribosomes / Q000737:chemistry / Q000378:metabolism*; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D013824:Thermus
D012338:RNA, Ribosomal, 23S; D012341:RNA, Ribosomal, 5S; D012269:Ribosomal Proteins; D010458:Peptidyl Transferases