Larger ribosomal subparticles (L-subparticles) of rabbit ribosomes were treated with either
ribonucleases (I or T1) or
proteinases (
trypsin or
chymotrypsin), and their capacity to function in
poly(U)-directed
polyphenylalanine synthesis and in the
puromycin reaction was investigated. The effects of pretreatment of L-subparticles on the reconstruction of active subparticles from core-particles derived by treatment with 2.75 M-NH4Cl/69 mM-MgCl2 and split-
protein fractions were also examined. The
protein moiety of
proteinase-treated L-subparticles was analysed by one-dimensional
sodium dodecyl sulphate/
polyacrylamide- and two-dimensional
polyacrylamide-gel electrophoresis. The introduction of 16--100 scissions in the
RNA moiety had no effect on the activity of the L-subparticles in
polyphenylalanine synthesis, and there was no effect on the stability of L-subparticles to high-
salt shock treatment and a marginal effect on the reconstruction of L-subparticles from high-
salt-
shock core-particles and split-
protein fractions. In contrast, L-subparticles treated with low amounts of
trypsin (0.56 ng of
trypsin/microgram of L-subparticle) were inactive in
polyphenylalanine synthesis, and their capacity to function in partial-reconstruction experiments was diminished. Activity in the
puromycin reaction was increased by 70% as a result of
trypsin treatment (280 ng of
trypsin/microgram of L-subparticle). At least two of the acidic
proteins implicated in the translocation function were not affected by
trypsin treatment.
Trypsin-treated L-subparticles had lost their capacity to bind the smaller ribosomal subparticle (S-subparticle). The
protein(s) needed for S-subparticle binding were shown to be present in high-
salt-
shock cores. At least six
proteins associated with the core-particles were attack during
trypsin treatment of L-subparticles. An examination of L-subparticles isolated from
trypsin-treated polyribosomes showed that the amount of
trypsin necessary to decrease the activity of the subparticle by 50% was about twice that needed in the treatment of L-subparticles alone. The largest
protein of rabbit L-subparticles (approx. 51 000 daltons) was cleaved in a stepwise fashion by
trypsin to fragments of approx. 40 000 daltons. This
protein was also cleaved by
chymotrypsin.