Isolation of a 5S RNA-Protein Complex from Mammalian Ribosomes

Abstract

(14). In rabbit reticulocytes, EDTA was effective only at pH 9.5. In this paper it is shown that 5S RNA is released from mammalian ribosomes by either EDTA or high concentrations of monovalent ions. The species released is not a naked molecule but a ribonucleoprotein, in which the 5S RNA is associated with a single protein. METHODS The procedures for the preparation of free ribosomes from rat liver and rabbit reticulocytes, the dissociation of ribosomes into biologically active subunits, and the subsequent separation of these subunits in sucrose gradients have been described (15,16). Gradient fractions were collected, made 10 mM in magnesium, and treated with 2 volumes of ethanol at -20°C. The resultant precipitate was recovered after 20 hr by centrifugation for 20 min at 2000 X g. Electrophoresis was performed in a vertical electrophoresis cell (E -C Apparatus Corp., Philadelphia, Pa.) in slabs 3-mm thick, each provided with eight slots.For the electrophoresis of 4S and 5S RNA, the procedure of Peacock and Dingman (17) was slightly modified as follows: a 5% acrylamide gel without agarose was used, and EDTA 1881 was omitted from both the gel and the buffer; instead, both contained 50 mM KCl and 2 mM MgCl2. The alcohol precipitates of the gradient fractions were dissolved in 15% sucrose, and 25-Al aliquots were layered into the slots. Electrophoresis was performed at 15°C for 4 hr at a constant current of 100 mA and at the end of the procedure the gels were stained with "Stains-all" (18).Ribosomal proteins were electrophoresed in sodium dodecyl sulfate gels by the procedure described by Maizel (19) in the E -C apparatus. After electrophoresis, the gels were stained in Coomassie Brilliant Blue. RESULTSDuring a study on the dissociation of mammalian ribosomes into biologically active subunits in high concentrations of monovalent ions in the presence of various magnesium concentrations, I noticed that below a critical divalent-cation concentration both ribosomal subunits were converted into slower-sedimenting, distinct derivatives (sharp symmetrical peaks). Table 1 summarizes the changes in sedimentation rates of both ribosomal subunits, when sedimented into sucrose gradients containing 500 mMI KCl and magnesium concentrations varying from 5.0 to 0.0 mM. Below 2.0 mM magnesium, the large ribosomal subunit (LO) forms a derivative (L') with a greatly reduced sedimentation rate, while the small subunit (SO) undergoes two distinct changes: a derivative (S1) that sediments slightly more slowly is formed at concentrations <2 mM magnesium and a derivative (S2) that sediments much more slowly appears below 0.5 mM magnesium. Ribosomal subunits, dissociated with EDTA (20), sediment with rates identical to L1 and S2. The derivatives of both ribosomal subunits are biologically inactive, i.e., they do not associate and do not synthesize polyphenylalanine in the presence of poly(U), whereas LO and S are active (15).The EDTA-induced conversion of the large ribosomal subunit from a 60S to a 47S form noted by Tashiro and Siekev...

Two Yeast La Motif-containing Proteins Are RNA-binding Proteins that Associate with Polyribosomes

“…When cell extracts from cycloheximide-treated cells were incubated with EDTA, both Sro9p and Slf1p shifted upward in the gradient; however, both Sro9p and Slf1p migrated further into the gradient after EDTA treatment than Rpl5p, which is released during EDTA treatment as a complex with 5S rRNA (Blobel, 1971). Thus, upon EDTA treatment, both Sro9p and Slf1p remain associated with other proteins or RNA species, or both.…”

Cooperation between the ribosomal proteins L5 and L11 in the p53 pathway

“…Furthermore, L5 and L11, together with the 5S rRNA form the central protrusion of the large ribosomal subunit (Harms et al, 2001;Spahn et al, 2001). The 5S rRNA and L5 can be released from the ribosome as a complex by chemical treatment of ribosomes (Blobel, 1971;Marion and Reboud, 1981), and the 5S rRNA and L5 form a pre-ribosomal complex that assembles into the ribosome together (Steitz et al, 1988). Thus it is possible that cells maintain a large pool of extraribosomal L5 and 5S rRNA for responding to a variety of stresses and that a ribosomal stress results in the release of L11, which then promote the binding of the 5S RNP complex to MDM2, resulting in p53 stabilization.…”

Biological significance of 5S rRNA import into human mitochondria: role of ribosomal protein MRP-L18

“…In the first study (Koc et al 2001b), 39S large mitoribosome subunits obtained in low-magnesium conditions were used. It is known though, that, after such a treatment, 5S rRNAprotein complexes are quantitatively liberated from ribosomes of eukaryotes (Blobel 1971), bacteria (Horne and Erdmann 1972), and archaea (McDougall and WittmannLiebold 1994). It should be also noted that, although MRP-L18 was first identified in this study, it seems utterly depleted since only one peptide was found.…”