Interaction of 5-S RNA, 5.8-S RNA and tRNA with Rat-Liver Ribosomal Proteins

Abstract

In order to ascertain 5-S RNA, 5.8-S RNA and tRNA binding proteins from eukaryotic ribosomes, affinity chromatography of the rat liver ribosomal 40-S and 60-S subunit proteins on immobilized RNAs was used. Rat liver 5-S RNA, 5.8-S RNA and tRNA were immobilized via 3 '-end ribose to adipic-acid-hydrazide-epoxy-activated Sepharose 6B. Using two-dimensional polyacrylamide gel electrophoresis in a urea-urea system, the bound proteins were identified as follows : a) 5-S RNA was found to form a complex containing mainly proteins L6 and L18 of the large ribosomal subunit, whereas proteins L7, L8 and L35 were present in the complex to a lesser degree.b) 5.8-S RNA bound proteins L5, L6, L7 and L18 from the 60-S subunit. c) tRNA formed a complex containing proteins S3', S6, S8, S9, S11, S14, S18 and S23/24 from the 40-S subunit and proteins L5, L6, L7, L8, L13, L14, L18, L19, L21, L25, L26, L27, L29, L31, L32, L35 and L36 from the 60-S subunit. From this list of proteins S6, S14 and S23/24 of the small and L6, L13, L19, L21, L26, L32, L35 and L36 of the large subunit gave stronger spots than the others.Proteins from the different complexes are overlapping to a certain extent. The results suggest that 5-S RNA and 5.8-S RNA are in a close neighbourhood in the rat liver ribosome. It is supposed that 5-S RNA, 5.8-S RNA and proteins L5, L6, L7, L8, L13, L19, L21, L26, L32, L35, L36 are at or near the eukaryotic peptidyltransferase centre.

The ternary complex consisting of rat liver ribosomal 5 S RNA, 5.8 S RNA and protein L5

“…In [5,6] and [7,8] affinity chromatography has been used to identify 5 S RNA and 5.8 S RNA-binding proteins of the rat liver ribosome. According to both experimental protocols, the formed complexes were dissociated and proteins eluted by high salt buffer containing EDTA [5-S].…”

“…To facilitate quantitative estimations, 5'-end labelled 32P 5.8 S RNA, prepared as in [9], was mixed with cold 5.8 S RNA. Preparation of the 5 S RNA-Sepharose gel and affinity chromatography of 60 S subunit proteins was as in [6]. The latter was performed in 10 mM buffer, containing 20 mM MgCl*, 300 mM KC1 and 6 mM 2-mercaptoethanol at 3-4'C.…”

“…Isolation of rat liver ribosomal subunit proteins and 5 S RNA was as in [6]. 5.8 S RNA was isolated according to [8].…”

“…For rat liver ribosomes it was identified as protein L5 [4]. However, affinity chromatography of rat ribosomal 60 S subunit proteins on the immobilized 5 S RNA did not reveal this protein in the formed complex [5-71, while it was present in a similar 5.8 S RNAprotein complex [5,6]. It was suggested that the immobilization technique itself (i.e., 3'-end oxidation of 5 S RNA with periodate and its subsequent linkage to hydrazide group) might prevent 5 S RNA-L5 interaction [7].…”

Differential utilization of poly(rC) binding protein 2 in translation directed by picornavirus IRES elements

“…The poliovirus S-L IV and the poly(rC) RNA affinity columns were prepared by methods adapted from Fahnestock and Nomura (1972) and Metspalu et al+ (1978)+ Briefly, 1 mg RNA [either S-L IV RNA or poly(rC)] in 0+1 M Na(CH 3 CO 2 ) (pH 5+0) was oxidized by treatment with a 120-fold molar excess of NaIO 4 + After incubation in the dark at room temperature for 1 h, a 10,000-fold molar excess of glycerol over NaIO 4 was added to inactivate the excess NaIO 4 + After inactivation for 20 min at room temperature, the RNA was precipitated with ethanol, collected by centrifugation, redissolved in 0+1 M Na(CH 3 CO 2 ), and precipitated again by the addition of ethanol+ The precipitated RNA was then collected by centrifugation, washed in 70% ethanol, and resuspended at a concentration of 1 mg RNA per milliliter 0+1 M Na(CH 3 CO 2 ) (pH 5+4)+ To prepare the affinity column resin, 2 mL of agaroseadipic acid hydrazide (Pharmacia) was washed with two volumes of wash buffer I [0+1 Na(CH 3 CO 2 ), pH 4+0, 0+5 M NaCl] followed by two volumes of wash buffer II [0+1 M Tris-acetate, pH 8, 0+5 M NaCl]+ These alternate washes were repeated two times followed by three two-volume washes with 0+1 M Na(CH 3 CO 2 ) (pH 5+4)+ During the final wash, the matrix was divided into two equal volumes+…”