In vitro nuclear transport of ribosomal ribonucleoprotein: temperature affects quantity but not quality of exported particles.

The in vitro export of ribosomal ribonucleoprotein (rRNP) from Tetrahymena nuclei was investigated at the optimal growth temperature of 28 degrees C and at the nonlethal temperature of 8 degrees C. At both temperatures, nuclei exported ribosomal precursor particles that revealed the same physical qualities of size, appearance in negative-staining electron microscopy, sedimentation coefficient, buoyant density, and rRNA pattern. Surprisingly, fewer rRNP particles were exported at 8 than at 28 degrees C, as was revealed by a lower saturation plateau in the export kinetics from nuclei prelabeled with [3H]uridine. Upon a temperature increase from 8 to 28 degrees C, additional rRNP particles were exported. We conclude that nuclei export only a defined portion of rRNP particles at a given temperature, although enough potentially transportable rRNP particles are present in nuclei. Obviously, the reactivity of at least one of the reactants involved directly or indirectly in rRNP export changes with temperature.     

Locking and unlocking of ribosomal motions

During the ribosomal translocation, the binding of elongation factor G (EF-G) to the pretranslocational ribosome leads to a ratchet-like rotation of the 30S subunit relative to the 50S subunit in the direction of the mRNA movement. By means of cryo-electron microscopy we observe that this rotation is accompanied by a 20 A movement of the L1 stalk of the 50S subunit, implying that this region is involved in the translocation of deacylated tRNAs from the P to the E site. These ribosomal motions can occur only when the P-site tRNA is deacylated. Prior to peptidyl-transfer to the A-site tRNA or peptide removal, the presence of the charged P-site tRNA locks the ribosome and prohibits both of these motions.     

Specific ribonucleoprotein fragments from 40-S ribosomal subunits.

Well-defined ribonucleoprotein fragments, resulting from the action of endogenous nuclease on 40-S subunits, were able to be separated when using high concentrations of LiCl. The ribonucleoproteins obtained sedimented at 12, 17 S, 23 S and 30 S and contained 8 S, 12 S and 17 S RNA, respectively, associated with a few proteins. The proteins extracted from the fragments were [3H] labeled by reductive methylation and their molar proportion was determined. The smallest fragment (12, 17 S) contained only three proteins, S8, S9 and S24. The 23-S and 30-S materials contained some proteins in common, S15, S19, S22, S25; S16 was found mainly in 30 S. Two proteins, S26 and "protein y" were found mainly in 23 S material. Thus, these results can give information on the relative location of certain proteins in the 40-S subunits.     

Functional modules in ribosomal protein L5 for ribonucleoprotein complex formation and nucleocytoplasmic transport

Ribosomal protein L5 forms a small, extraribosomal complex with 5 S ribosomal RNA, referred to as the 5 S ribonucleoprotein complex, which shuttles between nucleus and cytoplasm in Xenopus oocytes. Mapping elements in L5 that mediate nuclear protein import defines three separate such activities (L5-nuclear localization sequence (NLS)-1, -2, and -3), which are functional in both oocytes and somatic cells. RNA binding activity involves N-terminal as well as C-terminal elements of L5. In contrast to the full-length protein, none of the individual NLSs carrying L5 fragments are able to allow for the predominating accumulation in the nucleoli that is observed with the full-length protein. The separate L5-NLSs differ in respect to two activities. Firstly, only L5-NLS-1 and -3, not L5-NLS-2, are capable of promoting the nuclear transfer of a heterologous, covalently attached ribonucleoprotein complex. Secondly, only L5-NLS-1 is able to bind strongly to a variety of different import receptors; those that recognize L5-NLS-2 and -3 have yet to be identified.     

Limited nuclease digestion of heterogeneous nuclear ribonucleoprotein in nuclei.

We have used limited nuclease digestion of nuclei to probe the structure of nuclear ribonucleoprotein (nRNP). Analysis of [³H]uridine-labeled heterogeneous nuclear RNA isolated from nuclease digested nuclei revealed preferential generation of discrete bands of RNA ranging in size from 1.5 × 10⁵ to 6 × 10⁵ daltons. The nuclease digestion pattern of nRNP differed from the nuclease digestion pattern obtained with chromatin in that the RNA bands generated in these experiments were transient, appearing only early in the course of digestion, and no stable nRNP monomer size was evident. Therefore, although nRNP may be organized in a regular configuration, nRNP structure differs considerably from the repeating subunit structure of chromatin.     

In vitro synthesis of Escherichia coli ribosomal RNA

Synthesis of ribosomal RNA in a cell-free system was achieved using purified Escherichia coli RNA polymerase and bacterial DNA templates from E. coli, Proteus mirabilis and E. coli/P. mirabilis hybrid strains carrying an E. coli DNA enriched for ribosomal RNA genes.Both direct and indirect competition hybridization revealed that from 5 to 15% of the in vitro product, depending on the template used, had sequences homologous to rRNA. The level of synthesis of sequences homologous to rRNA was related directly to the proportion of rRNA genes in the template. The use of heterologous DNA during competition hybridization ensured at least a 100-fold greater sensitivity for the detection of rRNA sequences than from any messenger RNA sequence.     

In vitro assays of vesicular transport

Movement of proteins and lipids between the various compartments of eukaryotic cells is fundamental to the maintenance of cellular homeostasis, and an understanding of the molecular mechanisms that govern these processes remains a key goal of cell biological research. This aim has been greatly facilitated by the development of assays that recapitulate specific events in vitro. In the following article we provide an overview of some of the currently used assays that measure the movement of proteins within the exocytic and endocytic pathways, and provide a starting point for those wishing to establish their own systems to study other vesicular transport steps.     

Factors causing release of ribosomal subunits from isolated nuclei of regenerating rat liver in vitro.

Incubation medium II causes release of ribosomal subunits from isolated prelabeled nuclei of regenerating rat liver in vitro (Sato, T., Ishikawa, K. and Ogato, K. (1976) Biochim. Biophys. Acta 000, 000-000). The effects of individual components of this medium on release of subunits were studied and the following results were obtained. 1. Dialyzed cytosol was effective in causing release of total labeled RNA, but its effect on release of labeled ribosomal subunits was rather lower than that of low molecular yeast RNA. Spermidine inhibited the release of total labeled RNA as well as that of labeled ribosomal subunits. 2. Low molecular yeast RNA was the most effective component for inducing release of labeled ribosomal subunits. Homologous ribosomal RNA was as effective as yeast RNA. Cytoplasmic ribosomes, prepared by washing with solution of high salt concentration, and their subunits were also effective. 3. Transfer RNA was not so effective as yeast RNA and ribosomal RNA and even after heat treatment it had little effect. 4. Among the homopolyribonucleotides tested, polyuridylic acid had a strong effect but polyadenylic acid, polycytidylic acid and polyinosinic acid had no effect. 5. The effects of yeast RNA and polyuridylic acid in causing release of labeled ribosomal subunits were dependent upon their concentrations in the reaction mixture. The characteristics of the factors which cause release of labeled ribosomal subunits in vitro are discussed on the basis of the results.     

Characterization of ribonucleoprotein particles released from isolated nuclei of regenerating rat liver in two different in vitro systems.

The ribonucleoprotein particles released from isolated nuclei of regenerating rat liver in two in vitro systems were studied and the following results were obtained. 1. When the isolated nuclei of regenerating rat liver labeled in vivo with [14C] orotic acid were incubated in medium containing ATP and an energy-regenerating system (medium I) release of labeled 40-S particles was observed. Analysis of these 40-S particles showed that they contained heterogeneous RNA but no 18 S or 28 S ribosomal RNAs and their buoyant density in CsCl was 1.42-1.45 g/cm3, suggesting that they were nuclear informosome-like particles released during incubation. 2. When the same nuclei were incubated in the same medium fortified with dialyzed cytosol, spermidine and yeast RNA (medium II), release of labeled 60-S and 40-S particles was observed. Using CsCl buoyant density gradient centrifugation, two components were found in the labeled ribonucleoprotein particles released from nuclei in this medium. The labeled 60-S particles were found to contain 28-S RNA as the main component and their buoyant density in CsCl was 1.61 g/cm3, suggesting that they were labeled large ribosomal subunits. The labeled 40-S particles contained both 18 S RNA and heterogeneous RNA and they formed two discrete bands in CsCl, at 1.40 and 1.56 g/cm3, suggesting that they contained small ribosomal subunits and nuclear informosome-like particles. 3. These results clearly indicate that addition of dialyzed cytosol, spermidine and low molecular yeast RNA to medium I causes the release of ribosomal subunits or their precursors from isolated nuclei in the in vitro system.     

Uptake of ribosomal proteins by isolate HeLa nuclei.

The uptake of radioactive ribosomal proteins by isolated HeLa cell nuclei has been studied. Ribosomal proteins are taken up by nuclei $\text{in vitro}$ more rapidly than are cytosol proteins, suggesting that the uptake is selective. In addition, the ribosomal proteins are found associated with the nucleolus to a greater extent than are the cytosol proteins.