We have investigated the nuclear transport of U1 and U5 snRNPs by microinjection studies in oocytes from Xenopus laevis using snRNP particles prepared by reconstitution in vitro. Competition studies with snRNPs showed that the Sm core domain of U1 snRNPs contains a nuclear location signal that acts independently of the m3G cap. The transport of U1 snRNP can be blocked by saturation with competitor U1 snRNPs or by U5 snRNPs, which indicates that the signals on the respective Sm core domains interact with the same transport receptors. Further, by using a minimal U1 snRNP particle reconstituted in vitro and containing only the Sm core RNP domain and lacking stem‐loops I to III of U1 RNA, we show that this is targeted actively to the nucleus, in spite of the absence of the m3G cap. This indicates that under certain conditions the NLS in the Sm core domain not only is an essential, but may also be a sufficient condition for nuclear targeting. We propose that the RNA structure of a given snRNP particle determines at least in part whether the particle's m3G cap is required for nuclear transport or can be dispensed with.
In this paper we describe a method for preparing native, RNA-free, proteins from anti-m3G purified snRNPs (U1, U2, U4/U6 and U5) and the subsequent quantitative reconstitution of U1 and U2 snRNPs from purified proteins and snRNA. Reconstituted U1 and U2 snRNPs contained the full complement of core proteins, B, B', D1, D2, D3, E, F and G. Both the U1 and U2 reconstituted particles were stable in CsCl gradients and had the expected buoyant density of 1.4 g/cm3. Reconstituted RNP particle formation was not competited by a 50 fold molar excess of tRNA, as determined by gel retardation assays. However, U1 and U2 particle formation was reduced in the presence of an excess of cold U1 or U2 snRNA demonstrating a specific RNA-protein interaction. U1 and U2 snRNPs were also efficiently reconstituted in vitro, utilizing proteins prepared from mono Q purified U1 and U2 snRNPs. This suggests that for the assembly of snRNPs in vitro no auxiliary proteins other than bona fide snRNP proteins appear to be required. The potential of this reconstitution technique for investigating snRNP assembly and snRNA-protein interactions is discussed.
The yeast Saccharomyces cerevisiae mitochondrial release factor was expressed from the cloned MRF1 gene, purified from inclusion bodies, and refolded to give functional activity. The gene encoded a factor with release activity that recognized cognate stop codons in a termination assay with mitochondrial ribosomes and in an assay with Escherichia coli ribosomes. The noncognate stop codon, UGA, encoding tryptophan in mitochondria, was recognized weakly in the heterologous assay. The mitochondrial release factor 1 protein bound to bacterial ribosomes and formed a cross-link with the stop codon within a mRNA bound in a termination complex. The affinity was strongly dependent on the identity of stop signal. Two alleles of MRF1 that contained point mutations in a release factor 1 specific region of the primary structure and that in vivo compensated for mutations in the decoding site rRNA of mitochondrial ribosomes were cloned, and the expressed proteins were purified and refolded. The variant proteins showed impaired binding to the ribosome compared with mitochondrial release factor 1. This structural region in release factors is likely to be involved in codon-dependent specific ribosomal interactions.Release factors (RFs) 1 are proteins involved in the decoding of stop signals during translational termination. There are two prokaryotic class I RFs, RF1 and RF2, which recognize UAA/ UAG and UAA/UGA, respectively, within termination signals (1), and one class II RF, RF3, which is involved in recycling the other two factors (2). RF1 and RF2 are structurally related (3), whereas RF3 is distinct (4). In the eukaryotic cytosol, two RFs have been identified: eukaryotic RF1, which recognizes all three stop signals (5), and the G protein eukaryotic RF3 (6), which stimulates eukaryotic RF1 activity. They are structurally and functionally distinct compared with their prokaryotic counterparts (7). An organellar RF was first isolated from rat liver mitochondria (8), and genes coding for putative organellar RFs have been cloned from the yeasts Saccharomyces cerevisiae and Kluyveromyces lactis (9, 10) and from humans (11). The S. cerevisiae gene encodes a product of 413 amino acids with a calculated molecular mass of 46.74 kDa, a larger protein than either E. coli RF1 or RF2. This could be explained by a small number of insertions and the presence of extra amino acids at the N terminus (9). The N terminus derived from the gene sequence has a composition suggestive of a cleavable mitochondrial targeting sequence.The most functionally conserved sites between prokaryotic and mitochondrial ribosomes are the decoding site and peptidyltransferase center, and the most highly conserved primary sequence in the RFs might reflect an interaction with either of these two centers (12). The alignment of amino acid sequences of yeast mRF1 and class I prokaryotic RFs (9) shows the highest percentage of identity in the central and the C-terminal regions, particularly within a stretch of 43 amino acids between position 280 and 322 of mRF1 (79 and 7...
The 5‘-terminal TMG-capped triribonucleotide, m3 2,2,7G5‘pppAmpUmpA, has been synthesized by condensation of an appropriately protected triribonucleotide derivative of ppAmpUmpA with a new TMG-capping reagent. During this total synthesis, it was found that the regioselective 2‘-O-methylation of 3‘,5‘-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-N-(4-monomethoxytrityl)adenosine was achieved by use of MeI/Ag2O without affecting the base moiety. A new route to 2-N,2-N-dimethylguanosine from guanosine via a three-step reaction has also been developed by reductive methylation using paraformaldehyde and sodium cyanoborohydride. These key intermediates were used as starting materials for the construction of a fully protected derivative of pAmpUmpA and a TMG-capping reagent of Im-pm3 2,2,7G. The target TMG-capped tetramer, m3 2,2,7G5‘pppAmpUmpA, was synthesized by condensation of a partially protected triribonucleotide 5‘-terminal diphosphate species, ppAMMTrmpUmpA, with Im-pm3 2,2,7G followed by treatment with 80% acetic acid. The structure of m3 2,2,7G5‘pppAmpUmpA was characterized by 1H and 31P NMR spectroscopy as well as enzymatic assay using snake venom phosphodiesterase, calf intestinal phosphatase, and nuclease P1.
L2, L3, L4, L16 and L20 are proteins of the 50s ribosomal subunit of Escherichiu coli which are essential for the assembly and activity of the peptidyl transferase centre. These proteins have been modified with the histidinespecific reagent, diethylpyrocarbonate, while L17 and L18 were treated as controls. Each modified protein tested was able to participate in the reconstitution of a 50s particle when replacing its normal counterpart, although the particles assembled with modified L2 were heterogeneous. However, although they could support assembly, modified L16 and L20 were not themselves reconstituted stably, and modified L2 and L3 were found in less than stoichiometric amounts. Particles assembled in the presence of modified L16 retained significant peptidyl transferase activity (60 -70% at 10 mM diethylpyrocarbonate) whereas those reconstituted with modified L2, L3, L4 or L20 had low activity (10-30% at 10 mM diethylpyrocarbonate). The particles assembled with the modified control portein, L17, retained 80% of their peptidyl transferase activity under the same conditions. The histidine residues within the essential proteins therefore contribute to ribosome structure and function in three significant ways; in the correct assembly of the ribosomal subunit (L2), for the stable assembly of the proteins within the ribosomal particle (L20 and L16 in particular), and directly or indirectly for the subsequent activity of the peptidyl transferase centre (L2, L3, L4 and L20). The essential nature of the unmodified histidines for assembly events precludes the use of the chemical-modification strategy to test the proposal that a histidine on one of the proteins might participate in the catalytic activity of the centre.The enzymatic activity of the Escherichia coli ribosome is provided by the peptidyl transferase centre of the 50s subunit which catalyses not only peptide bonds between amino acid residues during elongation of a growing polypeptide chain [l] but also the release-factor-mediated peptidyl-tRNA hydrolysis when the polypeptide is completed [2]. Whether these enzymatic activities are a function of individual ribosomal proteins, the rRNA, or a combination of both types of macromolecules, has been the subject of much debate, but experimental evidence to support one of the three possibilities is lacking. There is good evidence that both ribosomal proteins and rRNA are required, however, for an active peptidyl transferase centre to be assembled [3]. Eight ribosomal components are essential for reconstitution of the enzymatic activity in E. coli, namely 23s rRNA and the seven proteins L2-L4, L15, L16, L20 and L24 [4].Proteins L20 [5, 61 and L24 [7] are exclusively involved in the early assembly events of the 50s subunits and therefore do not provide the activity of the centre. The existence of a mutant lacking L24 confirms a role for this proteins in assembly of the subunit rather than the peptidyl transferase Correspondence to W. P. Tate, Department of Biochemistry, University of Otago, P.O. Box 56, Dunedin, Ne...
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