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Query: UNIPROT:P06889 (Mol)
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The C proteins (C1 and C2) of HeLa 40S heterogeneous nuclear ribonucleoprotein particles copurify under native conditions as a stable complex with a fixed molar protein ratio (S.F. Barnett, W.M. LeStourgeon, and D.L. Friedman, J. Biochem. Biophys. Methods 16:87-97, 1988). Gel filtration chromatography and velocity sedimentation analyses of these complexes revealed a large Stokes radius (6.2 nm) and a sedimentation coefficient of 5.8S. On the basis of these values and a partial specific volume of 0.70 cm3/g based on the amino acid composition, the molecular weight of the complex was calculated to be 135,500. This corresponds well to 129,056, the sequence-determined molecular weight of a (C1)3C2 tetramer. Reversible chemical cross-linking with dithiobis(succinimidyl propionate) and analysis of cross-linked and cleaved complexes in sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirmed that the C proteins exist as tetramers, most or all of which are composed of (C1)3C2. The tetramer is stable in a wide range of NaCl concentrations (0.09 to 2.0 M) and is not dissociated by 0.5% sodium deoxycholate. This stability is not the result of disulfide bonds or interactions with divalent cations. The hydrodynamic properties of highly purified C-protein tetramers are the same for C-protein complexes released from intact particles with RNase or high salt. These findings support previous studies indicating that the core particle protein stoichiometry of 40S heterogeneous nuclear ribonucleoproteins is N(3A1-3A2-1B1-1B2-3C1-1C2), where N = 3 to 4, and demonstrate that the C-protein tetramer is a fundamental structural element in these RNA-packaging complexes. The presence of at least three tetramers per 40S monoparticle, together with the highly anisotropic nature of the tetramer, suggesting that one-third of the 700-nucleotide pre-mRNA moiety packaged in monoparticles is associated through a sequence-independent mechanism with the C protein.
Mol Cell Biol 1989 Feb
PMID:The C proteins of HeLa 40S nuclear ribonucleoprotein particles exist as anisotropic tetramers of (C1)3 C2. 256 30

Ribosomal protein L5 binds specifically to 5S rRNA to form a complex that is a precursor to 60S subunit assembly in vivo. Analyses in yeast cells, mammalian cells, and Xenopus embryos have shown that the accumulation of L5 is not coordinated with the expression of other ribosomal proteins. In this study, the primary structure and developmental expression of Xenopus ribosomal protein L5 were examined to determine the basis for its distinct regulation. These analyses showed that L5 expression could either coincide with 5S rRNA synthesis and ribosome assembly or be controlled independently of these events at different stages of Xenopus development. L5 synthesis during oogenesis was uncoupled from the accumulation of 5S rRNa but coincided with subunit assembly. In early embryos, the inefficient translation of L5 mRNA resulted in the accumulation of a stable L5-5S rRNA complex before ribosome assembly at later stages of development. Additional results demonstrated that L5 protein synthesized in vitro bound specifically to 5S rRNA.
Mol Cell Biol 1989 Dec
PMID:Developmental expression and 5S rRNA-binding activity of Xenopus laevis ribosomal protein L5. 258 20

The context requirements for recognition of an initiator codon were evaluated in vitro by monitoring the relative use of two AUG codons that were strategically positioned to produce long (pre-chloramphenicol acetyl transferase [CAT]) and short versions of CAT protein. The yield of pre-CAT initiated from the 5'-proximal AUG codon increased, and synthesis of CAT from the second AUG codon decreased, as sequences flanking the first AUG codon increasingly resembled the eucaryotic consensus sequence. Thus, under prescribed conditions, the fidelity of initiation in extracts from animal as well as plant cells closely mimics what has been observed in vivo. Unexpectedly, recognition of an AUG codon in a suboptimal context was higher when the adjacent downstream sequence was capable of assuming a hairpin structure than when the downstream region was unstructured. This finding adds a new, positive dimension to regulation by mRNA secondary structure, which has been recognized previously as a negative regulator of initiation. Translation of pre-CAT from an AUG codon in a weak context was not preferentially inhibited under conditions of mRNA competition. That result is consistent with the scanning model, which predicts that recognition of the AUG codon is a late event that occurs after the competition-sensitive binding of a 40S ribosome-factor complex to the 5' end of mRNA. Initiation at non-AUG codons was evaluated in vitro and in vivo by introducing appropriate mutations in the CAT and preproinsulin genes. GUG was the most efficient of the six alternative initiator codons tested, but GUG in the optimal context for initiation functioned only 3 to 5% as efficiently as AUG. Initiation at non-AUG codons was artifactually enhanced in vitro at supraoptimal concentrations of magnesium.
Mol Cell Biol 1989 Nov
PMID:Context effects and inefficient initiation at non-AUG codons in eucaryotic cell-free translation systems. 260 9

This paper describes in vitro experiments with two types of intramolecular duplex structures that inhibit translation in cis by preventing the formation of an initiation complex or by causing the complex to be abortive. One stem-loop structure (delta G = -30 kcal/mol) prevented mRNA from engaging 40S subunits when the hairpin occurred 12 nucleotides (nt) from the cap but had no deleterious effect when it was repositioned 52 nt from the cap. This result confirms prior in vivo evidence that the 40S subunit-factor complex, once bound to mRNA, has considerable ability to penetrate secondary structure. Consequently, translation is most sensitive to secondary structure at the entry site for ribosomes, i.e., the 5' end of the mRNA. The second stem-loop structure (hp7; delta G = -61 kcal/mol, located 72 nt from the cap) was too stable to be unwound by 40S ribosomes, hp7 did not prevent a 40S ribosomal subunit from binding but caused the 40S subunit to stall on the 5' side of the hairpin, exactly as the scanning model predicts. Control experiments revealed that 80S elongating ribosomes could disrupt duplex structures, such as hp7, that were too stable to be penetrated by the scanning 40S ribosome-factor complex. A third type of base-paired structure shown to inhibit translation in vivo involves a long-range interaction between the 5' and 3' noncoding sequences.
Mol Cell Biol 1989 Nov
PMID:Circumstances and mechanisms of inhibition of translation by secondary structure in eucaryotic mRNAs. 260 12

The mouse Surfeit locus, which contains a cluster of at least four genes (Surf-1 to Surf-4), is unusual in that adjacent genes are separated by no more than 73 base pairs (bp). The heterogeneous 5' ends of Surf-1 and Surf-2 are separated by only 15 to 73 bp, the 3' ends of Surf-1 and Surf-3 are only 70 bp apart, and the 3' ends of Surf-2 and Surf-4 overlap by 133 bp. This very tight clustering suggests a cis interaction between adjacent Surfeit genes. The Surf-3 gene (which could code for a basic polypeptide of 266 amino acids) is a highly expressed member of a pseudogene-containing multigene family. By use of an anti-peptide serum (against the C-terminal nine amino acids of the putative Surf-3 protein) for immunofluorescence and immunoblotting of mouse cell components and by in vitro translation of Surf-3 cDNA hybrid-selected mRNA, the Surf-3 gene product was identified as a 32-kilodalton ribosomal protein located in the 60S ribosomal subunit. From its subunit location, gel migration, and homology with a limited rat ribosomal peptide sequence, the Surf-3 gene was shown to encode the mouse L7a ribosomal protein. The Surf-3 gene is highly conserved through evolution and was detected by nucleic acid hybridization as existing in multiple copies (multigene families) in other mammals and as one or a few copies in birds, Xenopus, Drosophila, and Schizosaccharomyces pombe. The Surf-3 C-terminal anti-peptide serum detects a 32-kilodalton protein in other mammals, birds, and Xenopus but not in Drosophila and S. pombe. The possible effect of interaction of the Surf-3 ribosomal protein gene with adjacent genes in the Surfeit locus at the transcriptional or posttranscriptional level or both levels is discussed.
Mol Cell Biol 1989 Jan
PMID:Ribosomal protein L7a is encoded by a gene (Surf-3) within the tightly clustered mouse surfeit locus. 264 30

Yeast 60S ribosomal subunits have been dissociated by reversible modification with dimethylmaleic anhydride. Treatment with 40 mumol reagent/ml releases 35% of the protein, producing core particles inactive in polyphenylalanine synthesis, which are totally or highly deficient in 17 different proteins. This preparation of residual particles recovers 45% of the original activity upon incubation with the released proteins. The reconstituted particles can be isolated by centrifugation without loss of activity, having the protein composition of the original subunits.
Mol Cell Biochem 1989 Mar 16
PMID:Disassembly and reconstitution of yeast 60S ribosomal subunits. 266 49

A DNA segment approximately 200 base pairs upstream of the Xenopus laevis ribosomal promoter acts both as an upstream promoter element that augments transcription and as a transcription terminator. It is, however, unclear to what extent these two activities are related. A segment of the X. laevis ribosomal DNA, containing the terminator and the upstream promoter element, was subjected to point mutation, and the effects of the resulting mutations were investigated by oocyte microinjection. Analysis of 26 point mutants revealed not only sequences that augment 40S transcription but also those that repress it. The sequences that augmented transcription lay within the T3 homology box and also near the site of 3'-end formation. These sequences also played a role in termination. The sequences that repressed transcription lay within the G+C-rich DNA flanking the T3 box. It can be concluded that termination is probably essential but may not be sufficient for the activity of the upstream promoter element.
Mol Cell Biol 1989 Sep
PMID:The Xenopus laevis ribosomal gene terminator contains sequences that both enhance and repress ribosomal transcription. 277 66

The activity of a protein kinase specific to ribosomal protein S6 was determined in early loach embryos in basal conditions and after their treatment with epidermal growth factor (EGF). The cytosol of loach blastoderms isolated at the early gastrula stage possessed a high level of protein kinase activity catalysing incorporation of 0. 33 pmol.min-1.mg-1 of Pi into exogenous S6 protein of rat liver ribosomal 40S subunit. The treatment of embryos for 30 min with EGF (10 ng/ml) added to the incubation medium caused an 25% increase of total S6-kinase activity in cytosol compared with its counterpart in non-stimulated embryos. After chromatography of loach embryos cytosol on DE-52 three fractions possessing S6-kinase activity were revealed, which were eluted with 10 microM cAMP (I), 150 mM NaCl (II) and 300 mM NaCl (III), respectively. After treatment of blastoderms with EGF in the described conditions the enzymatic activity 2-fold decreased in fraction I, increased in fraction II 4-fold and remained practically unchanged in fraction III. The mitogen-stimulated kinase, apart from S6 protein, phosphorylated also casein and but not histone H1.
Mol Biol (Mosk)
PMID:[Epidermal growth factor stimulates protein kinase activity, specific for ribosomal protein S6 in loach embryo blastoderm]. 278 4

The ribonucleoprotein (RNP) structures of the pre-mRNA and RNA processing products generated during in vitro splicing of an SP6/beta-globin pre-mRNA were characterized by sucrose gradient sedimentation analysis. Early, during the initial lag phase of the splicing reaction, the pre-mRNA sedimented heterogeneously but was detected in both 40S and 60S RNP complexes. An RNA substrate lacking a 3' splice site consensus sequence was not assembled into the 60S RNP complex. The two splicing intermediates, the first exon RNA species and an RNA species containing the intron and the second exon in a lariat configuration (IVS1-exon 2 RNA species), were found exclusively in a 60S RNP complex. These two splicing intermediates cosedimented under a variety of conditions, indicating that they are contained in the same RNP complex. The products of the splicing reaction, accurately spliced RNA and the excised IVS1 lariat RNA species, are released from the 60S RNP complex and detected in smaller RNP complexes. Sequence-specific RNA-factor interactions within these RNP complexes were evidenced by the preferential protection of the pre-mRNA branch point from RNase A digestion and protection of the 2'-5' phosphodiester bond of the lariat RNA species from enzymatic debranching. The various RNP complexes were further characterized and could be distinguished by immunoprecipitation with anti-Sm and anti-(U1)RNP antibodies.
Mol Cell Biol 1986 Jul
PMID:Ribonucleoprotein complex formation during pre-mRNA splicing in vitro. 294 39

The Saccharomyces cerevisiae CRY1 gene encodes ribosomal protein rp59, a component of the 40S ribosomal subunit. Mutations in CRY1 can confer resistance to the alkaloid cryptopleurine, an inhibitor of the elongation step of translation. The nucleotide sequence of the cloned CRY1 gene was determined. The predicted amino acid sequence shows that CRY1 encodes a 14,561-dalton polypeptide that has 88% amino acid sequence homology to the hamster or human S14 ribosomal protein responsible for emetine resistance and 45% homology to Escherichia coli ribosomal protein S11. Analysis of the DNA sequences upstream from CRY1 revealed the presence of three sequences, HOMOL1 (consensus, A/TACATCC/TG/ATA/GCA), RPG (consensus, ACCCA/GTACATT/CT/A), and a thymine-rich sequence, found upstream of more than 20 other cloned yeast genes encoding components of the translational apparatus. We exploited the ability to assay the expression of CRY1 in vivo by using the cryptopleurine resistance phenotype to demonstrate that these three consensus sequences are necessary for the transcription of CRY1. We previously showed that the upstream promoter element of the yeast RP39A gene consists of these identical sequence motifs. Therefore, we suggest that these three sequences define a consensus promoter element for the genes encoding the yeast translational apparatus. CRY1 is one of several hundred yeast genes, including ribosomal protein genes, whose expression is transiently decreased 10-fold upon heat shock. We found that the HOMOL1 and RPG consensus sequences are not necessary for the heat shock response of CRY1.
Mol Cell Biol 1987 May
PMID:Structure and expression of the Saccharomyces cerevisiae CRY1 gene: a highly conserved ribosomal protein gene. 303 34


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