Showing papers on "Ribosomal protein published in 1986"
TL;DR: The set of 452 different sequences comprises all sequences that to the authors' knowledge had been published or were available from the sequence library file servers as of December 1, 1990, and that are either complete or cover a minimum of about 70% of the complete sequence.
Abstract: Table 1 lists data on 455 small ribosomal subunit RNA (further abbreviated as srRNA) sequences (references 1 —452) that have been published or submitted to the EMBL or GenBank nucleotide sequence libraries and that are presently stored in aligned form in our data base. The number identifying each sequence in the first column of Table 1 corresponds with the literature reference. If two or more closely related species share the same sequence, they bear the same number, followed by a different lower case character, and the common sequence is listed only once in our alignment. The set of 452 different sequences consists of 97 eukaryotic cytoplasmic, 19 archaebacterial, 276 eubacterial, 16 plastidial, and 44 mitochondrial srRNAs. It comprises all sequences that to our knowledge had been published or were available from the sequence library file servers as of December 1, 1990, and that are either complete or cover a minimum of about 70% of the complete sequence. Partial sequences are included because some of the methods now frequently used for srRNA sequencing preclude the determination of the structure at one or both of the termini. One such method consists of reverse transcription of the srRNA by means of primers complementary to conserved areas in the primary structure (453). In this case the 3'-terminal sequence cannot be found. Another approach (55, 328) involves amplification of the rDNA by means of the polymerase chain reaction (454), using primers binding to conserved areas near the termini, but within the sequence coding for the mature small subunit RNA. In this case both terminal sequences remain unknown. Both methods allow to establish a continuous sequence covering more than 95% of the structure, provided that a sufficient number of primers complementary to internal conserved areas is used. Some authors (e.g. 455), however, use a more limited set of primers and publish sequences that are not only partial but also discontinuous.
983 citations
TL;DR: Detailed investigation of the higher-order structure of 16 S ribosomal RNA and extensive protection of conserved, unpaired adenines upon formation of 30 S subunits suggests that they play a special role in the assembly process, possibly providing signals for protein recognition.
484 citations
TL;DR: The aim of this monograph is to provide a Discussion of the Foundations of Regulation of Ribosomal RNA Synthesis and its Applications to Ribosome Synthesis.
Abstract: INTRODUCTION . . . .. ... .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . ......... . . . . . . . . . . . . .. . . . . . . . . . . . 297 GENE STRUCTURE 298 Ribosomal RNA Genes. . . . . . . . . . . . . . . . . . . . . . . .. . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298 Ribosomal Protein Genes . . . . . . . . . . . . . . . . . . . . . . . .. ... .... .. . . . . . . . . . . . . . . ..... .. ...... 298 REGULATORY MECHANISMS ......... . . . . . . . . . .. .. . . . . . . . . . . . . . . . . . . ..... . . ... . . . . . 304 Regulation of Ribosomal RNA Synthesis . ...... . . . . . . . . . . . ......... . . . . . . . . . . . . . . . . . . . . . . . . . . 304 Regulation of Ribosomal Protein Synthesis.... .. . . . . . . . . . . . . . . . . . . . . . . . . . ..... . . . . . . . . . . . . . . 309 Autogenous Regulation of Selected R-Protein Operons . . . . . . . . . ' 314 ACCOUNTING FOR THE PHYSIOLOGICAL RESPONSES OF RIBOSOME SYNTHESIS " " " " , ,' 317
287 citations
TL;DR: Findings indicate that SLE anti-P antibodies react with a single sequential (linear) antigenic determinant on all three P proteins, but that additional antibodies recognize a conformational determinant(s).
Abstract: The characteristics of eukaryotic ribosomal proteins P0, P1, and P2 (P proteins) and their antigenic determinants were studied using the sera of patients with systemic lupus erythematosus (SLE). P0, P1, and P2 were isolated as a macromolecular complex by preparative isoelectric focusing and anion-exchange chromatography in the presence of 6 M urea. The apparent molecular size of the complex was 140 kDa as determined by gel filtration on a Sephadex G-200 column. P0 may, therefore, be the eukaryotic equivalent of Escherichia coli ribosomal protein L10. In addition, all three P proteins were detected in the postribosomal supernatant of HeLa cells, and P0 and P1 were found to be more acidic than their ribosome-bound counterparts. Partial proteolysis experiments revealed that SLE anti-P sera recognized one or both ends of the P2 equivalent protein from Artemia salina (eL12). Sixteen SLE sera containing antibodies to P0, P1, and P2 reacted with a carboxyl-terminal peptide 22 amino acids in length of eL12 and not with an amino-terminal peptide of 20 amino acids. Even though the carboxyl-terminal peptide completely inhibited the ability of the antiserum to react with all three proteins on an immunological blot, the same peptide produced only small decreases in binding of the SLE antibody to the native, nondenatured P proteins. These findings indicate that SLE anti-P antibodies react with a single sequential (linear) antigenic determinant on all three P proteins, but that additional antibodies recognize a conformational determinant(s).
230 citations
TL;DR: The availability of a purified S6 protein kinase should facilitate elucidation of the molecular mechanism of S6 phosphorylation during growth stimulation in Xenopus oocytes ribosomal protein S6.
179 citations
TL;DR: The distribution and orientation of the S13-like and “URF 1”-like sequences are discussed and the possibility that they are active genes is discussed.
Abstract: A transcribed segment of mitochondrial DNA (mtDNA) from Nicotiana tabacum contains the F0-ATPase subunit 9 gene, an open reading frame with homology to the E. coli small subunit ribosomal protein S13 and an open reading frame with homology to a portion of the mammalian “URF 1” protein, recently shown to be a component of the NADH:ubiquinone reductase complex (NADH:Q 1). The transcriptional patterns of the tobacco ATPase 9 gene and S13-like open reading frame share eight RNA species indicating the two sequences are part of the same transcriptional unit. A maize mtDNA fragment contains the S13 homologous sequence and the NADH:Q 1 homologous sequence in an orientation similar to tobacco. The S13-like sequence is present as a single copy in maize and tobacco, as two copies in wheat, and is absent in pea and bean. We discuss the distribution and orientation of the S13-like and “URF 1”-like sequences and the possibility that they are active genes.
154 citations
TL;DR: Human and Chinese hamster S14 protein sequences deduced from the cDNAs are identical, and human RPS14 contains a TATA sequence (TATACTT) upstream from exon 1, and three related short sequence motifs occur in this region of the R PS14 gene.
Abstract: Chinese hamster ribosomal protein S14 cDNA was used to recognize homologous human cDNA and genomic clones Human and Chinese hamster S14 protein sequences deduced from the cDNAs are identical Two overlapping human genomic S14 DNA clones were isolated from a Charon 28 placental DNA library A fragment of single-copy DNA derived from an intron region of one clone was mapped to the functional RPS14 locus on human chromosome 5q by using a panel of human X Chinese hamster hybrid cell DNAs The human S14 gene consists of five exons and four introns spanning 59 kilobase pairs of DNA Polyadenylated S14 transcripts purified from HeLa cell cytoplasma display heterogeneous 5' ends that map within noncoding RPS14 exon 1 This precludes assignment of a unique 5' boundary of RPS14 transcripts with respect to the cloned human genomic DNA Apparently HeLa cells either initiate transcription at multiple sites within RPS14 exon 1, or capped 5' oligonucleotides are removed from most S14 mRNAs posttranscription In contrast to the few murine ribosomal protein and several other mammalian housekeeping genes whose structures are known, human RPS14 contains a TATA sequence (TATACTT) upstream from exon 1 Three related short sequence motifs, also observed in murine and yeast ribosomal protein genes, occur in this region of the RPS14 gene RPS14 introns 3 and 4 both contain Alu sequences Interestingly, the Alu sequence in intron 3 is located slightly downstream from a chromosome 5 deletion breakpoint in one human X hamster hybrid clone analyzed
137 citations
TL;DR: It is shown that the sequences between nucleotides -256 and -170 upstream of RP39A are essential for expression of this gene, and the ability of a DNA fragment carrying these three sequence elements to substitute for the upstream activation site regions of CYC1 indicate that these three oligonucleotides may be essential to the transcription of yeast ribosomal protein genes.
Abstract: To initiate a genetic analysis of yeast ribosomal protein gene promoters, we have constructed a gene fusion between the yeast ribosomal protein gene RP39A and the Escherichia coli lacZ gene. This gene fusion contains approximately 1,030 nucleotides of the 5' flanking region and the first 49 1/3 codons of RP39A fused in frame to a large 3' end fragment of lacZ. Whether it is introduced into yeast cells on a moderately high-copy-number plasmid, or integrated into the yeast genome at the RP39A locus, this RP39A-lacZ gene directs the synthesis of a hybrid transcript which encodes beta-galactosidase activity. Deletions in the 5' flanking region of RP39A-lacZ were constructed by linker insertion and BAL 31 mutagenesis. The expression of the mutant genes in yeast cells was assayed by measuring RP39A-lacZ mRNA and beta-galactosidase levels. By these means we have shown that the sequences between nucleotides -256 and -170 upstream of RP39A are essential for expression of this gene. Three sequence motifs, HOMOL1, RPG, and a T-rich region, which were found in that order 5'----3' upstream of most yeast ribosomal protein genes, were present within this interval. We found that substitution of the CYC1-lacZ upstream activation site with the fragment from nucleotides -298 to -172 upstream of RP39A, containing the HOMOL1-RPG-T-rich motif in that 5'----3' orientation, fully restored expression of the CYC1-lacZ gene. The essentially of HOMOL1, the RPG sequence, and the T-rich region for wild-type levels of expression of RP39A, the conserved location and order of these sequence motifs in yeast ribosomal protein genes, and the ability of a DNA fragment carrying these three sequence elements to substitute for the upstream activation site regions of CYC1 indicate that these three oligonucleotides may be essential to the transcription of yeast ribosomal protein genes.
121 citations
TL;DR: The results are interpreted as indicative of loss of structure, and suggest that S4 binding causes disruption of adventitious pairing in these regions, possibly by stabilizing the geometry of the RNA such that these interactions are prevented from forming.
120 citations
TL;DR: The nucleotide sequence of 2.5 kbp from the cloned SalI fragments 8 and 11 of spinach plastid DNA has been determined and imply that plastids chromosomes code for components of the organelle transcription apparatus.
Abstract: The nucleotide sequence of 2.5 kbp from the cloned SalI fragments 8 and 11 of spinach plastid DNA has been determined. This region was found to encode three open reading frames for hydrophilic polypeptides of 77, 138, and 335 amino acids. Using the computer search algorithm of Lipman and Pearson (Science 227, 1435, 1985), these genes were identified as coding for homologues of E. coli initiation factor IF-1 (inFA), 30S ribosomal protein S11 (rps11), and the alpha-subunit of DNA-dependent RNA polymerase (rpoA). The spinach plastid gene organization is inFA - 381 bp spacer - rps11 - 72 bp spacer - rpoA. The genes are transcribed in vivo and appear to encode functional proteins. These findings imply that plastid chromosomes code for components of the organelle transcription apparatus.
117 citations
TL;DR: It is reported here in vivo S1 nuclease mapping and in vitro transcription experiments that demonstrate that rpsO and pnp are cotranscribed from a promoter P1, located 108 nucleotides upstream from rps O, and that another promoter P2, located between the two genes 158 nucleotide upstream from pnp, also directs the transcription of pnp.
01 Jan 1986
TL;DR: Ribosomal components from different organisms vary considerably in size and number, and they constitute ribosomes with sedimentation coefficients ranging from 55S to 80S (see chapter by Wittmann in this volume), which raises the question of how different species of ribosome can perform a common function despite of their drastic structural differences.
Abstract: Since ribosomes occur in all organisms ranging from bacteria to mammals, they are ideal objects for evolutionary studies. Although these organelles have maintained a common function in protein biosynthesis, their structure has undergone drastic changes throughout evolution. Ribosomal components from different organisms vary considerably in size and number, and they constitute ribosomes with sedimentation coefficients ranging from 55S to 80S (see chapter by Wittmann in this volume). This raises the uestions of how different species of ribosomes can perform a common function despite of their drastic structural differences and which parts of the individual ribosomal components are responsible for conservation of the basic function.
TL;DR: With modifications of Kaltschmidt and Wittmann's two dimensional gel electrophoresis, quantitative yield and reproducibility became better, and many faint spots disappeared not only at the high molecular weight side but also in the region containing primary spots of ribosomal proteins.
Abstract: Kaltschmidt and Wittmann's two dimensional gel electrophoresis was improved in the following points. Preruns using radical scavengers were carried out to eliminate free radicals remaining in gels. Gelation of sample solutions was not performed to avoid immobilization of proteins in the sample gels. Instead, for preparing sample gels, prior to the first dimension (1-D) electrophoresis, another electrophoresis was performed to charge proteins into gel pieces polymerized previously. Proteins migrated together with charged reductants to avoid formation of artificial disulfide bridges during migration. The second dimension (2-D) electrophoresis was carried out at a more acidic pH, 3.6, to get better separation of very small and basic proteins. With these modifications, quantitative yield and reproducibility became better, and many faint spots disappeared not only at the high molecular weight side but also in the region containing primary spots of ribosomal proteins. The proportionality of the migration distance to the logarithm of molecular weight was also increased. On the improved 2-D electrophoretogram of Escherichia coli ribosomal proteins, four new spots, called protein A, B, C, and D, were found in the basic region. Proteins A, B, and C belong to 50S subunits but D to 30S. Their molecular weights were determined electrophoretically as 6,400, 4,900, 8,200, and 5,900, respectively. Their copy numbers in crude ribosomes were estimated to be 0.6, 0.4, 0.3, and 0.1, respectively, by using 14C-labeling.
TL;DR: The gene for a yeast ribosomal protein, RPL32, contains a single intron and the product of this gene appears to participate in feedback control of the splicing of the intron from the transcript.
Abstract: The gene for a yeast ribosomal protein, RPL32, contains a single intron. The product of this gene appears to participate in feedback control of the splicing of the intron from the transcript. This autogenous regulation of splicing provides a striking analogy to the autogenous regulation of translation of ribosomal proteins in Escherichia coli.
TL;DR: The change in half-life observed during increased gene dosage is due to translational repression by L1 and is probably a consequence of L1 blocking translation of L11 mRNA and not due to some nucleolytic activity mediated by L2.
TL;DR: The results demonstrate that removal of one box has a detrimental effect on the level of transcription, whereas after the deletion of both boxes hardly any transcription can be observed.
Abstract: Previous studies have revealed the occurrence of two closely linked conserved sequence elements, designated as HOMOL 1 and RPG box, in front of most yeast ribosomal protein genes examined. To investigate whether these conserved nucleotide elements play a role in the regulation of ribosomal protein gene expression, we performed deletion analysis of the DNA region upstream of the gene encoding ribosomal protein L25. To that end we constructed a hybrid gene consisting of the pertinent 5'-flanking sequence and the Escherichia coli galK marker gene. The effects on the transcription of this fusion gene of Bal31-generated deletions were measured by Northern analysis of RNA isolated from the respective transformed yeast cells. The results demonstrate that removal of one box has a detrimental effect on the level of transcription, whereas after the deletion of both boxes hardly any transcription can be observed. Subsequently we inserted synthetic oligonucleotides in the upstream region of an L25 gene from which the original boxes had been removed. Expression of the inactivated hybrid gene turned out to be restored even by insertion of one RPG element. Moreover, the RPG box functions in both orientations, though not with equal efficiency.
TL;DR: Ribosomal and DNA binding proteins have been extracted from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius grown at 75–80°C and pH 4.8-4.5 and the DNA binding character of these proteins was verified using several binding assays.
TL;DR: It is proposed that the phosphorylation of S6 could alter the binding or positioning of the message on the 404 ribosomal subunit, thereby affecting translation, and this role in the regulation of protein synthesis is discussed.
Abstract: Publisher Summary Ribosomal protein S6 is a major phosphorylated ribosomal protein in every mammalian cell type and tissue. This chapter focuses on the phosphorylation of ribosomal protein S6 and aminoacyl-tRNA synthetases and discusses the role of these phosphorylation events in the regulation of protein synthesis. It proposes that the phosphorylation of S6 could alter the binding or positioning of the message on the 404 ribosomal subunit, thereby affecting translation. It is assumed that there are at least three classes of mRNA that are recognized differentially by 40-S ribosomal subunits: (1) proteins involved in growth—mRNA is preferentially translated in response to growth-promoting compounds when S6 is multiply phosphorylated by the mitogen-stimulated S6 kinase; (2) housekeeping proteins—mRNA is translated under all conditions; the phosphorylation of S6 has no effect; and (3) proteins involved in catabolism—mRNA is preferentially translated in response to elevated levels of cAMP where S6 is phosphorylated by cA kinase. The various classes of mRNA would be differentially recognized by the different phosphorylated states of the ribosome. This differential recognition of the mRNA could be mediated by the primary sequence and conformation of the leader region.
TL;DR: The nucleotide sequence of a tobacco (Nicotiana tabacum) chloroplast gene cluster that encodes eight proteins homologous to Escherichia coli ribosomal proteins L23, L2, S19, L22, S3, L16, L14, and S8 has been determined.
Abstract: The nucleotide sequence of a tobacco (Nicotiana tabacum) chloroplast gene cluster that encodes eight proteins homologous to Escherichia coli ribosomal proteins L23, L2, S19, L22, S3, L16, L14, and S8 has been determined. RNA gel blot hybridization revealed that all eight coding regions are expressed in the chloroplasts. The arrangement of the eight genes resembles that found in the E. coli S10 and spc operons. Among the eight genes, the L2 and L16 genes contain 666- and 1020-base-pair introns, respectively. These intron boundary sequences are consistent with the conserved boundary sequences of the chloroplast group III introns [Shinozaki, K., Deno, H., Sugita, M., Kuramitsu, S. & Sugiura, M. (1986) Mol. Gen. Genet. 202, 1-5].
TL;DR: The primary structures of the yeast recessive omnipotent suppressor gene SUP1 (SUP45) and one of its mutant alleles (sup1-ts36) was determined and the hypothetical function of this protein in control of translational fidelity is discussed.
Abstract: The primary structures of the yeast recessive omnipotent suppressor gene SUP1 (SUP45) and one of its mutant alleles (sup1-ts36) was determined. The gene codes for a protein of 49 kD. The mutant protein differs from the wildtype form in one amino acid residue (Ser instead of Leu) in the N-terminal part. The codon usage differs significantly from that of yeast ribosomal protein genes. However, an upstream element resembling a conserved oligonucleotide in the region 5' to ribosomal protein genes in S. cerevisiae has been found. A DNA probe internal to the SUP1 gene does not exhibit detectable homology to genomic DNA neither from higher eucaryotes nor from eu- or archaebacteria. The hypothetical function of this protein in control of translational fidelity is discussed.
TL;DR: During the nucleotide sequencing of chloroplast DNA from the liverwort, M. polymorpha, it was found that a coding sequence corresponding to the Esherichia coli ribosomal protein S12 gene (rps 12) is split into three exons.
TL;DR: It is demonstrated that ribosomes from the four suppressors SUP35, SUP44, SUP45 and SUP46 translate polyuridylate templates in vitro with higher errors than ribosome from the normal stain, and that this misreading is substantially enhanced by the antibiotic paromomycin.
TL;DR: The initiation factor IF3 is platinated with trans-diamminedichloroplatinum(II) and cross-linked to Escherichia coli 30S ribosomal subunit and two cross-linking sites are unambiguously identified on the 16S rRNA.
Abstract: The initiation factor IF3 is platinated with trans-diamminedichloroplatinum(II) and cross-linked to Escherichia coli 30S ribosomal subunit. Two cross-linking sites are unambiguously identified on the 16S rRNA: a major one, in the region 819-859 in the central domain, and a minor one, in the region 1506-1529 in the 3'-terminal domain. Specific features of these sequences together with their particular location within the 30S subunit lead us to postulate a role for IF3, that conciliates topographical and functional observations made so far.
Journal Article•
TL;DR: The 1,870-base-pair primary sequence of a human 18S rRNA gene is reported and a secondary structure based on this sequence and the general mammalian structure is proposed, which may make the small subunit rDNA the most highly conserved sequence known.
Abstract: We report the 1,870-base-pair primary sequence of a human 18S rRNA gene and propose a secondary structure based on this sequence and the general mammalian structure A basic secondary structure for the small subunit rRNA has been preserved throughout evolution by compensatory and neutral base changes in double-stranded regions The molecule contains eight regions that can vary in structure and that comprise 432 bases, while 1,438 bases belong to regions of conserved structure among all species tested The conserved regions show a remarkably low sequence divergence rate of 01% between the human and mouse genes over the approximately 80 million years since the mammalian radiation This value may make the small subunit rDNA the most highly conserved sequence known Sequence conservation in higher eukaryotes with multiple copies of the gene is probably achieved by the combination of strong selection and the correction of tandem genes by unequal homologous exchange
TL;DR: The nucleotide sequence of a tobacco chloroplast gene coding for a protein homologous to Escherichia coli ribosomal protein S16 (rps16) has been determined and its deduced amino acid sequence shows 34% homology (46% conservative replacements) with that of E. coli Ribosomal Protein S16.
Abstract: The nucleotide sequence of a tobacco chloroplast gene coding for a protein homologous to Escherichia coli ribosomal protein S16 (rps16) has been determined. It is located 690 bp upstream from the gene for tRNALys (UUU) on the same DNA strand. This gene has an 860 bp intron. The intron boundary sequences are similar to those of the tobacco chloroplast split genes for tRNAGly (UCC), tRNALys (UUU), tRNAVal (UAC) and ribosomal proteins L2 and S12. We classified these introns as a new group, designated group III, which is different from groups I and II proposed by Michel and Dujon (1983). The conserved boundary sequences of the group III introns are GTGCGNY at the 5′ ends and ATCNRY(N)YYAY at the 3′ ends. The rps16 coding region contains 255 bp (85 codons) and its deduced amino acid sequence shows 34% homology (46% conservative replacements) with that of E. coli ribosomal protein S16. The primary transcript of rps16 is 1.3 kb long.
TL;DR: It is concluded that accumulation of L25 is not controlled by an autogenous (pre-)mRNA-targeted mechanism similar to that operating in bacteria, but rather by extremely rapid degradation of excess protein produced.
Abstract: When the gene dosage for the primary rRNA-binding ribosomal protein L25 in yeast cells was raised about 50-fold, the level of mature L25 transcripts was found to increase almost proportionally. The plasmid-derived L25 transcripts were structurally indistinguishable from their genomic counterparts, freely entered polysomes in vivo and were fully translatable in a heterologous in vitro system. Nevertheless, pulse-labelling for periods varying from 3–20 min did not reveal a significant elevation of the intracellular level of L25 protein. When pulse-times were decreased to 10–45 s, however, we did detect a substantial over production of L25. We conclude that, despite the strong RNA-binding capacity of the protein, accumulation of L25 is not controlled by an autogenous (pre-)mRNA-targeted mechanism similar to that operating in bacteria, but rather by extremely rapid degradation of excess protein produced.
TL;DR: The structure of ribosomal protein L30 from Bacillus stearothermophilus has been solved to a resolution of 2.5 A and it is proposed that these reflect sites of protein-protein and protein-RNA interaction, respectively.
Abstract: The structure of ribosomal protein L30 from Bacillus stearothermophilus has been solved to a resolution of 2.5 A. The molecule is somewhat elongated and contains two helices and a three-stranded, anti-parallel beta-pleated sheet. The protein fold, in which helices pack on the same side of the sheet, generates a simple helix-sheet, two-layered motif. It is possible to distinguish three hydrophobic patches on the molecular surface, and one end has six isolated arginine and lysine residues. It is proposed that these reflect sites of protein-protein and protein-RNA interaction, respectively. The protein fold is very similar to that of the only other known ribosomal protein structure, L7/L12 from Escherichia coli, and, based on this similarity, an attempt is made to align the amino acid sequences of the two proteins.
TL;DR: Complexes containing rat liver 80S ribosomes treated with puromycin and high concentrations of KCl, elongation factor 2 (EF-2) from pig liver, and guanosine 5'-[beta, gamma-methylene]triphosphate were prepared and new neighboring protein pairs in large subunits, LA33-L11 and LA 33-L12 were found.
Abstract: Complexes containing rat liver 80S ribosomes treated with puromycin and high concentrations of KCl, elongation factor 2 (EF-2) from pig liver, and guanosine 5'-[beta, gamma-methylene]triphosphate were prepared. Neighboring proteins in the complexes were cross-linked with the bifunctional reagent 2-iminothiolane. Proteins were extracted and then separated into 22 fractions by chromatography on carboxymethylcellulose of which seven fractions were used for further analyses. Each protein fraction was subjected to diagonal polyacrylamide/sodium dodecyl sulfate gel electrophoresis. Nine cross-linked protein pairs between EF-2 and ribosomal proteins were shifted from the line formed with monomeric proteins. The spots of ribosomal proteins cross-linked to EF-2 were cut out from the gel plate and labelled with 125I. The labelled protein was extracted from the gel and identified by three kinds of two-dimensional gel electrophoresis, followed by autoradiography. The following proteins of both large and small subunits were identified: L9, L12, L23, LA33 (acidic protein of Mr 33000), P2, S6 and S23/S24, and L3 and L4 in lower yields. The results are discussed in relation to the topographies of ribosomal proteins in large and small subunits. Furthermore we found new neighboring protein pairs in large subunits, LA33-L11 and LA33-L12.
TL;DR: A cDNA clone specific for rat ribosomal protein L31 was isolated by hybrid-selected translation from a cDNA library made for 8-9-S poly(A)-rich RNA from regenerating rat liver as mentioned in this paper.
Abstract: A cDNA clone, specific for rat ribosomal protein L31, was isolated by hybrid-selected translation from a cDNA library made for 8-9-S poly(A)-rich RNA from regenerating rat liver. The nucleotide sequence of the cDNA was determined. It consists of 25 base pairs from the 5' leading sequence, the entire coding sequence of 378 base pairs and the 3' trailing sequence of 36 base pairs besides the poly(A) tail. The primary structure of protein L31 was deduced from the nucleotide sequence. It consists of 124 amino acids with a relative molecular mass of 14,331. The calculated amino acid composition is consistent with the reported composition determined for the hydrolysate of L31. The amino acid sequence showed marked homology with that of yeast ribosomal protein L34.
TL;DR: There appears to be a correlation between the efficiency of an RBS to function in the expression of the fused gene and the lack of secondary structure, involving the Shine and Dalgarno nucleotides (SDnts) and/or the initiation codon.
Abstract: Using a previously described vector (pKL203) we fused several heterologous ribosomal binding sites (RBSs) to the lacZ gene of E. coli and then studied the variation in expression of the fusions. The RBSs originated from bacteriophage Q beta and MS2 genes and the E. coli genes for elongation factor EF-Tu A and B and ribosomal protein L11 (rplK). The synthesis of the lacZ fusion proteins was measured by an immuno precipitation method and found to vary at least 100-fold. Lac-specific mRNA synthesis follows the variation in protein production. It appears that there is a correlation between the efficiency of an RBS to function in the expression of the fused gene and the lack of secondary structure, involving the Shine and Dalgarno nucleotides (SDnts) and/or the initiation codon. This efficiency is context dependent. The sequence of the SD nts and the length and sequence of the spacer region up to the initiation codon alone are not able to explain our results. Deletion mutations, created in the phage Q beta replicase RBS, reveal a complex pattern of control of expression, probably involving the use of a "false" initiation site.