Ribosomal protein L15 as a probe of 50 S ribosomal subunit structure

L15, a 15 kDa protein of the large ribosomal subunit, interacts with over ten other proteins during 50 S assembly in vitro. We have probed the interaction L15 with 23 S rRNA in 50 S ribosomal subunits by chemical footprinting, and have used localized hydroxyl radical probing, generated from Fe(II) tethered to unique sites of L15, to characterize the three-dimensional 23 S rRNA environment of L15. Footprinting of L15 was done by reconstituting purified, recombinant L15 with core particles derived from Escherichia coli 50 S subunits by treatment with 2 M LiCl. The cores migrate as compact 50 S-like particles in sucrose gradients, contain 23 S and 5 S rRNA, and lack a subset of the 50 S proteins, including L15. Using both Fe(II).EDTA and dimethyl sulfate, we have identified a strong footprint for L15 in the region spanning nucleotides 572-654 in domain II of 23 S rRNA. This footprint cannot be detected when L15 is incubated with "naked" 23 S rRNA, indicating that formation of the L15 binding site requires a partially assembled particle.Protein-tethered hydroxyl radical probing was done using mutants of L15 containing single cysteine residues at amino acid positions 68, 71 and 115. The mutant proteins were derivatized with 1-[p-(bromo-acetamido)benzyl]-EDTA. Fe(II), bound to core particles, and hydroxyl radical cleavage was initiated. Distinct but overlapping sets of cleavages were obtained in the footprinted region of domain II, and in specific regions of domains I, IV and V of 23 S rRNA. These data locate L15 in proximity to several 23 S rRNA elements that are dispersed in the secondary structure, consistent with its central role in the latter stages of 50 S subunit assembly. Furthermore, these results indicate the proximity of these rRNA regions to one another, providing constraints on the tertiary folding of 23 S rRNA.     

Localization of proteins HL29 and HL31 from Haloarcula marismortui within the 50 S ribosomal subunit by chemical crosslinking

Isolated 50 S ribosomal subunits from the halophilic archaebacterium Haloarcula marismortui were treated in situ with the homobifunctional and cleavable crosslinking reagent dithiobis(succinimidyl propionate) (12 A). Several crosslinked complexes were obtained. Among these were the protein pairs HmaL4-HL29 and HmaL18-HL31; HL29 and HL31 are ribosomal proteins without any equivalent in eubacterial ribosomes. The crosslinked protein pairs were isolated on a preparative scale by combining conventional ion-exchange chromatography and reverse phase high-pressure liquid chromatography. The monomeric proteins involved in crosslink formation were unambiguously identified by two-dimensional gel electrophoresis and N-terminal or internal protein sequencing. Due to the homology between HmaL4 and HmaL18 and their Escherichia coli counterparts, and the roughly known location of these proteins within the 50 S subunit, our results demonstrate that HL29 is probably located in the centre of the large subunit in the vicinity of the peptidyltransferase domain, whereas HL31 must be situated within the central protuberance close to the region of the 5 S RNA.     

Neighborhood of 16S rRNA nucleotides U788/U789 in the 30S ribosomal subunit determined by site-directed crosslinking

Site-specific photo crosslinking has been used to investigate the RNA neighborhood of 16S rRNA positions U788/ U789 in Escherichia coli 30S subunits. For these studies, site-specific psoralen (SSP) which contains a sulfhydryl group on a 17 A side chain was first added to nucleotides U788/U789 using a complementary guide DNA by annealing and phototransfer. Modified RNA was purified from the DNA and unmodified RNA. For some experiments, the SSP, which normally crosslinks at an 8 A distance, was derivitized with azidophenacylbromide (APAB) resulting in the photoreactive azido moiety at a maximum of 25 A from the 4' position on psoralen (SSP25APA). 16S rRNA containing SSP, SSP25APA or control 16S rRNA were reconstituted and 30S particles were isolated. The reconstituted subunits containing SSP or SSP25APA had normal protein composition, were active in tRNA binding and had the usual pattern of chemical reactivity except for increased kethoxal reactivity at G791 and modest changes in four other regions. Irradiation of the derivatized 30S subunits in activation buffer produced several intramolecular RNA crosslinks that were visualized and separated by gel electrophoresis and characterized by primer extension. Four major crosslink sites made by the SSP reagent were identified at positions U561/U562, U920/U921, C866 and U723; a fifth major crosslink at G693 was identified when the SSP25APA reagent was used. A number of additional crosslinks of lower frequency were seen, particularly with the APA reagent. These data indicate a central location close to the decoding region and central pseudoknot for nucleotides U788/U789 in the activated 30S subunit.     

Gene expression evidence indicates that nucleotides 507-513 and 1434-1440 in 16S rRNA are organized in close proximity on the Escherichia coli 30S ribosomal subunit

Biotinylation has become a popular alternative to radioiodination for labeling cell surface proteins, whereas labeling of the total cellular protein pool is usually achieved metabolically with [35S]methionine and [35S]cysteine. In this paper we describe a new technique in which total cellular lysate proteins that have been affinity bound to a solid phase are labeled efficiently with biotin. This labeling technique is preferable to direct biotinylation of cell lysate since the unreacted biotin can be readily removed from the sample by washing. The affinity step permits preselection of the molecules to be labeled, thereby decreasing the potential for nonspecific binding during subsequent immunoprecipitation. We applied this affinity biotinylation method to a human cellular lysate in order to preselect the total glycoprotein pool for subsequent immunoprecipitation of HLA class I. Following immunoprecipitation, SDS-PAGE, and Western blot, the biotinylated protein could be readily revealed by enhanced chemiluminescence. The results were comparable to those obtained by radiometabolic labeling and Western blot using a monoclonal antibody probe. Overall, the affinity biotinylation method is faster and more practical than conventional radiolabeling, without any loss in sensitivity.     

Ribosomal protein L9 interactions with 23 S rRNA: the use of a translational bypass assay to study the effect of amino acid substitutions

During translation of bacteriophage T4 gene 60 mRNA, ribosomes bypass 50 nucleotides with high efficiency. One of the mRNA signals for bypass is a stem-loop in the first part of the coding gap. When the length of this stem-loop is extended by 36 nucleotides, bypass is reduced to 0.35% of the wild-type level. Bypass is partially restored by a mutation in the C-terminal domain of Escherichia coli large ribosomal subunit protein L9. Previous work has shown that L9 is an elongated protein with an alpha-helix that connects and orients the N and C-terminal domains that both contain a predicted RNA binding site. We have determined two binding sites of L9 on 23 S rRNA. A 778 nucleotide RNA fragment encompassing domain V (nucleotides 1999 to 2776) of the 23 S rRNA is retained on filters by L9 and contains both sites. The N and C-terminal domains of L9 were shown to interact with nucleotides just 5' to nucleotide 2231 and 2179 of the 23 S rRNA, respectively, using the toeprint assay. These L9 binding sites on 23 S rRNA suggest that L9 functions as a brace across helix 76 to position helices 77 and 78 relative to the peptidyl transferase center. In this study, bypass on a mutant gene 60 mRNA has been used as an assay to probe the importance of particular L9 amino acids for function. Amino acid substitutions in the C-terminal domain are shown to partially restore bypass. These mutant L9 proteins have reduced binding to a 23 S rRNA fragment (nucleotides 1999 to 2274) containing domain V, to which L9 binds. They partially retain both the N and C-terminal domain interactions. On the other hand, substitutions of amino acids in the N-terminal domain, which greatly reduce RNA binding, do not restore bypass. The latter mutants have completely lost the N-terminal domain interaction. Addition of an amino acid to the alpha-helix also restores gene 60 bypass. RNA binding by this mutant is similar to that observed for the C-terminal domain mutants that partially restore bypass.     

In vitro protein folding by ribosomes from Escherichia coli, wheat germ and rat liver: the role of the 50S particle and its 23S rRNA

Ribosomes from a number of prokaryotic and eukaryotic sources (e.g. Escherichia coli, wheat germ and rat liver) can refold a number of enzymes which are denatured with guanidine/HC1 prior to incubation with ribosomes. In this report, we present our observations on the refolding of denatured lactate dehydrogenase from rabbit muscle and glucose-6-phosphate dehydrogenase from baker's yeast by ribosomes from E. coli, wheat germ and rat liver. The protein-folding activity of E. coli ribosomes was found to be present in 50S particles and in 23S rRNA. The 30S particle or 16S rRNA did not show any protein-folding activity. The protein-folding activity of 23S rRNA may depend on its tertiary conformation. Loss of tertiary structure, by incubation with low concentrations of EDTA, inhibited the protein-folding activity of 23S rRNA. This low concentration of EDTA had no effect on folding of the denatured enzymes by themselves.     

The exosome subunit Rrp43p is required for the efficient maturation of 5.8S, 18S and 25S rRNA

The Saccharomyces cerevisiae protein Rrp43p co-purifies with four other 3'-->5' exoribonucleases in a complex that has been termed the exosome. Rrp43p itself is similar to prokaryotic RNase PH. Individual exosome subunits have been implicated in the 3' maturation of the 5.8S rRNA found in 60S ribosomes and the 3' degradation of mRNAs. However, instead of being deficient in 60S ribosomes, Rrp43p-depleted cells were deficient in 40S ribosomes. Pulse-chase and steady-state northern analyses of pre-RNA and rRNA levels revealed a significant delay in the synthesis of both 25S and 18S rRNAs, accompanied by the stable accumulation of 35S and 27S pre-rRNAs and the under-accumulation of 20S pre-rRNA. In addition, Rrp43p-depleted cells accumulated a 23S aberrant pre-rRNA and a fragment excised from the 5' ETS. Therefore, in addition to the maturation of 5.8S rRNA, Rrp43p is required for the maturation 18S and 25S rRNA.     

The isolation of eukaryotic ribosomal proteins. The purification and characterization of the 40 S ribosomal subunit proteins S2, S3, S4, S5, S6, S7, S8, S9, S13, S23/S24, S27, and S28

The proteins of the small subunit of rat liver ribosomes were separated into five groups by stepwise elution from carboxymethylcellulose with LiCl at pH 6.5 (Collatz, E., Lin, A., St?ffler, G., Tsurugi, K., and Wool, I.G., (1976) J. Biol. Chem. 251, 1808-1816). From the several groups, 12 proteins (S2,S3, S4, S5, S6, S7, S8, S9, S13, S23/S24, S27, and S28) wereisolated by ion exchange chromatography on carboxymethylcellulose, by chromatography on sulfopropyl-Sephadex, and by gel filtration through Sephadex G-75. The amount of protein obtained varied from 1 to 9 mg depending on the number of steps required for the preparation; several proteins had no detectable contamination and the impurities in the others were no greater than 9%. The molecular weight of the proteins was estimated by polyazrylamide gel electrophoresis in sodium dodecyl sulfate; the amino acid composition was determined.     

hnRNP proteins and B23 are the major proteins of the internal nuclear matrix of HeLa S3 cells

The nuclear matrix is the structure that persists after removal of chromatin and loosely bound components from the nucleus. It consists of a peripheral lamina-pore complex and an intricate internal fibrogranular structure. Little is known about the molecular structure of this proteinaceous internal network. Our aim is to identify the major proteins of the internal nuclear matrix of HeLa 53 cells. To this end, a cell fraction containing the internal fibrogranular structure was compared with one from which this structure had been selectively dissociated. Protein compositions were quantitatively analyzed after high-resolution two-dimensional gel electrophoresis. We have identified the 21 most abundant polypeptides that are present exclusively in the internal nuclear matrix. Sixteen of these proteins are heterogeneous nuclear ribonucleoprotein (hnRNP) proteins. B23 (numatrin) is another abundant protein of the internal nuclear matrix. Our results show that most of the quantitatively major polypeptides of the internal nuclear matrix are proteins involved in RNA metabolism, including packaging and transport of RNA.     

Structure of 5S rRNA within the Escherichia coli ribosome: iodine-induced cleavage patterns of phosphorothioate derivatives

The protection patterns of 5S rRNA in solution, within the ribosomal 50S subunit, 70S ribosomes, and functional complexes, were assessed with the phosphorothioate method. About 20% of the analyzed positions (G9-G107) showed strong assembly defects: A phosphorothioate at one of these positions significantly impaired the incorporation of 5S rRNA into 50S particles. The reverse has also been observed: A phosphorothioate is preferred over a phosphate residue in the assembly process at a few positions. The results further demonstrate that 5S rRNA undergoes conformational changes during the assembly in the central protuberance of the 50S subunit and upon association with the small ribosomal subunit forming a 70S ribosome. In striking contrast, when the 70S ribosomes are once formed, the contact pattern of the 5S rRNA is the same in various functional states such as initiation-like complexes and pre- and posttranslocational states.     

The importance of base pairing in the penultimate stem of Escherichia coli 16S rRNA for ribosomal subunit association

The influence of base pairing in the penultimate stem of Escherichia coli 16S rRNA (defined as nt 1409-1491) on ribosome function has been addressed by the construction of mutations in this region of rRNA. Two sets of mutations were made on either side of a structurally conserved region in the penultimate stem that disrupted base pairing, while a third set of mutations replaced the wild-type sequence with other base pair combinations. The effects of these mutations were analyzed in vivo and in vitro . The mutations that disrupted base pairing caused significant increases in cell doubling times as well as a severe subunit association defect and a modest increase in frame shifting and stop codon read-through. Restoration of base pairing restored wild-type growth rates, decoding and subunit association, indicating that base pairing in this region is essential for proper ribosome function.     

Variable region V1 of Saccharomyces cerevisiae 18S rRNA participates in biogenesis and function of the small ribosomal subunit

The role of helix 6, which forms the major portion of the most 5'-located expansion segment of Saccharomyces cerevisiae 18S rRNA, was studied by in vivo mutational analysis. Mutations that increased the size of the helical part and/or the loop, even to a relatively small extent, abolished 18S rRNA formation almost completely. Concomitantly, 35S pre-rRNA and an abnormal 23S precursor species accumulated. rDNA units containing these mutations did not support cell growth. A deletion removing helix 6 almost completely, on the other hand, had a much less severe effect on the formation of 18S rRNA, and cells expressing only the mutant rRNA remained able to grow, albeit at a much reduced rate. Disruption of the apical A.U base pair by a single point mutation did not cause a noticeable reduction in the level of 18S rRNA but did result in a twofold lower growth rate of the cells. This effect could not be reversed by introduction of a second point mutation that restores base pairing. We conclude that both the primary and the secondary structure of helix 6 play an important role in the formation and the biological function of the 40S subunit.     

Specific PCR primers from the 16S-23S rRNA spacer region for the rapid detection and identification of Actinobacillus seminis

Actinobacillus seminis is a common cause of ovine epididymitis and ram infertility. The ability to detect and identify this organism promptly is important commercially for the quality control of ram semen samples. Actinobacillus seminis is a fastidious and slow-growing bacterium and primary isolation and presumptive identification can be difficult and time-consuming. In this study, two ribosomal operons, termed rrnA and rrnB, have been characterized in the A. seminis genome, and these contain one and two tRNAs, respectively, in the spacer region between the 16S and 23S rRNA genes. Species-specific primers for A. seminis were developed from the sequence of the spacer region of rrnB for the identification and detection of A. seminis by PCR. The PCR assay was specific for A. seminis and gave no amplification products with phenotypically similar organisms such as Histophilus ovis. Storage solution used to preserve semen for long-term storage was found to inhibit the PCR. Therefore, for diagnostic purposes, the assay would best be performed after primary isolation or perhaps on fresh semen prior to storage if obvious contamination is indicated.     

Overexpression of the thiostrepton-resistance gene from Streptomyces azureus in Escherichia coli and characterization of recognition sites of the 23S rRNA A1067 2'-methyltransferase in the guanosine triphosphatase center of 23S ribosomal RNA

The thiostrepton-resistance gene encoding the 23S rRNA A1067 methyltransferase from Streptomyces azureus has been overexpressed in Escherichia coli using a T7-RNA-polymerase-dependent expression vector. The protein was efficiently expressed at levels up to 20% of total soluble protein and purified to near homogeneity. Kinetic parameters for S-adenosyl-L-methionine (Km = 0.1 mM) and an RNA fragment containing nucleotides 1029-1122 of the 23S ribosomal RNA from E. coli (Km = 0.001 mM) were determined. S-Adenosyl-L-homocysteine showed competitive product inhibition (Ki = 0.013 mM). Binding of either thiostrepton or protein L11 inhibited methylation. RNA sequence variants of the RNA fragment with mutations in nucleotides 1051-1108 were tested as substrates for the methylase. The experimental data indicate that methylation is dependent on the secondary structure of the hairpin including nucleotide A1067 and the exact sequence U(1066)-A(1067)-G(1068)-A(1069)-A(1070) of the single strand.     

Characterization of cyanobacteria by SDS-PAGE of whole-cell proteins and PCR/RFLP of the 16S rRNA gene

Childhood neuroblastoma, an embryonal neoplasm of sympathetic nervous system progenitors, occurs in a familial form with an autosomal dominant mode of inheritance. Genetic susceptibility to this disorder is thought to arise via a germline mutation affecting a tumor suppressor gene, in accord with the two-hit model established for familial and sporadic retinoblastoma. Surprisingly, the familial neuroblastoma predisposition locus does not map to chromosome band 1p36, a genomic region likely to contain one or more neuroblastoma suppressor genes. We reasoned that inherited point mutations affecting one allele would be unmasked in many cases by somatically acquired deletions of the second allele that included the target gene in the tumor cells from these patients. Thus, to identify chromosomal regions that might contain suppressor genes important in hereditary neuroblastoma, we analyzed six familial tumors by comparative genomic hybridization. Recurrent losses of genetic material were detected on chromosome arms 3p (consensus region, 3p24-pter), 10p (consensus, 10p12-p13), 10q (consensus, 10q25-qter), 16q (consensus, 16q12-q22), and 20q (consensus, 20q13.3-qter), in addition to the regions commonly deleted in sporadic neuroblastomas (1p36 and 11q). These chromosomal sites may harbor novel tumor suppressor genes that could aid in our understanding of the predisposition to and pathogenesis of familial neuroblastoma and potentially sporadic tumors as well.     

Evaluation of intraspecies genetic variation within the 16S rRNA gene of Mycoplasma hominis and detection by polymerase chain reaction

Mycoplasma hominis is a heterogeneous species with DNA-DNA hybridization values ranging from 51 to 100%. We report here the sequencing of the 16S rRNA gene of a strain (183) that greatly differs from the type strain (PG21) of this species. Comparison of 16S rDNA sequences from these two strains showed limited differences, indicating that the two strains belong to the same rRNA species complex. Using these nucleotide sequence data, we established a rapid method for the detection of M. hominis by using polymerase chain reaction. This method was shown to be sensitive and specific when tested with reference strains and clinical isolates.     

Multiubiquitin chain binding and protein degradation are mediated by distinct domains within the 26 S proteasome subunit Mcb1

The 26 S proteasome is a multisubunit proteolytic complex responsible for degrading eukaryotic proteins targeted by ubiquitin modification. Substrate recognition by the complex is presumed to be mediated by one or more common receptor(s) with affinity for multiubiquitin chains, especially those internally linked through lysine 48. We have identified previously a candidate for one such receptor from diverse species, designated here as Mcb1 for Multiubiquitin chain-binding protein, based on its ability to bind Lys48-linked multiubiquitin chains and its location within the 26 S proteasome complex. Even though Mcb1 is likely not the only receptor in yeast, it is necessary for conferring resistance to amino acid analogs and for degrading a subset of ubiquitin pathway substrates such as ubiquitin-Pro-beta-galactosidase (Ub-Pro-beta-gal) (van Nocker, S., Sadis, S., Rubin, D.M., Glickman, M., Fu, H., Coux, O., Wefes, I., Finley, D., and Vierstra, R. D. (1996) Mol. Cell. Biol. 16, 6020-28). To further define the role of Mcb1 in substrate recognition by the 26 S proteasome, a structure/function analysis of various deletion and site-directed mutants of yeast and Arabidopsis Mcb1 was performed. From these studies, we identified a single stretch of conserved hydrophobic amino acids (LAM/LALRL/V (ScMcb1 228-234 and At-Mcb1 226-232)) within the C-terminal half of each polypeptide that is necessary for interaction with Lys48-linked multiubiquitin chains. Unexpectedly, this domain was not essential for either Ub-Pro-beta-gal degradation or conferring resistance to amino acid analogs. The domain responsible for these two activities was mapped to a conserved region near the N terminus. Yeast and Arabidopsis Mcb1 derivatives containing an intact multiubiquitin-binding site but missing the N-terminal region failed to promote Ub-Pro-beta-gal degradation and even accentuated the sensitivity of the yeast delta mcb1 strain to amino acid analogs. This hypersensitivity was not caused by a gross defect in 26 S proteasome assembly as mutants missing either the N-terminal domain or the multiubiquitin chain-binding site could still associate with 26 S proteasome and generate a complex indistinguishable in size from that present in wild-type yeast. Together, these data indicate that residues near the N terminus, and not the multiubiquitin chain-binding site, are most critical for Mcb1 function in vivo.