Ribosomal protein-dependent orientation of the 16 S rRNA environment of S15

Ribosomal protein S15 binds specifically to the central domain of 16 S ribosomal RNA (16 S rRNA) and directs the assembly of four additional proteins to this domain. The central domain of 16 S rRNA along with these five proteins form the platform of the 30 S subunit. Previously, directed hydroxyl radical probing from Fe(II)-S15 in small ribonucleoprotein complexes was used to study assembly of the central domain of 16 S rRNA. Here, this same approach was used to understand the 16 S rRNA environment of Fe(II)-S15 in 30 S subunits and to determine the ribosomal proteins that are involved in forming the mature S15-16 S rRNA environment. We have identified additional sites of Fe(II)-S15-directed cleavage in 30S subunits compared to the binary complex of Fe(II)-S15/16 S rRNA. Along with novel targets in the central domain, sites within the 5' and 3' minor domains are also cleaved. This suggests that during the course of 30S subunit assembly these elements are positioned in the vicinity of S15. Besides the previously determined role for S8, roles for S5, S6+S18, and S16 in altering the 16 S rRNA environment of S15 were established. These studies reveal that ribosomal proteins can alter the assembly of regions of the 30 S subunit from a considerable distance and influence the overall conformation of this ribonucleoprotein particle.     

Chemical inactivation of Escherichia coli 30-S ribosomes by iodination. Identification of proteins involved in tRNA binding.

30-S ribosomal subunits are inactivated by iodination for both enzymic fMet-tRNA and non-enzymic Phe-tRNA binding activities. This inactivation is due to modification of the protein moiety of the ribosome. Reconstitutions were performed with 16-S RNA and mixtures of total protein isolated from modified subunits and purified proteins isolated from unmodified subunits. This allowed identification of the individual proteins which restore tRNA binding activity. S3, S14 and S19 were identified as proteins involved in fMet-tRNA binding. S1, S2, S3, S14 and S19 were identified as proteins involved in Phe-tRNA binding. Modified particles shown normal sedimentation constants and complete protein compositions both before and after reconstitution. This suggests that the loss of activity is due to modification of one or more of the actual binding sites located on the 30-S subunit and that restoration of activity is due to structural correction at this site rather than to correction of an assembly defect.     

The effect of modification of cytosines in Escherichia coli 16-S rRNA on reconstitution and function of 30-S ribosomes.

O-Methylhydroxylamine (methoxyamine) was used for selective modification of cytosine residues in Escherichia coli 16-S rRNA. It was shown that cytosines accessible for methoxyamination are randomly distributed along the 16-S rRNA chain. Preparations of methoxyaminated 16-S rRNA, containing 2--130 modified cytosines/chain, still retained the ability to bind 30-S proteins, but the physical assembly of reconstituted particles was incorrect. The protein compositions of the reconstituted and native particles did not differ qualitatively from each other. However, the amount of protein in reconstituted particles decreased with an increasing number of methoxyaminated cytosines in 16-S rRNA. The particles obtained sedimented slower than native 30-S subunits, lost their ability to associate with 50-S ribosomes and to bind native phage f2 RNA. In contrast, modification of 16-S rRNA did not affect binding of poly(U) by reconstituted particles.     

The 5-S RNA binding protein from yeast (Saccharomyces cerevisiae) ribosomes. Evolution of the eukaryotic 5-S RNA binding protein.

The ribonucleoprotein complex between 5-S RNA and its binding protein (5-S RNA . protein complex) of yeast ribosomes was released from 60-S subunits with 25 mM EDTA and the protein component was purified by chromatography on DEAE-cellulose. This protein, designated YL3 (Mr = 36000 on dodecylsulfate gels), was relatively insoluble in neutral solutions (pH 4--9) and migrated as one of four acidic 60-S subunit proteins when analyzed by the Kaltschmidt and Wittman two-dimensional gel system. Amino acid analyses indicated lower amounts of lysine and arginine than most ribosomal proteins. Sequence homology was observed in the N terminus of YL3, and two prokaryotic 5-S RNA binding proteins, EL18 from Escherichia coli and HL13 from Halobacterium cutirubrum: Ala1-Phe2-Gln3-Lys4-Asp5-Ala6-Lys7-Ser8-Ser9-Ala10-Tyr11-Ser12-Ser13-Arg14-Phe15-Gln16-Tyr17-Pro18-Phe19-Arg20-Arg21-Arg22-Arg23-Glu24-Gly25-Lys26-Thr27-Asp28-Tyr29-Tyr35; of particular interest was homology in the cluster of basic residues (18--23). Since the protein contained one methionine residue it could be split into two fragments, CN1 (Mr = 24700) and CN2 (Mr = 11300) by CNBr treatment; the larger fragment originated from the N terminus. The N-terminal amino acid sequence of CN2 shared a limited sequence homology with an internal portion of a second 5-S RNA binding protein from E. coli, EL5, and, based also on the molecular weights of the proteins and studies on the protein binding sites in 5-S RNAs, a model for the evolution of the eukaryotic 5-S RNA binding protein is suggested in which a fusion of the prokaryotic sequences may have occurred. Unlike the native 5-S RNA . protein complex, a variety of RNAs interacted with the smaller CN2 fragment to form homogeneous ribonucleoprotein complexes; the results suggest that the CN1 fragment may confer specificity on the natural 5-S RNA-protein interaction.     

Assembly of the central domain of the 30S ribosomal subunit: roles for the primary binding ribosomal proteins S15 and S8

Assembly of the 30S ribosomal subunit occurs in a highly ordered and sequential manner. The ordered addition of ribosomal proteins to the growing ribonucleoprotein particle is initiated by the association of primary binding proteins. These proteins bind specifically and independently to 16S ribosomal RNA (rRNA). Two primary binding proteins, S8 and S15, interact exclusively with the central domain of 16S rRNA. Binding of S15 to the central domain results in a conformational change in the RNA and is followed by the ordered assembly of the S6/S18 dimer, S11 and finally S21 to form the platform of the 30S subunit. In contrast, S8 is not part of this major platform assembly branch. Of the remaining central domain binding proteins, only S21 association is slightly dependent on S8. Thus, although S8 is a primary binding protein that extensively contacts the central domain, its role in assembly of this domain remains unclear. Here, we used directed hydroxyl radical probing from four unique positions on S15 to assess organization of the central domain of 16S rRNA as a consequence of S8 association. Hydroxyl radical probing of Fe(II)-S15/16S rRNA and Fe(II)-S15/S8/16S rRNA ribonucleoprotein particles reveal changes in the 16S rRNA environment of S15 upon addition of S8. These changes occur predominantly in helices 24 and 26 near previously identified S8 binding sites. These S8-dependent conformational changes are consistent with 16S rRNA folding in complete 30S subunits. Thus, while S8 binding is not absolutely required for assembly of the platform, it appears to affect significantly the 16S rRNA environment of S15 by influencing central domain organization.     

Photo-induced protein-RNA cross-linking in mammalian 60-S ribosomal subunits.

Rat liver 60-S ribosomal subunits were submitted to increasing doses of radiation (253.7 nm), at 4 degrees C and 25 degrees C, as previously reported fro 40-S subunits. The existence of protein-RNA cross-linking was demonstrated by two methods. The first consisted in the separation of protein-RNA complex; the second was indirect, and took into account alteration either in the electrophoretic mobility of cross-linked proteins or the separability of 28-S RNA in a 4 M urea/3 M LiCl buffer. The peptide synthetase activity and the sedimentation characteristics of the particles irradiated at 4 degrees C were well preserved, but at 25 degrees C the large subunits were progressively inactivated and unfolded for doses higher than 2 x 10(18) quanta. The dose-dependent variations of protein cross-linkage determined by two-dimensional gel electrophoresis allowed us to distinguish those proteins which reacted at the lowest doses with a first-order reaction from those which cross-linked to RNA after a subtle modification of the subunit structure. At 25 degrees C, all proteins became low-dose reactive. The curve obtained for 28-S RNA cross-linkage was similar to that of the total protein moiety, while those obtained fro the 5-S and 5.8-S RNA (which were parallel) suggest a lower reactivity of these RNAs. As a general rule, proteins from the large subunits were more reactive to RNA than those from the small subunits. This could indicate differences in the organisation of the two subunits.     

A structural model for the assembly of the 30S subunit of the ribosome

The order in which proteins bind to 16S rRNA, the assembly map, was determined by Nomura and co-workers in the early 1970s. The assembly map shows the dependencies of binding of successive proteins but fails to address the relationship of these dependencies to the three-dimensional folding of the ribosome. Here, using molecular mechanics techniques, we rationalize the order of protein binding in terms of ribosomal folding. We determined the specific contacts between the ribosomal proteins and 16S rRNA from a crystal structure of the 30S subunit (1FJG). We then used these contacts as restraints in a rigid body Monte-Carlo simulation with reduced-representation models of the RNA and proteins. Proteins were added sequentially to the RNA in the order that they appear in the assembly map. Our results show that proteins nucleate the folding of the head, platform, and body domains, but they do not strongly restrict the orientations of the domains relative to one another. We also examined the contributions of individual proteins to the formation of binding sites for sequential proteins in the assembly process. Binding sites for the primary binding proteins are generally more ordered in the naked RNA than those for other proteins. Furthermore, we examined one pathway in the assembly map and found that the addition of early binding proteins helps to organize the RNA around the binding sites of proteins that bind later. It appears that the order of assembly depends on the degree of pre-organization of each protein's binding site at a given stage of assembly, and the impact that the binding of each protein has on the organization of the remaining unoccupied binding sites.     

The formation of a defective small subunit of the mitochondrial ribosomes in petite mutants of Saccharomyces cerevisiae

The involvement of mitochondrial protein synthesis in the assembly of the mitochondrial ribosomes was investigated by studying the extent to which the assembly process can proceed in petite mutants of Saccharomyces cerevisiae which lack mitochondrial protein synthetic activity due to the deletion of some tRNA genes and/or one of the rRNA genes on the mtDNA. Petite strains which retain the 15-S rRNA gene can synthesize this rRNA species, but do not contain any detectable amounts of the small mitochondrial ribosomal subunit. Instead, a ribonucleoparticle with a sedimentation coefficient of 30 S (instead of 37 S) was observed. This ribonucleoparticle contained all the small ribosomal subunit proteins with the exception of the var1 and three to five other proteins, which indicates that the 30-S ribonucleoparticle is related to the small mitochondrial ribosomal subunit (37 S). Reconstitution experiments using the 30-S particle and the large mitochondrial ribosomal subunit from a wild-type yeast strain indicate that the 30-S particle is not active in translating the artificial message poly(U). The large mitochondrial ribosomal subunit was present in petite strains retaining the 21-S rRNA gene. The petite 54-S subunit is biologically active in the translation of poly(U) when reconstituted with the small subunit (37 S) from a wild-type strain. The above results indicate that mitochondrial protein synthetic activity is essential for the assembly of the mature small ribosomal subunit, but not for the large subunit. Since the var1 protein is the only mitochondrial translation product known to date to be associated with the mitochondrial ribosomes, the results suggest that this protein is essential for the assembly of the mature small subunit.     

Assembly of two independent populations of flock house virus particles with distinct RNA packaging characteristics in the same cell

Flock House virus (FHV; Nodaviridae) is a positive-strand RNA virus that encapsidates a bipartite genome consisting of RNA1 and RNA2. We recently showed that specific recognition of these RNAs for packaging into progeny particles requires coat protein translated from replicating viral RNA. In the present study, we investigated whether the entire assembly pathway, i.e., the formation of the initial nucleating complex and the subsequent completion of the capsid, is restricted to the same pool of coat protein subunits. To test this, coat proteins carrying either FLAG or hemagglutinin epitopes were synthesized from replicating or nonreplicating RNA in the same cell, and the resulting particle population and its RNA packaging phenotype were analyzed. Results from immunoprecipitation analysis and ion-exchange chromatography showed that the differentially tagged proteins segregated into two distinct populations of virus particles with distinct RNA packaging phenotypes. Particles assembled from coat protein that was translated from replicating RNA contained the FHV genome, whereas particles assembled from coat protein that was translated from nonreplicating mRNA contained random cellular RNA. These data demonstrate that only coat proteins synthesized from replicating RNA partake in the assembly of virions that package the viral genome and that RNA replication, coat protein translation, and virion assembly are processes that are tightly coupled during the life cycle of FHV.     

Ty3 Retrotransposon Hijacks Mating Yeast RNA Processing Bodies to Infect New Genomes

Retrotransposition of the budding yeast long terminal repeat retrotransposon Ty3 is activated during mating. In this study, proteins that associate with Ty3 Gag3 capsid protein during virus-like particle (VLP) assembly were identified by mass spectrometry and screened for roles in mating-stimulated retrotransposition. Components of RNA processing bodies including DEAD box helicases Dhh1/DDX6 and Ded1/DDX3, Sm-like protein Lsm1, decapping protein Dcp2, and 5’ to 3’ exonuclease Xrn1 were among the proteins identified. These proteins associated with Ty3 proteins and RNA, and were required for formation of Ty3 VLP retrosome assembly factories and for retrotransposition. Specifically, Dhh1/DDX6 was required for normal levels of Ty3 genomic RNA, and Lsm1 and Xrn1 were required for association of Ty3 protein and RNA into retrosomes. This role for components of RNA processing bodies in promoting VLP assembly and retrotransposition during mating in a yeast that lacks RNA interference, contrasts with roles proposed for orthologous components in animal germ cell ribonucleoprotein granules in turnover and epigenetic suppression of retrotransposon RNAs.     

The 5-S RNA . protein complex from an extreme halophile, Halobacterium cutirubrum. Purification and characterization.

A 5-S RNA . protein complex has been isolated from the 50-S ribosomal subunit of an extreme halophile, Halobacterium cutirubrum. The 50-S ribosomal subunit from the extreme halophile requires 3.4 M K+ and 100 mM Mg2+ for stability. However, if the high K+ concentration is maintained but the Mg2+ concentration lowered to 0.3 mM, the 5-S RNA . protein complex is selectively extracted from the subunit. After being purified on an Agarose 0.5-m column the complex had a molecular weight of about 80000 and contained 5-S RNA and two proteins, HL13 and HL19, with molecular weights (by sedimentation equilibrium) of 18700 and 18000, respectively. No ATPase or GTPase activity could be detected in the 5-S RNA . protein complex. The amino acid composition and electrophoretic mobility on polyacrylamide gels indicated both proteins were much more acidic than the equivalent from Escherichia coli or Bacillus stearothermophilus. Partial amino acid sequence data suggest HL13 is homologous to EL18 and HL19 to EL5.     

Assembly of the archaeal box C/D sRNP can occur via alternative pathways and requires temperature-facilitated sRNA remodeling

Archaeal dual-guide box C/D small nucleolar RNA-like RNAs (sRNAs) bind three core proteins in sequential order at both terminal box C/D and internal C'/D' motifs to assemble two ribonuclear protein (RNP) complexes active in guiding nucleotide methylation. Experiments have investigated the process of box C/D sRNP assembly and the resultant changes in sRNA structure or "remodeling" as a consequence of sRNP core protein binding. Hierarchical assembly of the Methanocaldococcus jannaschii sR8 box C/D sRNP is a temperature-dependent process with binding of L7 and Nop56/58 core proteins to the sRNA requiring elevated temperature to facilitate necessary RNA structural dynamics. Circular dichroism (CD) spectroscopy and RNA thermal denaturation revealed an increased order and stability of sRNA folded structure as a result of L7 binding. Subsequent binding of the Nop56/58 and fibrillarin core proteins to the L7-sRNA complex further remodeled sRNA structure. Assessment of sR8 guide region accessibility using complementary RNA oligonucleotide probes revealed significant changes in guide region structure during sRNP assembly. A second dual-guide box C/D sRNA from M. jannaschii, sR6, also exhibited RNA remodeling during temperature-dependent sRNP assembly, although core protein binding was affected by sR6's distinct folded structure. Interestingly, the sR6 sRNP followed an alternative assembly pathway, with both guide regions being continuously exposed during sRNP assembly. Further experiments using sR8 mutants possessing alternative guide regions demonstrated that sRNA folded structure induced by specific guide sequences impacted the sRNP assembly pathway. Nevertheless, assembled sRNPs were active for sRNA-guided methylation independent of the pathway followed. Thus, RNA remodeling appears to be a common and requisite feature of archaeal dual-guide box C/D sRNP assembly and formation of the mature sRNP can follow different assembly pathways in generating catalytically active complexes.     

RNA and ribosomal protein patterns during aerial spore germination in Streptomyces granaticolor

Disruption of the external sheath of Streptomyces granaticolor aerial spores and subsequent cultivation in a rich medium result in a synchronous germination. This method was used to analyze RNA and protein patterns during the germination. The germination process took place through a sequence of time-ordered events. RNA and protein synthesis started during the first 5 min and net DNA synthesis at 60-70 min of germination. Within the first 10 min of germination, synthesis of RNA was not sensitive to the inhibitory effect of rifamycin. During this period rRNA and other species including 4-5-S RNA were synthesized. Dormant spores contained populations of ribosomes or ribosomal precursors that were structurally and functionally defective. The ribosomal particles bound a sporulation pigment(s) of the melanine type. The ribosomal proteins complexed to the pigments formed insoluble aggregates which were easily removed from the ribosomes by one wash with 1 M NH4Cl. During the first 10 min of germination, pigment(s) were liberated from the complexes with the ribosomes and protein extracts of the washed ribosomes had essentially the same pattern as the extracts of ribosomes of vegetative cells. These structural alterations were accompanied by enhancement of the ribosome activities in polypeptide synthesis in vivo and in vitro. When the spores were incubated with a 14C-labelled amino acid mixture in the presence of rifamycin, only three proteins (GS1, GL1 and GS9) were identified to be radiolabelled in the extracts from the washed ribosomes. These experiments indicate that liberation of the sporulation pigment(s) from the complexes with ribosomal proteins and assembly of de novo synthesized proteins and proteins from a preexisting pool in the spore are involved in the reactivation of the ribosomes of dormant spores of S. granaticolor.     

The topography of the 5' end of 16-S RNA in the presence and absence of ribosomal proteins S4 and S20

A ribonucleoprotein prepared by strong ribonuclease digestion of a complex of 16-S ribosomal RNA and proteins S4 and S20 from Escherichia coli has been characterized; its nucleotide sequence, the positions of enzyme cuts and the sequence excisions have been placed in the completed sequence of 16-S RNA. The positions and yields of enzyme cuts, and excisions of sequence, are compared with those of various ribonucleoproteins prepared with S4 or S20 alone, and with the ribonuclease-resistant S4 RNA prepared from renatured 16-s RNA in the absence of ribosomal protein. These data yield important information on the topography and organisation of the 5' third of the 16-s RNA which is selectively maintained in its native conformation by the bound proteins; they also provide criteria for testing secondary structural models of this region of 16-S RNA.     

Quantitative analysis of the interaction between the envelope protein domains and the core protein of human hepatitis B virus

Interaction between preformed nucleocapsids and viral envelope proteins is critical for the assembly of virus particles in infected cells. The pre-S1 and pre-S2 and cytosolic regions of the human hepatitis B virus envelope protein had been implicated in the interaction with the core protein of nucleocapsids. The binding affinities of specific subdomains of the envelope protein to the core protein were quantitatively measured by both ELISA and BIAcore assay. While a marginal binding was detected with the pre-S1 or pre-S2, the core protein showed high affinities to pre-S with apparent dissociation constants (K(D)(app)) of 7.3+/-0.9 and 8.2+/-0.4microM by ELISA and BIAcore assay, respectively. The circular dichroism analysis suggested that conformational change occurs in pre-S through interaction with core protein. These results substantiate the importance of specific envelope domains in virion assembly, and demonstrate that the interaction between viral proteins can be quantitatively measured in vitro.     

Kinetics of utilization of Sendai virus RNA and protein in the process of virion assembly.

The synthesis of the 50S genomic RNA and strucural proteins of Sendai virus was examined with respect to their utilization in virus assembly. It was found that during a single cycle of infection, 50S RNA was synthesized before the structural proteins and that both RNA and protein were synthesized 2 to 4 h before their appearance in released virions. Pulse-chase labeling indicated that the NP and P proteins synthesized early and the M and F proteins synthesized late were preferentially incorporated into virus relative to the other viral proteins. The kinetics of incorporation of pulse-labeled NP protein suggested that it was withdrawn from a relatively large pool whereas the M protein appeared to be present in a relatively small pool in the cytoplasm. Further, it was possible to chase pulse-labeled M protein, but not NP protein, from the cell during an 8-h time period.     

RNA Granule Assembly and Disassembly Modulated by Nuclear Factor Associated with Double-stranded RNA 2 and Nuclear Factor 45

RNA granules are large messenger ribonucleoprotein complexes that regulate translation and mRNA translocation to control the timing and location of protein synthesis. The regulation of RNA granule assembly and disassembly is a structural basis of translational control, and its disorder is implicated in degenerative disease. Here, we used proteomic analysis to identify proteins associated with RNA granule protein 105 (RNG105)/caprin1, an RNA-binding protein in RNA granules. Among the identified proteins, we focused on nuclear factor (NF) 45 and its binding partner, nuclear factor associated with dsRNA 2 (NFAR2), and we demonstrated that NF45 promotes disassembly of RNA granules, whereas NFAR2 enhances the assembly of RNA granules in cultured cells. The GQSY domain of NFAR2 was required to associate with messenger ribonucleoprotein complexes containing RNG105/caprin1, and it was structurally and functionally related to the low complexity sequence domain of the fused in sarcoma protein, which drives the assembly of RNA granules. Another domain of NFAR2, the DZF domain, was dispensable for association with the RNG105 complex, but it was involved in positive and negative regulation of RNA granule assembly by being phosphorylated at double-stranded RNA-activated kinase sites and by association with NF45, respectively. These results suggest a novel molecular mechanism for the modulation of RNA granule assembly and disassembly by NFAR2, NF45, and phosphorylation at double-stranded RNA-activated kinase PKR sites.     

Specific ribosomal RNA recognition by a fragment of E. coli ribosomal protein S4 missing the C-terminal 36 amino acid residues.

We have previously investigated the role of the N-terminal region of ribosomal protein S4 to participate in 30S ribosome assembly and function (1-3). In this report we extend these studies to the two fragments produced by the chemical cleavage of protein S4 at the tryptophan residue 167. We find that the carboxyl terminal fragment (168-203) does not bind 16S RNA nor does it participate in assembly with the other 20 proteins from the 30S ribosome. In contrast, the larger fragment (1-167), does bind 16S RNA specifically. If the S4-fragment (1-167) is used to replace protein S4 in the complete 30S assembly reaction, all 20 of the other 30S proteins are incorporated. We conclude that the carboxyl terminal section of the protein S4 is not directly involved in binding 16S RNA or in the assembly of any of the other 30S proteins.