Asymmetry in the 50S Ribosomal Subunit of Escherichia coli

A substantial separation of the centers of mass of the protein and RNA portions of the 50S ribosomal subunit has been achieved using neutron scattering. This separation reveals that the subunit has a protein-rich side, a finding which is inconsistent with many models proposed for the structure. By analyzing the variation of the radius of gyration of the subunit under conditions in which the contributions of the protein and RNA were separately enhanced by deuteration and comparing these radii to the undeuterated subunit radius, we have obtained the following values. The radius of gyration of the whole particle is 78.0 +/- 0.95 A, that of the RNA moiety is 72.5 +/- 1.5 A, and that of the protein is 73.4 +/- 2.0 A. These data give a separation of the centers of mass of the RNA and protein distributions of 57.7 +/- 10 A.     

Effect of Initiation Factor 3 Binding on the 30S Ribosomal Subunits of Escherichia coli

Under certain conditions, initiation factor 3 (IF-3) can cause the release of aminoacyl-tRNA bound to 30S ribosomal subunits of E. coli. It is shown that this IF-3-induced aminoacyl-tRNA release cannot be attributed to either nucleolytic attack or competition between IF-3 and aminoacyl-tRNA for the same ribosomal binding site. It was found that the 30S-aminoacyl-tRNA-codon complexes formed in the absence of IF-3 are intrinsically different from those prepared in the presence of IF-3. In the absence of IF-3, the ribosomal binding of aminoacyl-tRNA is a virtually irreversible process, since the bound aminoacyl-tRNA can neither be spontaneously released upon dilution nor exchanged for unbound aminoacyl-tRNA. In the presence of IF-3, the binding of one molecule of IF-3 per 30S ribosome renders the binding of aminoacyl-tRNA reversible upon dilution and promotes exchange between bound and unbound aminoacyl-tRNA. It is suggested that this difference is due to a conformational transition of the 30S ribosomal subunit induced by the binding of IF-3. The possible implications of this finding in relation to the mechanism of action of IF-3 and its functional role in the cell are discussed.     

Role of 5S RNA in the Functions of 50S Ribosomal Subunits

50S ribosomal subunits from Bacillus stearothermophilus can be reconstituted from their dissociated components, namely a 5S RNA-free protein fraction, a 5S RNA-free 23S ribosomal RNA fraction, and purified 5S RNA. The biological activity of reconstituted particles in polypeptide synthesis is dependent on the presence of 5S RNA. In the absence of 5S RNA, particles are produced that have greatly reduced activity in (a) polypeptide synthesis directed by synthetic, as well as natural, messenger RNA, (b) peptidyl transferase assay, (c) [(3)H]UAA binding dependent on peptide chain termination factor R1, (d) G factor-dependent [(3)H]GTP binding, and (e) codon-directed tRNA binding assayed in the presence of 30S subunits. Thus, 5S RNA is an essential 50S ribosomal component.     

Localization of the Stringent Protein of Escherichia coli on the 50S Ribosomal Subunit

The "stringent" protein of the ribosome, required for its synthesis of (p)ppGpp, is readily lost during zonal centrifugation. However, enough can be retained to permit its qualitative localization. It is found in native 50S subunits, runoff 70S ribosomes, and polysomes, but not in native 30S subunits. This protein, therefore, appears to be attached to the 50S moiety of the ribosome, and it may be a constant (though easily removed) component of that structure rather than a factor that joins and leaves during the ribosome cycle.     

A New Relaxed Mutant of Escherichia coli with an Altered 50S Ribosomal Subunit

A new relaxed mutant called rel C has been isolated from a rel A(+)/rel A(+) partial diploid strain. The rel C mutant is unable to synthesize ppGpp or pppGpp in vivo in response to amino acid starvation or in vitro, but can synthesize these nucleotides in a shift-down. Rel C maps near rif. Studies in vitro demonstrate the lesion to be probably in one of the 50S ribosomal proteins that can be removed by 1.0 M LiCl.     

Interaction of Escherichia coli 30S Ribosomal Subunits with MS2 Phage RNA in the Absence of Initiation Factors

MS2 RNA binds at 0 degrees to 30S subunits from E. coli and, to a smaller extent, to those of a Pseudomonas species, as judged by filtration on nitrocellulose membranes; this mRNA does not bind to 30S subunits from Bacillus brevis or Caulobacter crescentus. Binding does not depend on the presence of initiation factors; it is sensitive to aurintricarboxylic acid but insensitive to edeine and is competitive with such synthetic polynucleotides as poly(U) and poly(AUG). Complex formation can also be detected by electrophoresis on polyacrylamide-agarose gels. By this procedure, E. coli 30S subunits are separated into two major components. Only the more slowly moving component, which contains the ribosomal protein S1, interacts with the RNA.     

Topography of the Escherichia coli 30S Ribosomal Subunit and Streptomycin Binding

Treatment of Escherichia coli 30S ribosomal subunits with trypsin sequentially removes a number of different ribosomal proteins, as revealed by polyacrylamide gel electrophoresis. Proteins that are removed early by trypsin correlate well with those that are added last during reconstitutive assembly of the 30S subunit from 16S ribosomal RNA and the total protein complement. Proteins that are resistant to removal from the subunit by the highest trypsin concentration used correlate with those that are added early during assembly. Six proteins can be removed from the subunit with trypsin without affecting its ability to bind the antibiotic streptomycin. A decline in the ability of the 30S subunit to bind streptomycin is correlated with the removal of either one, or both, of two proteins, neither one of which is the gene product of the streptomycin locus. The implications of these findings for the topography and assembly of the 30S subunit are considered.     

Ribosomal Proteins, XII. Number of Proteins in Small and Large Ribosomal Subunits of Escherichia coli as Determined by Two-Dimensional Gel Electrophoresis

Two-dimensional gel electrophoresis separates all of the component proteins of the ribosomal subunits of Escherichia coli. This method shows 21 proteins in the 30S, and 34 proteins in the 50S, subunit.     

Association Factor of Ribosomal Subunits from Bacillus stearothermophilus

The isolation of a new factor, which can cause the in vitro association of 30S and 50S ribosomal subunits at low Mg⁺⁺ concentration, is described. The association factor is eluted together with the dissociation protein when ribosomes of Bacillus stearothermophilus are washed with salt solutions of high concentration. The association activity is heat-stable, whereas dissociation factor is inactivated after 10 min at 80°C. This treatment allows the separation of both factors. Several properties rule out the possibility that uncharged, amino-acyl-, or peptidyl-tRNA are responsible for the association process described in this report. Digestion with trypsin shows that the association factor contains at least two components, one of which is a protein.     

Functional Correspondence Between 30S Ribosomal Proteins of Escherichia coli and Bacillus stearothermophilus

30S ribosomal proteins from Bacillus stearothermophilus (B. proteins) have been fractionated and characterized with respect to their ability to replace various E. coli 30S proteins (E. proteins) in the E. coli 30S ribosome reconstitution system. The functional counterparts of all the E. proteins, except S1, S6, S9, and S13, have been tested. In all cases, B. proteins can substitute for E. proteins. Several purified B. proteins are chemically different from their functionally homologous E. proteins. Five B. proteins are immunochemically related to E. proteins; this set includes two proteins that could not be tested in the reconstitution system (S9 and S13). Thus most, if not all, of the E. proteins have functionally equivalent counterparts among B. proteins, even though properties of the two ribosomes are different in several respects. These results suggest that the fundamental structural organization of ribosomes may be the same throughout prokaryotic organisms.     

Structure and Function of Bacterial Ribosomes, XI. Dependence of 50S Ribosomal Assembly on Simultaneous Assembly of 30S Subunits

Some spectinomycin-resistant mutants of Escherichia coli are cold-sensitive. They are unable to assemble both 30S and 50S ribosomal subunits at low temperatures. They accumulate two kinds of incomplete particles, related to 30S and 50S subunits respectively. A single mutation, causing an alteration in a 30S ribosomal component, is responsible for these phenotypes. These results show that assembly of 50S subunits in vivo is dependent on simultaneous assembly of 30S subunits. On the other hand, the assembly of 30S subunits appears to be independent of 50S assembly.     

Shape of the 50S subunit of Escherichia coli ribosomes.

Extrapolation of a series of low-angle neutron scattering curves to infinitely high contrast gives a scattering function IC(kappa) which is dependent on the shape of the solute molecule. For the 50S subunit of E. coli ribosomes, the first part of the structure determination by neutron scattering, namely the determination of the molecular shape from IC(kappa), is reported. The result is in good agreement with models of the 50S subunit determined by electron microscopy.     

Effect of Colicin E3 upon the 30S Ribosomal Subunit of Escherichia coli

The properties of 30S ribosomal subunits from untreated and from colicin E3-treated (E3-30S) bacteria have been compared. Polyacrylamide gel electrophoresis of ribosomal proteins revealed no difference, but several studies indicated that the 16S RNA from E3-30S particles was modified. E3-16S RNA showed slightly increased resistance to heat-induced degradation and had a reduced (15 S) sedimentation coefficient on sucrose gradients. Fingerprint analyses of E3-16S RNA revealed that the 3'-terminus of the molecule had been deleted. It was concluded that a primary effect of colicin E3 is the activation of a highly specific RNase that degrades 30S ribosomal RNA in situ, and that the resulting fragment(s) are probably retained within the 30S particle.     

Sequence Differences of Escherichia coli 30S Ribosomal Proteins as Determined by Immunochemical Methods

Antisera specific for each of the 21 homogenous ribosomal proteins from 30S subunits of Escherichia coli were used to investigate, by immunodiffusion and immunoprecipitation, whether there are any extensive sequence homologies among these proteins.No immunological crossreactions were detected between any of the proteins. Therefore, we conclude that no significant common sequences exist among any of the 21 30S ribosomal proteins of E. coli.     

Alteration of a 30S Ribosomal Protein Accompanying the ram Mutation in Escherichia coli

The functional peculiarities of ram mutants correlate with an observed alteration in chromatographic mobility of P4(a), a specific protein of the 30S ribosomal subunit. This finding is supported by ribosomal reconstitution experiments. These facts, together with the known location of the ram mutational site in the vicinity of other 30S genetic determinants, suggest that ram is the structural gene for P4(a).The known contrasting roles of ram and strA in determining translational efficiency require that the function of P4(a) should be explained in relation to P10 (the 30S-subunit protein defined by strA). One consequence of altering P4(a), a key protein in ribosome assembly, might be to change the interaction of P10 with the 30S subunit. The functional interrelationship of P4(a) and P10 is discussed in terms of the possible roles of these two proteins in regulating access of tRNA molecules to the decoding site.     

Alteration of Ribosomal Protein S4 by Mutation Linked to Kasugamycin-Resistance in Escherichia coli

An alteration in the chromatographic mobility of 30S ribosomal protein S4 from two kasugamycin-resistant (ksgA) strains of E. coli was observed. The locus determining this alteration frequently cotransduced with ksgA but could be separated from it by transduction and conjugation. Since the structural gene for protein S4 is thought to lie some 30 min from ksgA on the E. coli chromosome, the product of the newly-identified gene may modify protein S4. We propose to designate this gene ramB.Reconstitution of 30S subunits from RNA and protein components of ksgA ramB and ksgA(+)ramB(+) strains demonstrated that ribosomal resistance to kasugamycin was due to altered 16S RNA and not to altered protein S4. The altered 16S RNA was undermethylated, and a methylating enzyme that acts on 16S RNA from ksgA strains was present in ksgA(+) but not in ksgA strains.     

Cellular Site of Escherichia coli Ribosomal RNA Synthesis

Escherichia coli, grown for a generation after the addition of [(14)C]uracil and for one minute after a pulse of [(3)H]uracil, were lysed in the presence of 6 mM Mg(++), and divided into a 30,000 x g "supernatant" and sediment. The sediment was resuspended in buffer containing no divalent cations, and again centrifuged to give the "Mg(++) sediment" and the remaining "sediment". The ratio of (3)H (one-minute pulse RNA) to (14)C (Stable RNA) in the supernatant is 1/6 to 1/10 that of the sediment RNA, and the ratio of (3)H:(14)C in the Mg(++) sediment is 1/2 to 1/6 that in the sediment RNA. Gel electrophoresis of RNA from the different fractions shows that ribosomal RNA precursors first appear in the sediment. It is concluded that the site of ribosomal RNA synthesis is associated with a sedimentable structure, which is probably cell membrane.     

Role of 5S RNA in assembly and function of the 50S subunit from Escherichia coli.

Total reconstitution experiments performed under various conditions revealed that 5S RNA plays an important role during the last assembly step in vitro leading to an active 50S particle. For the preceding steps this RNA species is dispensable. However, 50S RNA can be integrated efficiently during any of the assembly steps in vitro. The 47S particle, reconstituted in two steps and lacking 5S RNA, shows low but significant activity in many functional tests. High activity could be obtained by incubating this particle with 5S RNA alone, demonstrating the importance of the 5S RNA in generating an active ribosomal conformation. In particular, the activity of the peptidyltransferase (peptidyl-tRNA:aminoacyl-tRNA N-peptidyltransferase; EC 2.3.2.12) center is drastically influenced by 5S RNA. No significant factor-dependent tRNA binding to the A-site was observed with the 47S particle, in contrast to the corresponding P-site binding. The elongation factor G dependent GTPase activity was not affected by the lack of 5S RNA.     

Drastic Alteration of Ribosomal RNA and Ribosomal Proteins in Showdomycin-Resistant Escherichia coli

In a mutant of Escherichia coli resistant to showdomycin, both the 50S and 30S ribosomal subunits contain RNA species in which the purine concentration greatly exceeds that of pyrimidines. The same is true for total rapidly-labeled RNA. The modified ribosomal RNA hybridizes poorly with homologous DNA, which is apparently unchanged in base composition. Acrylamide gel electrophoresis of mutant ribosomal proteins shows a highly altered protein pattern for both ribosomal subunits, although the activity of these ribosomes is not decreased.     

Initiator proteins for the assembly of the 50S subunit from Escherichia coli ribosomes.

An rRNA-binding protein that binds to the rRNA independently of other proteins during the course of ribosomal assembly is termed "assembly initiator protein." In spite of the large number of rRNA-binding proteins (more than 17 out of 32 proteins have been identified in the case of the large ribosomal subunit), only a very small number of proteins should actually initiate ribosomal assembly for theoretical reasons. Here we demonstrate that only two of the L proteins derived from the large subunit (50S) function as assembly initiator proteins. Two different techniques are used to identify these initiator proteins: reconstitution experiments with purified proteins and pulse-chase experiments during in vitro assembly. Both methods independently identify L24 and L3 as initiator proteins for the 50S assembly. The existence of two initiator proteins (not just one) resolves an apparent contradiction--namely, that on the one hand, rRNA is synthesized in excess under unfavorable growth conditions, whereas on the other hand, rRNA-binding proteins should be available for translational control.