Escherichia coli ribosomes bind to non-initiator sites of Q beta RNA in the absence of formylmethionyl-tRNA.

Escherichia coli ribosomes and Qβ [³²P]RNA were incubated with or without fMet-tRNA under protein initiation conditions, treated with RNase A, and centrifuged through a sucrose density gradient. The sample incubated with fMet-tRNA gave a main radioactivity peak in the 70 S region, which consisted predominantly of coat cistron initiator fragments. After incubation without fMet-tRNA, equal amounts of radioactivity were found in the 70 S and the 30 S regions, but in both peaks almost all of the radioactivity was duo to three RNase A-resistant oligonucleotides, A-G-A-G-G-A-G-G-Up (P-2a), A-G-G-G-G-G-Up (P-15) and G-G-A-A-G-G-A-G-Cp (P-4). These three oligonucleotides are derived from three different RNA regions, none of which is close to a protein initiation site. All three fragments show striking complementarity to the 3′-terminal region of E. coli 16 S RNA. Ribosomes incubated with an RNase A digest of Qβ [³²P]RNA bound almost exclusively oligonucleotide P-2a; treatment with cloacin DF13 cleaved off a complex consisting of a 49-nucleotide long segment of 16 S rRNA and oligonucleotide P-2a. These experiments show that the interaction of 30 S ribosomes with the “Shine-Dalgarno” region preceding the initiator codon of the Qβ coat cistron is insufficient to direct correct placement of the ribosome on the viral RNA, and that an additional contribution from the interaction of fMet-tRNA with the initiator triplet is required for ribosome binding to the initiator region.     

Specificity of protein synthesis by bacterial ribosomes and initiation factors: Absence of change after phage T4 infection

Using several natural messenger RNA's—f2 RNA, Qβ RNA, T7 RNA, T4 early mRNA, T4 late mRNA and Escherichia coli RNA—ribosomes isolated from cells either 5 or 12 minutes after T4 infection direct synthesis of only 35 to 70% as much protein as do ribosomes from uninfected cells. However, with poly(U) or formaldehyde-treated f2 RNA message, both types of ribosomes work equally well. Experiments mixing salt-washed ribosomes and initiation factors from these cells show, in agreement with work of others, that the reduction with natural messages is due only to changes in the initiation factors.     

Ribosome-binding sites on chloroplastrbcL andpsbA mRNAs and light-induced initiation of D1 translation

Chloroplast ribosome-binding sites were identified on the plastidrbcL andpsbA mRNAs using toeprint analysis. TherbcL translation initiation domain is highly conserved and contains a prokaryotic Shine-Dalgarno (SD) sequence (GGAGG) located 4 to 12 nucleotides upstream of the initiator AUG. Toeprint analysis ofrbcL mRNA associated with plastid polysomes revealed strong toeprint signals 15 nucleotides downstream from the AUG indicating ribosome binding at the translation initiation site.Escherichia coli 30S ribosomes generated similar toeprint signals when mixed withrbcL mRNA in the presence of initiator tRNA. These results indicate that plastid SD sequences are functional in chloroplast translation initiation. ThepsbA initiator region lacks a SD sequence within 12 nucleotides of the initiator AUG. However, toeprint analysis of soluble and membrane polysome-associatedpsbA mRNA revealed ribosomes bound to the initiator region.E. coli 30S ribosomes did not associate with thepsbA translation initiation region.E. coli and chloroplast ribosomes bind to an upstream region which contains a conserved SD-like sequence. Therefore, translation initiation onpsbA mRNA may involve the transient binding of chloroplast ribosomes to this upstream SD-like sequence followed by scanning to localize the initiator AUG. Illumination 8-day-old dark-grown barley seedlings caused an increase in polysome-associatedpsbA mRNA and the abundance of initiation complexes bound topsbA mRNA. These results demonstrate that light modulates D1 translation initiation in plastids of older dark-grown barley seedlings.     

Identification of homologues of a functionally important 50 S ribosomal protein in different bacterial species.

Functional homology between the 50 S ribosomal protein L3 from Bacillus stearothermophilus and a protein from each of three other species of Bacillaceae (Bacillus licheniformis, Bacillus megaterium, and Bacillus subtilis) is demonstrated by substituting each of the proteins for B. stearothermophilus L3 in active reconstituted ribosomes. The structurally related protein from Escherichia coli, L2, cannot replace B, stearothermophilus L3, nor can any other E. coli protein.     

Two ribosome binding sites from the gene 0.3 messenger RNA of bacteriophage T7

Ribosome-protected regions have been isolated and analyzed from the bacteriophage T7 gene 0.3 mRNA labeled in vivo. Two discrete sites which are nearly equally protected by ribosomes are obtained from what was previously assumed to be a monocistronic message. Use of appropriate T7 deletion mutant RNAs has allowed mapping of both ribosome-recognized regions. Site a is positioned very close to the 5′ terminus of the mRNA and is apparently the initiator region for the major gene 0.3 protein, which acts to overcome the host DNA restriction system. Site b is located within several hundred nucleotides of the 3′ end of the RNA and probably initiates synthesis of a small polypeptide of unknown function. Both ribosome binding sites exhibit features common to other initiator regions from Escherichia coli and bacteriophage mRNAs. The proximity of site a to the RNase III cleavage site at the left end of gene 0.3 may explain why processing by RNase III is required for efficient translation of the major gene 0.3 protein.     

Molecular Basis for Repressor Activity of Qβ Replicase

WITH the purification and characterization of viral replicases, a novel feature of nucleic acid polymerases—stringent template specificity—was recognized^1,2. Qβ replicase, the most extensively studied viral RNA polymerase^2–8, is now known to replicate Qβ RNA², the complementary Qβ minus strand⁹, RNA molecules described as “variants” of Qβ RNA^10,11 and a set of small RNAs of unknown origin which accumulate in Qβ-infected Escherichia coli, collectively designated as “6S RNA”¹². On the other hand, the RNA from phages related distantly, if at all, to Qβ^13,14, such as MS2 or R17 and of other viruses such as TMV² or AMV (Diggelmann and Weissmann, unpublished results) are completely inert as templates, as are ribosomal and tRNA from E. coli². Poly C and C-rich synthetic copolymers at high concentrations elicit synthesis which, however, remains restricted to the formation of a strand complementary to the template^15,16.     

Translation of bacteriophage R17 and Qbeta RNA in a mammalian cell-free system

The polycistronic RNAs from both bacteriophage R17 and Qβ are translated in a mammalian cell-free system of purified and partially purified components. The requirement of one of the partially purified initiation factors (IF-E₃ from rabbit reticulocytes) for the phage RNA translation is strikingly different from that for rabbit globin messenger RNA translation. The phage RNA-directed products are characterized by acrylamide gel electrophoresis and compared with those synthesized in an Escherichia coli cell-free system. There is good agreement between the respective coat proteins and the presumptive synthetase proteins. R17 RNA directs the synthesis of two additional defined polypeptides. However, their possible relationship with the A-protein cistron has not yet been investigated. The RNA from the amB2 mutant of R17, which carries an amber triplet at position 6 in the coat protein cistron, directs the synthesis of the same polypeptides as the wild-type RNA with the exception of the coat protein which is completely abolished. This identifies the product made with wild-type RNA as coat protein and provides a direct in vitro assay for the suppression of nonsense mutations in eukaryotic cells.     

Replication of RNA viruses: specific binding of the Q RNA polymerase to Q RNA

The specific binding in vitro of the Qβ RNA polymerase to Qβ RNA has been detected by the formation of an enzyme-Qβ RNA complex that did not exchange bound RNA molecules and was not dissociated by 0.8 m NaCl. Formation of this nondissociating complex required GTP and two host protein factors, but not ATP, CTP, UTP, or Mg²⁺ ions. GDP, GMP, dGTP, ITP, and β,γ-methylene GTP did not replace GTP in the reaction. Complex formation at 0 °C was not observed, and the rates of the reaction at 30 °C and 25 °C were 41% and 23%, respectively, of the rate at 37 °C. The reaction occurred with intact Qβ RNA and with polycytidylic acid template but not with bacterial or other bacteriophage RNA. With limiting amounts of enzyme, the amount of Qβ RNA bound in the nondissociating complex was the same as the amount of [γ-³²P]GTP incorporated into nascent RNA chains, indicating a close relationship between complex formation and the initiation of RNA synthesis. The two reactions appear to be separate, however, because in the absence of Mg²⁺ ions, when complex formation occurred readily, no RNA synthesis could be detected either by incorporation of labeled substrate into acid-insoluble material or by formation of short RNA chains still attached to the enzyme. In the presence of factor protein and GTP, a maximum of one active enzyme molecule was bound per molecule of Qβ RNA template, as determined by a liquid polymer phase-separation procedure. These results suggest that formation of the nondissociating complex measures recognition by the Qβ RNA polymerase of a single Qβ RNA site utilized for the initiation of synthesis.     

Reconstitution of active 50 S ribosomal subunits from Bacillus licheniformis and Bacillus subtilis.

Active 50 S ribosomal subunits from Bacillus licheniformis and Bacillus subtilis can be reconstituted in vitro from dissociated RNA and proteins. The reconstituted 50 S sub-units are indistinguishable from native 50 S subunits in sedimentation on sucrose gradients and in protein composition. The procedure used is similar to that developed for reconstitution of Bacillus stearothermophilus 50 S subunits, though the optimal conditions are somewhat different. Hybrid ribosomes can be reconstituted with 23 S RNA and proteins from different sources (B. stearothermophilus and B. licheniformis or B. subtilis). The thermal stability of these ribosomes depends on the source of the proteins, and not on the source of 23 S RNA.     

Protein synthesis in Bacillus subtilis. II. Selective translation of natural mRNAs and its possible relation to the species-specific inhibition of protein synthesis by lincomycin and erythromycin.

Vacant ribosomal couples from Bacillus subtilis W168 incorporate only very small amounts of amino acids into polypeptides in response to Escherichia coli cellular RNA or bacteriophage f2 RNA, but are observed to form initiation complexes in the presence of f2 RNA. Vacant ribosomal couples from E. coli acquire pressure-resistance, but do not bind fMet-tRNA, when incubated with B. subtilis RNA in the absence of ribosomal wash fraction. The implied mRNA binding in the absence of salt wash fraction, taken with previously reported observations of salt wash-independent translation of mRNAs from Grampositive bacteria, suggests that mRNAs from Gram-positive bacteria have an active functional character which is masked or absent in mRNAs from Gram-negative sources. It is proposed that this property of B. subtilis mRNAs is required by B. subtilis ribosomes for some translational function subsequent to the formation of the 70 S initiation complex, and that f2 RNA, while it is bound by B. subtilis ribosomes in initiation complexes, is not translated because it lacks this feature.The antibiotic lincomycin has been found to inhibit translation of natural mRNAs in vitro in systems from Gram-positive bacteria at concentrations 10 to 100 times lower than those necessary to inhibit translation in systems from Gram-negative species. Lincomycin does not inhibit formation of initiation complexes by vacant couples from B. subtilis or E. coli. Taken with the published findings of other investigators, these results are interpreted as indicating that the first translocation step following assembly of the initiation complex may coincide with a transition between distinct “initiating” and “elongating” states of the ribosome, and that this transition may involve structural elements, and possibly mechanisms, which are different in Gram-positive systems than in Gram-negative systems.A comprehensive model is constructed to account for the results of these studies and for the published findings of other investigators. It is proposed that some feature of Gram-positive mRNA, perhaps a vestige of early protein synthetic systems, is required by the ribosomes of Gram-positive bacteria to facilitate the transition between initiating and elongating ribosomal states. Inhibition of protein synthesis by lincomycin and the similarly species-specific macrolide antibiotic erythromycin is interpreted as an allosteric effect on the transition between initiating and elongating ribosomal states, in which the different binding affinities of ribosomes from Gram-positive and Gram-negative bacteria for the drugs are related to the functional differences between the two types of systems at this critical step. The implications of this interpretation of interspecies translational specificity for mechanisms of translational control in the cell and for the nature of the divergence of bacterial protein synthesis systems into Gram-positive and Gram-negative types are discussed.     

Structure of methemerythrin at 5 A resolution.

Rabbit globin messenger RNA was labelled in vitro with ¹²⁵I to specific activities in the range 20 to 200 × 10⁶ cts/min per μg. This ¹²⁵I-labelled mRNA bound to rabbit reticulocyte ribosomes with the kinetics and sensitivity to inhibitors expected from its participation in the normal process of the initiation of protein synthesis. Furthermore, when modified in 25% of its cytidine residues with unlabelled iodide, the mRNA coded for the same series of initiation peptides as did the unmodified mRNA. Using the techniques of RNA fingerprinting, the binding reaction was shown to select against contaminants and against “globin mRNA” molecules which lack a particular oligonucleotide implicated in the initiation process. When the ¹²⁵I-labelled mRNA was bound to ribosomes, both the initiating 40 S subunits and the 80 S ribosomes protected a fraction of the mRNA from digestion by pancreatic ribonuclease. Fingerprint analysis showed that highly specific regions of the mRNA were protected by the 40 S subunits and 80 S ribosomes and that these two protected regions were not identical.     

Ribosomal proteins

Summary The number of specific binding sites for homogenous single ribosomal proteins on 16S E. coli ribosomal RNA was investigated. The capacity of each of the twenty-one 30S subunit proteins to bind to the RNA was estimated by two newly developed methods, namely immunoprecipitation and a polyacrylamide gel method. Five proteins, namely S4, S7, S8, S15 and S20 bound specifically. One, S17, bound nonspecifically. No binding of the other proteins was detected. The binding proteins bound simultaneously to the RNA, with stimulated binding of proteins S7 and S8. Evidence is provided for the similarity of the chemistry of the binding sites of the binding proteins in Escherichia coli and in Bacillus stearothermophilus ribosomes.     

Studies on rDNA from the extreme thermophilic eubacterium Thermus thermophilus HB8: The 23 S rDNA region D

23 S ribosomal ribonucleic acid gene from the extreme thermophile eubacterium Thermus thermophilus HB8 has been cloned in pBR322, and the nucleotide sequence of region D has been determined, which encompasses 873 nucleotides at the 3′-end of the RNA. We compare the primary and secondary structure of this region with the respective part of the 23 S rRNA from Escherichia coli and Bacillus stearothermophilus. A high level of structural conservation can be observed, throughout the RNA domain, albeit the usage of G/C basepairs is substantial even in comparison with another thermophilic eubacterium B. stearothermophilus. It is surprising that, in contrast to the usage of ^3′U-G^5′, the occurrence of ^3′G-U^5′ is comparable in E. coli as well as in B. stearothermophilus and T. thermophilus. Furthermore, it is most remarkable that the use of ^3′A-U^5′ and ^3′U-A^5′ is, compared to E. coli, only slightly reduced in B. stearothermophilus, but drastically decreased in T. thermophilus.     

Effect of a Purified Initiation Factor F3 (B) on the Selection of Ribosomal Binding Sites on Phage MS2 RNA

STUDIES with T4 mRNA showed that initiation factor F2 (C) promotes the attachment of ribosomes to mRNA¹. On the 30S ribosomal subunit this effect is independent of the function of F2 in the binding of formylmethionyl tRNA², whereas formation of a 70S-mRNA complex depends on the binding of fMet-tRNA³. Template competition experiments⁴ showed that, with F2 (C), the ribosome seems to have the same affinity for synthetic polynucleotides as for natural mRNA. Addition of initiation factor F3 (B), however, leads to preferential binding of ribosomes to the natural mRNA. This suggests⁴ that while factor F2 (C) binds the ribosome to any site on the mRNA, the function of factor F3 (B) is to recognize some specific signal in natural mRNA corresponding, perhaps, to the beginning of a cistron. Fractionation of initiation factor F3 (B) into several species differing in their specificity for different mRNA templates⁵ gave further support to the hypothesis that this protein can select binding sites. An excellent system to demonstrate this effect of F3 (B) would be the binding of ribosomes to RNA from E. coli RNA bacteriophages, since Steitz⁶ has analysed and determined the nucleotide sequence of the three binding sites corresponding to the three cistrons of R17 mRNA. Experiments were thus undertaken to study the effect of a purified fraction of F3 (B) on the binding of ribosomes to the different sites of such a phage RNA.     

The inhibition of nucleic acid-binding proteins by aurintricarboxylic acid

Qβ replicase, $\text{Escherichia coli}$ RNA polymerase, and T7 RNA polymerase are inhibited by low concentrations of the dye aurintricarboxylic acid (ATA). In each case initiation by the enzyme was preferentially inhibited. The elongation of initiated polynucleotide chains by Qβ replicase was insensitive to ATA in the range of concentrations required to inhibit initiation. Treatment of Qβ replicase, RNA polymerase and $\text{lac}$ repressor with ATA prevented enzymemediated binding of the templates to nitrocellulose filters. We propose that the inhibitor combines with the template binding site of these proteins to prevent initiation.     

Nondiscrimination of RNA viral message in binding to 30S ribosomes derived from T4 phage infected Escherichia coli

The effect of T4 phage on ribosomes in terms of their ability to bind RNA viral template is examined. It is found that the 30S subunits of T4 ribosomes bind MS2 RNA as efficiently as do the subunits of uninfected $\text{E. coli}$ ribosomes. On the other hand, analyses of the formation of 70S initiation complex, presumably from MS2 RNA-30S ribosome complex, using both labeled MS2 RNA and initiator tRNA, reveal that T4 ribosomes are only about half as active as $\text{E. coli}$ ribosomes. The latter phenomenon has been reported previously. These results suggest that, following T4 infection, ribosomes are modified in such a way that the attachment of fMet-tRNA_f to MS2 RNA-30S subunit complex is impaired.     

Function of bacteriophage Qbeta replicase containing an altered subunit IV

In order to elucidate the function of elongation factor Ts in Qβ replicase, enzyme was obtained from a Qβ-infected Escherichia coli mutant HAK88, which carries an altered EFTs² with a thermolabile catalytic activity. HAK88 Qβ replicase was found to be quite unstable at 42 °C. Further studies indicated that the mutant enzyme exhibits temperature sensitivity with regard to GTP binding ability but not with Qβ RNA and poly(C) binding. These results suggest that the function of EFTs in Qβ replicase is closely related to the binding of GTP to the enzyme.A defect in Qβ replicase also appears when it is reconstituted from the Qβ replicase subunit complex I–II and the HAK88 EFTu-EFTs complex. Several lines of evidence obtained by using the reconstituted enzyme suggest strongly that the EFTs function is involved specifically in initiation of RNA synthesis, but not in the elongation reaction.     

A complex of acidic ribosomal proteins. Evidence of a four-to-one complex of proteins in the Bacillus stearothermophilus ribosome

Two acidic proteins from the 50 S subunit of Bacillus stearothermophilus ribosomes, namely B-L13 (homologous to Escherichia coli protein $\text{L}\text{7}\text{L}\text{12}$) and B-L8, form a complex. Radioactive B-L13, added to ribosomes before dissociation, does not appear in the complex after electrophoresis, so the (B-L13 · B-L8) complex must exist in the ribosome before dissociation. Digestion of B. stearothermophilus ribosomes with polyacrylamide-bound trypsin causes the appearance of new B-L8 and B-L13 spots on two-dimensional polyacrylamide gel electrophoresis, in a pattern which suggests that single molecules of B-L13 are being sequentially cleaved from a four-to-one complex of B-L13 and B-L8.     

Expression of small RNAs by Bacillus sp. strain PS3 and B. subtilis cells during sporulation

A small RNA sequence identified in an rRNA-tRNA cluster from the thermophilic Bacillus sp. strain PS3 was examined. An oligonucleotide probe specific for the RNA bound to multiple restriction fragments in Bacillus sp. strain PS3 DNA, thus several copies of this sequence occur in its genome. Similar findings were observed using DNA from B. subtilis, B. stearothermophilus, Escherichia coli, Staphylococcus aureus, Haemophilus influenzae and Thermus thermophilus. This sequence apparently is widespread in the eubacteria. Northern analysis of RNA from sporulating Bacillus sp. strain PS3 and B. subtilis cells revealed RNA species homologous to the probe in both bacteria. Expression of the small RNA in B. subtilis depended on σ^H.     

On the accessibility and selection of the initiator site of mRNA in protein synthesis.

The specificity of the cell-free system of Escherichia coli for mRNA was examined, and the "accessibility" of some natural and synthetic RNAs to the ribosomes was determined by measurement of AcPhe-tRNA and fMet-tRNA binding, AcPhe-puromycin and fMet-puromycin formation, and polypeptide synthesis. The E. coli system effectively initiates the translation of various synthetic RNAs with AcPhe-tRNA or fMet-tRNA under conditions optimal for the translation of viral RNA. Poly(A,G,U) is accessible to the ribosomes according to all of the above criteria. Poly(A,C,G,U), 23 S rRNA, R17 RNA, and MS2 RNA, on the other hand, show limited accessibility when tested for initiator tRNA binding, or for AcPhe-puromycin and fMet-puromycin formation. MS2 and R17 RNA, but not poly(A,C,G,U) and 23 S rRNA, show accessibility when measured by polypeptide synthesis. The results suggest that, except at initiator sites of natural mRNA, an RNA containing about equal amounts of all four bases is inaccessible to E. coli ribosomes for polypeptide synthesis. Rate constants obtained for fMet-tRNA binding with MS2 RNA, poly(A,G,U), and poly(C,G,U) indicate that the ribosomes do not have any special affinity for the viral RNA. Thus, the selection of the initiator site in protein synthesis may be critically determined more by the accessibility of the initiator codon than by ribosomal recognition of the site.