R17 RNA Research Articles

The effect of ribonuclease on the penetration of R17 phage A-protein and RNA.

In an attempt to throw further light on the relationship of R17 phage RNA and A-protein during the early stages of infection, studies were carried out to determine the effect of ribonuclease (ribonuclease I, EC 3.1.4.22) on the ability of these two phage components to penetrate into host bacteria. It was found that the penetration of phage RNA is affected by ribonuclease concentrations as low as 0.1 mug/ml, while the penetration of phage A-protein was unaffected by ribonuclease concentrations as high as 20 mug/ml. In addition, it was found that a significant fraction of the phage RNA is resistant to the ribonuclease effect. This RNase-resistant portion of the phage population increased with increasing phage concentrations, and gave rise to the penetration of intact, 28S RNA molecules that produced the expected number of infectious centers. These findings are discussed in terms of a model for phage RNA injection in which the A-protein functions both as an attachment organelle and a pilot protein that guides the RNA from the capsid to the exterior surface of the F pilus, and thence into the host bacterium.

Site-specific interaction of Qbeta host factor and ribosomal protein S1 with Qbeta and R17 bacteriophage RNAs.

We have studied the interaction of the host factor (HF) required for bacteriophage Qbeta RNA replication and of ribosomal protein S1, a subunit of Qbeta replicase, with Qbeta and R17 RNA. Both proteins bind to both Qbeta and R17 RNA; HF has a higher affinity than S1 for these phages RNAs. HF binds to a single site in R17 RNA located in the replicase cistron, and to two sites of Qbeta RNA, one of which is located approximately 60 nucleotides from the 6' end of Qbeta RNA. The three HF binding sites all have portions rich in adenylate residues; all are bound by HF when contained in oligonucleotides which are predicted to exist only in single-stranded form. S1 selects a single site in Qbeta RNA, also near the 6' end, but binds to a large number of sites in R17 RNA. These results suggest that HF and possibly S1, through their interaction with the 3'-terminal region of Qbeta RNA, are directly involved in the recognition of the 6' end of Qbeta RNA by Qbeta replicase. Under conditions where specific protein-R1M RNA complexes are formed, we have also tested host factor and S1 for cistron-specific interference with ribosome binding to R17 RNA. Although S1 and HF lower the efficiency of initiation complex formation as described previously, we detect no discrimination against any particular cistron. We therefore conclude that translational interference exhibited by the two proteins probably reflects simply their high affinity for RNA and certain defined polynucleotides.

The interaction of fusidic acid with peptidyl-transfer-ribonucleic-acid - ribosome complexes.

The inhibitory action of fusidic acid on peptide-chain elongation was studied with systems in vitro directed by either polyuridylic acid or endogenous messenger (Escherichia coli polysomes washed with 1 M NH4Cl) or R17 RNA, and supplemented with either crude or purified elongation factors. In all cases strong inhibition of synthesis required high concentrations of the antibiotic (approx. 1 mM), while a similar inhibition of the EF-G-plus-ribosome-dependent GTP hydrolysis required between 10 and 100 times less antibiotic. Since most of the GTP hydrolysis observed was presumably due to free ribosomes (without aminoacyl-tRNA or peptidyl-tRNA), fusidic acid seemed to interact far more easily with these ribosomes than with ribosomes engaged in peptide-chain elongation. The role of the GDP-EF-G-ribosome-fusidic acid complex in the inhibition of polypeptide synthesis was assessed by measuring formation of this complex on polysomes engaged in peptide-chain elongation. Using purified elongation factors the complex formed on only 25-35% of ribosomes, as measured either by retention of [3H]GDP or by hydrolysis of [3H, gamma-32P]GTP. In contrast, with crude factors (S 100 extract) it formed on more than 70% of ribosomes. The results are compatible with the postulated role of the complex in polypeptide synthesis inhibition (blockade of the ribosomal acceptor site and subsequent inhibition of aminoacyl-tRNA binding) and indicate that formation of the complex takes place by overriding the control that prevents interaction of EF-G when the donor site is occupied by peptidyl-tRNA. In the polyuridylic-acid-directed system for synthesis of oligophenylalanine the antibiotic inhibits every round of peptide elongation, including dipeptide formation, to roughly the same extent.

Translational repression of a viral messenger RNA by a host protein.

It is shown that factor i, a bacterial protein, specifically inhibits that step in the initiation of R17 bacteriophage RNA translation that involves the attachment of native R17 RNA to 30 S ribosomal subunits carrying fMet-tRNA. This inhibition by factor i is relieved by the addition of excess R17 RNA, but not by the addition of excess 30 S subunits. That R17 RNA is the only target of the inhibition is demonstrated further by the fact that in a cell-free extract containing all components for protein synthesis, factor i-mediated inhibition of exogenous R17 RNA translation can be overcome only by the addition of excess R17 RNA and not by excess cell-free extract. Upon relief of inhibition, phage coat protein synthesis is restored; enhancement of formation of other cistron products is not seen. While initiation of R17 RNA translation is blocked by factor i, chain elongation is not affected. Although foactor i inhibits the IF-3-dependent binding of R17 RNA to fMet-tRNA-30 S complexes, under conditions of initiation of protein synthesis formation of stable complexes between factor i and IF-3 could not be detected, and factor i did not interfere with the binding of IF-3 to free, native R17 RNA. Instead of affecting the function of IF-3 or ribosomes, factor i exerts its inhibition by binding to R17 RNA and acting as a translational repressor. Factor i prefers intact R17 RNA to fragments generated by autoradiolysis; its binding to R17 RNA is specific in that little competition is observed by transfer RNA, ribosomal RNA or poly(A). However, factor i has a high affinity for poly(U) sequences.

The molecular size of the RNA genome of Pseudomonas aeruginosa bacteriophage PP7

The molecular size of Pseudomonas aeruginosa bacteriophage PP7 RNA has been estimated by electron microscopy and polyacrylamide gel electrophoresis. The length of the genome is estimated to be 1.16 μm, similar to R17 RNA (1.22 μm). After formaldehyde treatment under conditions in which R17 RNA has an estimated molecular weight of 1.3 × 10 6, the molecular weight of the PP7 genome is 1.24 × 10 6. Without formaldehyde treatment, under conditions in which R17 RNA has an apparent molecular weight of 1.1 × 10 6, PP7 RNA molecular weight is calculated as 1.03 × 10 6. PP7 RNA is therefore a very similar size to Group I Escherichia coli RNA bacteriophage genomes, being only 6% smaller than R17 RNA, but about 20% smaller than Group III Qβ RNA. This molecular size is consistent with the suggestion that the PP7 genome has only three cistrons.

Initiation of protein synthesis. Binding of messenger RNA.

Complexes between 30 S ribosomal subunits and fMet-tRNA are formed during incubation of 30 S subunits with fMet-tRNA and all other components for initiation of protein synthesis, except R17 bacteriophage RNA. That these complexes serve as intermediates in the binding of messenger RNA is demonstrated directly by the finding that upon addition of R17 RNA, fMet-tRNA in preformed fMet-RNA-30 S complexes preferentially enters fMet-tRNA-30 S-R17 RNA complexes. On the other hand, incubation of 30 S ribosomal subunits with R17 RNA and all other components for initiation except fMet-tRNA does not yield 30 S-R17 RNA complexes that can act subsequently as functional intermediates in the binding of fMet-tRNA: formation of fMet-tRNA-30 S-R17 RNA complexes does not occur when fMet-tRNA is added and further binding of R17 RNA to 30 S subunits is prevented by specific inhibitors. These experiments lead to an unambiguous order of events in the sequence of initiation, in which binding of fMet-tRNA to the small ribosomal subunit must occur before messenger RNA can be bound and phased correctly. Complexes between fMet-tRNA and 60 S subunits are in rapid equilibrium with the free components, and have a half-life of less than 2 min at 37 degrees. This explains why such complexes are not detected in sucrose gradients, unless they are first fixed with glutaraldehyde. Attachment of R17 RNA, however, results in formation of an fMet-tRNA-30 S-R17 RNA complex that is stabilized greatly; fMet-tRNA in this complex exchanges only very slowly with free fMet-tRNA. Initiation factor IF-3 has two functions in initiation. The first is to direct the binding of messenger RNA to the 30 S-fMet-tRNA complex. This function is not needed when initiation complex formation occurs on ApUpG triplets, in which case the second function of IF-3 is detected, that of providing free 30 S subunits for initiation. The ability of IF-3 to bind directly to R17 RNA may be related to its requirement in messenger RNA recognition. However, since IF-3 exhibits a greater affinity for the 30 S subunit than for R17 RNA, it appears that the recognition function of IF-3 is expressed while IF-3 is associated with the 30 S subunit.

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A new procedure for R17 RNA preparation was devised using a 300 liters fermenter. The key factor in processing such a large quantity is the purification of phage particles prior to RNA extraction. The method involves the preparation of 250 liters of crude lysate, condensation of phage particles by the partition method, purification by DEAE-cellulose column, and removal of adherent proteins by a series of high-salt washes. The method permits preparation of approximately 3 g of phage particles free of ribosomal fragments and RNase. Phage RNA extracted in gram quantities using conventional methods often contain phenol. Thus repeated extraction of R17 RNA with salt-alcohol mixture is required.

The host factor required for RNA phage Qbeta RNA replication in vitro. Intracellular location, quantitation, and purification by polyadenylate-cellulose chromatography.

The Qbeta host factor, a heat-stable protein necessary in concert with Qbeta replicase for phage Qbeta RNA replication in vitro, has been localized in Escherichia coli and found to be associated primarily with ribosomes. This location has been established both by complement fixation assays with highly specific antiserum directed against the host factor, and by in vitro stimulation of Qbeta RNA replication by the Qbeta replicase. The complement fixation assay has provided the estimate that there are approximately 2500 copies of the host factor polypeptide per cell. The host factor is released from the ribosomes by a 1 M NH4Cl wash and concentrated by ammonium sulfate precipitation. It can be purified to apparent homogeneity in one further step by chromatography on poly(A)-cellulose. Ribosomal protein S1 subunit I of Qbeta replicase) also binds to the poly(A)-cellulose column and elutes before the host factor. In agreement with previous reports, we find that the host factor has a monomer molecular weight of 12,000 as judged by sodium dodecyl sulfate-polyacrylamide gels, and a native molecular weight of 72,000 as judged by the stoichiometric interaction of the host factor with Qbeta RNA, by sedimentation in sucrose velocity gradients, and by sodium dodecyl sulfate gel mobility when incompletely disaggregated. The Qbeta host factor is a potent inhibitor of an in vitro poly(A)-directed polylysine protein-synthesizing system, but has less effect on the in vitro translation of poly(U), R17 RNA, late T7 mRNA, or endogenous E. coli mRNA. The amino acid composition and NH2- terminal sequence rule out the host factor as one of the known 30 S or 50 S E. coli ribosomal proteins. The finding that the Qbeta host factor is associated with ribosomes in vivo completes the demonstration that all of the host-supplied proteins required for phage Qbeta RNA replication in vitro are either associated with ribosomes or are involved in the protein-synthetic machinery of the cell.

Synthesis of viral and rRNA in bacteriophage R17 infection of a stringent strain of Escherichia coli.

A previous paper (1973) indicated that infection with bacteriophage R17 permits the synthesis of RNA and spermidine in Escherichia coli (CP78 in the absence of the exogenous essential amino acid, arginine. We have now isolated RNA formed under such conditions and analyzed the newly synthesized species by agarose-acrylamide electrophoresis. It has been shown that infection of the stringent cells in the absence of exogenous arginine resulted in a marked incorporation of uracil into rRNA, as well as into R17 RNA. It was shown that, although the organism was nonauxotrophic for uracil, addition of [-14C]uracil resulted in the rapid formation of TUP, the specific radioactivity of which approached that of the exogenous uracil. This indicated that the incorporation of exogenous uracil into rRNA in R17 infection of the stringent strain reflected a true stimulated synthesis of this nucleic acid. Infection of the essentially isogenic relaxed strain, CP79, under the same conditions inhibited the RNA synthesis to a much less extent than the inhibition caused during the normal infection. These observations provide another example of the close correlation between synthesis of spermidine and of host RNA, even in cells infected by an RNA bacteriophage.

Structural dynamics of bacterial ribosomes: III. Quantitative conversion of vacant ribosome couples into an initiation complex with R17 RNA as messenger

Conditions for the optimum conversion of ribosomes into an initiation complex with R17 RNA as messenger have been examined on a quantitative basis, and at limiting ribosome concentrations, in order to establish structural and functional criteria for ribosome activity. Ribosomes that are in the associated state at Mg 2+ concentrations ≥ 5 m m (“tight couples”) can be converted into an initiation complex with an efficiency of ≈ 85% with crude, and with an efficiency of about 60% with pure initiation factors. For tight couples, the concentration of crude initiation factors and Mg 2+ is most critical. Crude initiation factors contain both RNAase and ribosomal proteins. Because degradation of messenger RNA and ribosomes by RNAase competes with initiation, maximum initiation requires dilution of the factor preparation to an optimal factor/ribosome ratio, while keeping the R17 RNA/ ribosome ratio constant at a molar ratio of 2.5. Pure initiation factors, although nearly free of RNAase, are less efficient apparently because lacking ribosomal proteins they cannot reconstitute a fraction of deficient ribosomes. The Mg 2+ concentration is critical because it determines the rate of dissociation of ribosomes into subunits as well as the rate of reassociation and messenger binding. Initiation complex formation depends stoichiometrically on the presence of fMet-tRNA which, as shown by titration, is bound with extremely high affinity. Close agreement was found in the quantitative results obtained by several independent measurements based on (i) specific activity of f-[ 3H]Met-tRNA bound to the 70 S complex, (ii) absorbance of stable 70 S initiation complex in gradients under conditions at which contaminating vacant ribosomes are dissociated, (iii) recovery of ribosomes converted into initiation complex ralative to total ribosome input, and (iv) specific activity and average molecular weight of 3H-labeled mRNA bound to initiation complex. Computation of recoveries of ribosomes from gradients are complicated by losses due to adsorption to the wall of the centrifuge tubes. Empirical calibration data are given to correct for this “wall effect”.

Total reconstitution of functionally active 50S ribosomal subunits from Escherichia coli.

Total reconstitution of 50S subunits from E. coli was achieved by a two-step incubation procedure. In the first step, 23S RNA, 5S RNA, and the total proteins from 50S subunits were incubated for 20 min at 40 degrees in the presence of 4 mM Mg(++) and 400 mM NH(4)Cl. In the second step, the Mg(++) concentration was raised to 20 mM and the incubation was performed for 90 min at 50 degrees . No requirement for 30S subunits or other components (e.g., polyamine) was found. The reconstituted particle has the same sedimentation coefficient as the native 50S subunit and is highly active in protein synthesis with natural (R17 RNA) and artificial [poly(U)] messengers as well as in tests for peptidyltransferase (fragment assay) and for binding of antibiotics (chloramphenicol).

Translation of R17 RNA by Escherichia coli ribosomes : Initiator transfer RNA-directed binding of 30 S subunits to the starting codon of the coat protein gene

1. (1) Active ribosomes (tight vacant 50 S/30 S couples) not contaminated with subunits require factors IF-2 and IF-3 for initiation. 2. (2) Requirements for the formation of a 40 S initiation complex consisting of intact R17 RNA and fMet-tRNA bound to the 30 S subunit are fully satisfied by IF-3 alone, if 50 S subunits are absent. The 40 S complex, obtained in yields of up to 30% of the 30 S subunits added, is stable in sucrose gradients at 15 m m-Mg 2+ without fixation with glutaraldehyde. In the presence of 50 S subunits and GTP, the 40 S complex is converted quantitatively at 0 °C into a 76 S complex that is fully reactive with puromycin. 3. (3) In the presence of strongly associating 50 S-a subunits, IF-2 and GTP in addition to IF-3 are required for formation of the 40 S complex because IF-2 and GTP prevent reassociation of subunits by enhancing the binding of fMet-tRNA and IF-3 to the 30 S subunit in the absence of messenger. Optimal formation of the mRNA-free 30 S · fMet-tRNA complex, demonstrable on sucrose gradients even without fixation by glutaraldehyde, requires all three initiation factors and GTP. 4. (4) Binding of mRNA to 30 S subunits in the absence of initiator tRNA, observed on sucrose gradients after fixation, appears to be unspecific because it is not dependent on initiation factors. The results are compatible only with a model (Noll & Noll, 1972) in which the binding of initiator tRNA to the 30 S subunit directs the binding of the mRNA initiator codon. From these and earlier findings we visualise the sequence of assembly of an initiation complex between Escherichia coli ribosomes and R17 RNA as follows: (i) 70 S + IF-1 → 30 S · IF-1 + 50 S (Noll, 1972; Noll & Noll, 1972), (ii) 30 S · IF-1 + IF-3 → 30 S · IF-1,3, (iii) 30 S · IF-1,3 + fMet-tRNA · IF-2 · GTP → 30 S · IF-1,2,3 · GTP · fMet-tRNA, (iv) 30 S · IF-1,2,3 · GTP · fMet-tRNA + mRNA → 40 S 1 + IF-3, (v) 40 S 1 + 50 S → 76 S 1 + IF-1,2 + GDP.

Controlled Cleayage of Phage R17 RNA within the Virion by Treatment with Ascorbate and Copper (II)

A method is described for the degradation of R17 phage RNA in situ using vitamin C and copper (II). The procedure permits the controlled cleavage of the phage genome without affecting in any gross way the properties of the capsid proteins nor the secondary structure of the RNA. Losses of infectivity of the order of 6–7 logs were readily obtained without causing any detectable changes in the attachment capacity of phage nor in the structural integrity of the virion. Studies on the effect of oxygen, catalase, and superoxide dismutase on this reaction suggested that hydroxyl radicals generated from H2O2 may be the reactive species responsible for the cleavage of phage RNA.

Identification and RNA-binding properties of an initiation factor capable of relieving translational inhibition induced by heme deprivation or double-stranded RNA

An initiation factor from rabbit reticulocytes can overcome the block in initiation of protein synthesis occurring in reticulocyte lysates when exogenous hemin is not present, or when double-stranded RNA is added. This factor has been identified with IF-MP, an initiation factor capable of forming ternary complexes with GTP and methionyl-tRNA F. Initiation factor IF-M3 by itself is unable to overcome the block in initiation, but appears to stimulate this activity of IF-MP. IF-MP binds to single-stranded R17 RNA as well as to double-stranded RNA, while IF-M3 only binds to double-stranded RNA. The protein synthetic activity of IF-MP is sensitive to N-ethylmaleimide, but its ability to bind RNA is resistant.

Resistance of bacterial protein synthesis to double-stranded RNA

Double-stranded RNA fails to inhibit the formation of translation initiation complexes on R17 bacteriophage RNA, overall synthesis of R17 proteins, or the ability of bacterial initiation factor IF-3 to prevent the association of 30S and 50S ribosomal subunits into single ribosomes. Yet, IF-3 can form complexes with double-stranded RNA. However, IF-3 binds to double-stranded RNA with lower apparent affinity than to either R17 RNA or 30S ribosomal subunits; this may explain the resistance of bacterial protein synthesis to double-stranded RNA.

Translation of virus mRNA: Synthesis of bacteriophage PP7 proteins in cell-free extracts from Pseudomonas aeruginosa

The translation of Pseudomonas phage PP7 RNA has been investigated, using cell-free extracts from Pseudomonas aeruginosa, strain 1. S-30 extracts translated PP7 phage RNA quite efficiently, but the isolated ribosomes incubated with an Escherchia coli S-100 supernatant preparation, were even more active. Three distinct products are made which correspond to in vivo labeled PP7 phage polypeptides judged by polyacrylamide-SDS gels. It is suggested that the PP7 genome has at least three cistrons. The major product of in vitro synthesis, synthesized in great excess of the other products, coelectrophoreses with authentic coat protein prepared from labeled phage. Tryptic peptide analysis of this product yielded leucine labeled tryptic peptides very similar to those of PP7 coat protein. It is concluded that this in vitro polypeptide is the product of the coat protein cistron. This product was also efficiently synthesized when PP7 RNA was translated by E. coli ribosomes, but the other PP7 genome products are not made, except occasionally in very small amounts. This result, and the fact that Pseudomonas ribosomes translate PP7 RNA more efficiently than R17 and Qβ RNA, indicates differential cistron recognition between Pseudomonas and E. coli ribosomes or their initiation factors.

Sequence of events in initiation of translation: a role for initiator transfer RNA in the recognition of messenger RNA.

It is shown that initiation of translation involves several steps. (i) Binding of fMet-tRNA(fMet) to the bacterial 30S ribosomal subunit in the absence of messenger RNA, yielding a 34S complex. This binding is rapid and dependent on initiation factor 2 but not on initiation factor 3. (ii) Binding of messenger RNA to the 34S complex. This binding is slower and depends on initiation factor 3. If R17 RNA is used as messenger, the resulting complex sediments at 46 S. (iii) Joining of a 50S subunit to yield a complete initiation complex. Binding of fMet-tRNA(fMet) not only precedes, but is necessary for, correct binding of messenger RNA to ribosomes. Thus, initiator tRNA may play an active role in the selection of initiation sites in messenger RNA.

Inhibition of Synthetic and Natural Messenger Translation: I. PURIFICATION AND PROPERTIES OF A PROTEIN ISOLATED FROM ESCHERICHIA COLI MRE 600 RIBOSOMES

Abstract An acidic protein isolated from the 1.0 m NH4Cl wash of Escherichia coli MRE 600 ribosomes was found to inhibit translation of R17 RNA or poly(U) and initiation complex formation with R17 RNA. The effect of this protein on poly(U) translation was used as the basis of a simple and specific assay for its purification. The factor was purified to homogeneity by chromatography on DEAE-cellulose, hydroxyapatite, and Sephadex G-150, and its molecular weight was determined to be 69,000. Factor activity was rapidly lost above 50°, and was not inactivated by treatment with p-hydroxymercuribenzoate. An affinity of the purified protein for RNA was shown by the formation of an RNA-protein complex which could be retained on Millipore filters. On the basis of molecular weight, mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and inhibitory activity in R17 RNA and poly(U) translation, the factor was identified as subunit I of Qβ replicase.

Inhibition of Synthetic and Natural Messenger Translation: II. SPECIFICITY AND MECHANISM OF ACTION OF A PROTEIN ISOLATED FROM ESCHERICHIA COLI MRE 600 RIBOSOMES

Abstract The effect of subunit I, an acidic protein isolated from the 1.0 m NH4Cl wash of Escherichia coli MRE 600, on the translation of synthetic and natural mRNA was investigated. In addition to R17 RNA and poly(U) translation, subunit I strongly inhibited amino acid incorporation directed by synthetic polynucleotides containing a high proportion of pyrimidines. Inhibition of translation was independent of the time of addition of this factor to the reaction. The amount of inhibition by subunit I of either poly(U) or R17 RNA translation was a sensitive function of the messenger concentration in the assay. A stable complex of subunit I and ribosomes was isolated and was found to have reduced activity in messenger translation. No interaction of subunit I with IF-3 could be demonstrated either in messenger translation or in dissociation of single ribosomes. Endogenous E. coli mRNA translation and ApUpG-dependent binding of f[14C]Met-tRNA to ribosomes were unaffected by this protein.

Purification and Properties of Chain Initiation Factor 3 from T4-infected and Uninfected Escherichia coli MRE 600: STIMULATION OF TRANSLATION OF SYNTHETIC AND NATURAL MESSENGERS

Abstract The requirement for chain initiation factor 3 in the translation of synthetic polynucleotides lacking an ApUpG codon was examined. Highly purified IF-3 stimulates amino acid incorporation 6- to 10-fold in the presence of 4 µg of poly(U). Based on this observation, a simple and specific assay was developed for the purification of IF-3 from the 1.0 m NH4Cl wash of Escherichia coli MRE 600 ribosomes. At all steps of purification, IF-3 activity detected by the poly(U) assay coincided with that determined by R17 RNA-dependent lysine incorporation. The purified factor was required for maximal rates of amino acid incorporation with poly(U), poly(A), and other synthetic polynucleotides. Samples of purified IF-3 isolated from uninfected and T4-infected E. coli MRE 600 migrated as one major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with a molecular weight of 22,600. Both standard IF-3 and T4-IF-3 were found to promote the translation of R17 RNA and late T4 RNA with equal efficiency.