Qβ RNA Research Articles

A sequence of 83 nucleotides containing the replicase cistron ribosome binding site of phage Qbeta RNA.

70‐S initiation complexes of Escherichia coli ribosomes and 32P‐labelled Qβ RNA were digested with T1 ribonuclease at 0°C, and the ribosome‐protected Qβ initiator fragments resolved by poly‐acrylamide gel electrophoresis. The sequence of one of the fragments (83 nucleotides long) has been determined. It begins with the sequence of 32 nucleotides already determined for the ribosome binding site of the replicase cistron. The sequence of the remaining 51 nucleotides establishes the order of a further 17 amino acids in the phage‐coded replicase subunit. Seven out of the first 11 amino acids in the replicase subunits of phages MS2 and Qβ are identical.

The inhibition of nucleic acid-binding proteins by aurintricarboxylic acid

Qβ replicase, 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 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.

Cell-free protein synthesis resulting in active phage Qbeta replicase.

SEVERAL steps in the replication of the small phage Qβ can be reconstructed under cell-free conditions. Net synthesis of biologically active RNA “plus” strands has been achieved in vitro using RNA from virus particles as a template and RNA dependent RNA polymerase or “replicase” from infected E. coli cells1. This reaction proceeded through complementary RNA or “minus” strands as intermediate templates2. The plus strand-dependent reaction required a “host factor” HF I in addition to purified replicase3–6,7. Furthermore, three of four phage coded proteins can be synthesized in vitro using Qβ RNA as an mRNA and an extract from uninfected E. coli as a protein synthesizing system8,9. Coat protein and the phage specific subunit of replicase (R protein) when synthesized in vitro became bound to Qβ RNA8,10 as would be expected for the authentic proteins. Cell-free synthesis of enzymatically active Qβ replicase, however, has not been reported so far.

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The effects of natural and synthetic polyribonucleotides on both the polycytidylic acid-dependent polyguanylic acid polymerase (poly (G) polymerase) and Qβ replicase reactions have been investigated. Among the polymers examined, poly (U) and Qβ RNA strongly inhibited the poly (G) polymerase, but f2 RNA, E. coli tRNA, poly (A) did not. Poly (U) was found to compete with poly (C) for the enzyme, while Qβ RNA did not compete with poly (C), but reduced the Vmax, suggesting that Qβ RNA affects the poly (G) polymerase activity noncompetitively. Inhibition by Qβ RNA of the poly (G) polymerase was restored by the addition of HFI. The degree of recovery depended upon the ratio of HFI to Qβ RNA. Complete restoration of the poly (G) polymerase activity was attained at a molar ratio of 2 to 10 HFI molecules per molecule of Q/8 RNA. Poly (C) was found to inhibit the Qβ replicase activity noncompetitively. Similar but somewhat less strong inhibition occurred on the addition of poly (U), poly (A), poly (A+2U), and poly (I). Following kinetic studies, it was concluded that the poly (C) binding site was not identical with that of Qβ RNA.

Replication of RNA viruses: Specific binding of the Qβ RNA polymerase to Qβ 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 Mg 2+ 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 Mg 2+ 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.

Replication of RNA viruses: Analysis by liquid polymer phase partition of the binding of Qβ RNA polymerase to nucleic acid

The number of RNA sites bound by the Qβ RNA polymerase and the affinity of the enzyme for different RNA sites was measured by equilibrium partition of enzyme and enzyme-nucleic acid complexes between two liquid polymer phases. At 0 °C and in the absence of other components required for RNA synthesis the enzyme bound to many regions of Qβ RNA, f2 RNA, 16S rRNA, double-stranded RNA, and circular DNA. Under conditions of enzyme excess, the maximum numbers of enzyme molecules bound per molecule of Qβ RNA, f2 RNA, and 16S rRNA were 32, 26, and 12, respectively. The enzyme bound to most, if not all, of the Qβ RNA sites with the same affinity. Nevertheless, the association constant for enzyme binding to Qβ RNA was more than 10-fold greater than for binding to f2 RNA over a wide range of salt concentrations.

Resynchronization of RNA synthesis by coliphage Qβ replicase at an internal site of the RNA template

In previous work Qβ replicase has been used to synthesize labelled 5′ terminal segments of Qβ plus or minus strands of defined length. A procedure has now been developed which allows resynchronization of Qβ replicase at an internal position and synthesis of a labelled minus-strand segment complementary to the coat cistron ribosome binding site and the intercistronic region between the A 2 (maturation) and the coat cistron. Resynchronization is accomplished by binding a ribosome to Qβ RNA and allowing Qβ replicase to initiate and elongate up to the ribosome, using unlabelled ribonucleoside triphosphates. The ribosome is dissociated by EDTA treatment and the EDTA is removed. The replicating complex remains functional after this treatment, and addition of labelled substrates leads to synchronized elongation. The radioactive part of the product recovered after a short elongation period with labelled substrates was shown to be complementary to the coat protein ribosome binding site.

A sequencing algorithm for Qβ RNA

A computer algorithm for sequencing bacteriophage Qβ RNA is described. It uses data on pancreatic and T1 ribonuclease digested fragments which were obtained by Weissmann, Billeter and collaborators from synchronized synthesis of Qβ RNA in vitro. Conventional methods of sequences reconstruction from fragment data require many man hours and may miss multiple solutions. It is shown that within the limits of experimental accuracy many multiple solutions may occur and the algorithm described here reconstructs all solutions exhaustively and efficiently. The algorithm has been successfully tested on artificial data as well as on actual data for the first 175 nucleotide segment of Qβ RNA.

Translation of bacteriophage R17 and Qβ 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-E 3 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.

Host proteins in the replication of bacteriophage RNA.

In recent years, the bacteriophage Qβ RNA polymerase reaction has become a highly characterized system for studying the synthesis of genetic material. An outstanding feature of this reaction is the actual replication of infectious bacteriophage RNA, as first shown by Spiegelman et al. (1). All of the reaction components have now been isolated as discrete species: (a) The enzyme, containing four proteins. (b) A host RNA binding factor, composed of a single protein. (c) A basic protein factor, of which there are many active species. (d) Qβ RNA. (e) Q β RNA complementary strand.

Specific recognition of non-initiator regions in RNA bacteriophage messengers by ribosomes of Bacillus stearothermophilus

The interaction between ribosomes of Bacillus stearothermophilus and the RNA genomes of R17 and Qβ bacteriophage has been studied. Whereas Escherichia coli ribosomes can initiate the synthesis of all three RNA phage-specific proteins in vitro, ribosomes of B. stearothermophilus were previously shown to recognize only the A (or maturation) protein initiation site of f2 or R17 RNA. Under these same conditions, a Qβ region is bound and protected from nuclease digestion. Qβ RNA, however, does not direct the synthesis of any formylmethionyl dipeptide in the presence of B. stearothermophilus ribosomes, nor does the binding of either this Qβ region or the R17 A protein initiation site to these ribosomes show the same fMet-tRNA requirement for recognition of initiator regions as that previously established with E. coli ribosomes. Analysis of a 38-nucleotide sequence in the protected Qβ region reveals no AUG or GUG initiator codon. These observations suggest that messenger RNA may be recognized and bound by B. stearothermophilus ribosomes quite independently of polypeptide chain initiation. Binding experiments using R17 RNA and mixtures of components from B. stearothermophilus and E. coli ribosomes confirm the conclusion drawn by Lodish (1970 a) that specificity in the selection of authentic phage initiator regions by the two species resides in the ribosomal subunit(s). However, anomalous attachment of B. stearothermophilus ribosomes to R17 RNA, which is observed upon lowering the incubation temperature of the binding reaction, is clearly a property of the initiation factor fraction. The results are discussed with respect to current ideas on the role of ribosomes and initiation factors in determining the specificity of polypeptide chain initiation.

Estimation of the double-helical content in various single-stranded nucleic acids by treatment with a single strand-specific nuclease

Single-stranded nucleic acids from various sources were digested at low temperature with nuclease S1-specific for single-stranded polymers. The order of S1-resistance was : (double-stranded DNA) > poly(A) · poly(U) ⪢ ribosomal RNA , transfer RNA, Qβ RNA, MS2 RNA, TMV RNA > f1 DNA ⪢ heat-denatured DNA ⪢ poly(U) . In an experiment using intact f1 DNA and fragmented f1 DNA, the S1 resistance coincided well with the degree of hyperchromicity exhibited by the tested DNA. The S1 resistance was changeable depending on the reaction temperature and the ionic strength in the reaction mixture.

In vitro initiation of coliphage T7 mRNA

The synthesis of two classes of late T7 proteins appears to be directed by several polycistronic messenger RNAs. To understand how they are translated in appropriate order during phage infection, we have isolated late mRNA from T7-infected cells and mRNA that was synthesized in vitro with the T7 RNA polymerase and T7 DNA. Using a purified cell-free system in which ribosomal interaction with the message is restricted to the authentic phage initiation sites, we find that late T7 mRNA directs the synthesis of two initiation dipeptides, formylmethionine-alanine and formylmethionine-serine. These dipeptides account for 80% of the incorporated fMet and are synthesized in systems derived from both uninfected and infected female Escherichia coli cells. However, ribosomes isolated from T7-infected female cells apparently contain mRNA corresponding to these same T7 initiation regions since they also direct the endogenous synthesis of formylmethionine-alanine and formylmethionine-serine. This capability increases as a function of time after infection. In contrast, neither uninfected host ribosomes, ribosomes from T7 am23 (gene 1 mutant lacking T7 RNA polymerase) infected cells, ribosomes from T7-infected male host cells nor ribosomes from cells infected with Qβ RNA phage direct the synthesis of these or other formylmethionine-dipeptides. It is concluded that the ribosomes after T7 infection seem to have a high affinity for the late viral message and that the regions coding for these formylmethionine-dipeptides may represent preferred sites for their entrance on to the message.

Replication of RNA viruses: Structure of a 6 s RNA synthesized by the Qβ RNA polymerase

The in vitro synthesis of RNA catalyzed by the Qβ RNA polymerase has been studied using a single-stranded 6 s RNA template. Whereas Qβ RNA replication results in the synthesis predominantly of single-stranded Qβ RNA, the predominant reaction product of 6 s RNA replication was found to be double stranded. When treated with formaldehyde to dissociate complementary base pairs, 6 s RNA exhibited a decrease in molecular weight as indicated by its slower sedimentation rate and faster electrophoretic mobility. 6 s RNA also exhibited a hyperchromic thermal transition indicative of double-stranded RNA and differing markedly from that of single-stranded RNA. The T m of this transition increased linearly with the logarithm of ionic strength. Renaturation of 6 s RNA below the T m occurred slowly and was also dependent upon ionic strength. Further analysis indicated that a small amount of RNA was in a singlestranded configuration. The frequencies of complementary dinucleotides in purified 6 s RNA were not equal and 10 to 20% of the RNA was hydrolyzed by pancreatic ribonuclease or by Neurospora crassa endonuclease. The electrophoretic mobilities of native and nuclease-treated 6 s RNA indicated that the single-stranded RNA may be present as “tails” at one or both termini of the molecule. Template activity of 6 s RNA was observed only with heat-denatured RNA and was lost upon renaturation.

Replication of viral nucleic acids: II. Inhibition of Qβ replicase by sulfhydryl-blocking reagents

1. Four SH-blocking reagents were used to examine whether the Qβ replicase contains SH groups which have any role in the function of the enzyme. 2. All of these reagents strongly inhibited the replicase activity which was measured with both Qβ RNA and poly(C) as template. 3. Inhibition caused by a mercaptide-forming reagent such as PCMB and HgCl 2 was reversed by incubating the inhibited enzyme with an excess amount of dithiothreitol and 2-mercaptoethanol, suggesting that the replicase contains SH groups which participate, in some fashion, in the function of the enzyme. 4. The PCMB-inhibited enzyme, unlike native enzyme, is unable to synthesize RNA, but still maintains template-binding ability. It was demonstrated that upon the treatment with PCMB, a component of molecular weight of 70 000–85 000 exhibiting template-binding activity dissociates from the enzyme. 5. As a conclusion, it is likely that the SH groups of the replicase must be involved, directly or indirectly, in the binding among the subunits.

A replicating RNA molecule suitable for a detailed analysis of extracellular evolution and replication.

The aim of the present study is to make available a replicating molecule of known sequence. Accordingly, we sought a molecule that has the following properties: (a) replicates in vitro in a manner similar to phage Qbeta RNA; (b) produces antiparallel complementary strands that can be separated from one another; and (c) is small enough to yield its sequence with reasonable effort. We report here the isolation of a replicating RNA molecule that contains 218 nucleotides and possesses the other features desired for a definitive analysis of the replicating mechanism. Despite its small size, this molecule can mutate to previously determined phenotypes. It will, therefore, permit the precise identification of the base changes required to mutate from one phenotype to another in the course of extracellular Darwinian selection experiments.

Characterization of extended sequences around the coat and replicase cistron ribosome binding sites in phage Qβ RNA

Characterization of extended sequences around the coat and replicase cistron ribosome binding sites in phage Qβ RNA

Properties of Q -replicase protein synthesized in a cell-free system.

The largest polypeptide obtained as a product of Qβ RNA‐stimulated cell‐free protein synthesis is the replicase (R) protein. The R polypeptide synthesized in vitro has a chain length of approximately 550 residues and is identical in size to the β chain of purified replicase. From its electrophoretic behavior at pH 8.5, a net charge of ‐27 and a cysteine content of > 15 residues can be deduced. A portion of R polypeptide synthesized in vitro binds to added Qβ RNA and this binding is more resistant to increased ionic strength than the binding of the positively charged coat protein to Qβ RNA. Another portion of the R polypeptide binds to some constituent of the Escherichia coli extract to form a 20‐S complex.

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. As shown by peptide mapping of protein made in vitro and labeled with N-formyl [ 35S]methionyl-transfer RNA, T4 and control ribosomes direct initiation, using f2 RNA, Qβ RNA, early and late T4 mRNA and T7 RNA, of the same polypeptides, in about the same relative proportions. We conclude that there is no change in the specificity of initiation factors after T4 infection; alterations in the amounts of factors are probably not essential either for shut-off of host protein synthesis or for the transition from early to late T4 protein synthesis.

Histone or Bacterial Basic Protein required for Replication of Bacteriophage RNA

IN spite of the apparent simplicity of RNA bacteriophage, several proteins, both phage and bacterial, are required for the synthesis of Qβ RNA in vitro. The polymerase complex alone contains one phage-coded and three host proteins1,2. The specific role of these proteins in Qβ RNA replication is unknown, but because they demonstrate an associative interaction and are always found with active enzyme, it has been suggested that all four contribute to polymerase activity1.