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PubMed1993

Template-directed and template-free RNA synthesis by Q beta replicase.

In the absence of extraneously added templates, Q beta replicase produces different RNA species after long lag times spontaneously in vitro. The sequences of the spontaneous products are short (30 to 45 nucleotides) and bear little sequence relation to one another and no detectable sequence homology to Q beta virus RNA or to the host. Their replication rates are much lower than those of optimized products. Incubation without template in long closed capillaries produces after long lag times many separate RNA growth foci with heterogeneous kinetics. The template-free reaction is strongly dependent on the conditions: lowering the enzyme or the triphosphate concentrations abolishes the template-free RNA synthesis without affecting the template-dependent synthesis. An explanation of the emerging RNA species in template-free reactions by residual RNA contaminants in the incubation mixture is very unlikely in the light of the experimental evidence; however, the experimental evidence is fully compatible with a de novo mechanism (which may include instruction by non-replicatable oligonucleotides).

Artifacts↗
PubMed1979

RNA replication: required intermediates and the dissociation of template, product, and Q beta replicase.

Replication complexes containing only one molecule of Q beta replicase and one strand of midivariant RNA (MDV-1 RNA) template were prepared by incubating the replicase with an excess of MDV-1 (-) RNA. In the presence of excess minus strands, these monoenzyme replication complexes were shown to synthesize essentially pure MDV-1 (+) RNA in both the first and second cycles of replication. When an equivalent concentration of mutant MDV-1 (-) RNA was added to this reaction before completion of the first cycle of replication, only wild-type MDV-1 (+) RNA was produced in the first cycle, but both mutant and wild-type MDV-1 (+) RNA were produced in the second cycle of replication. These results demonstrate that a monoenzyme complex is competent to synthesize RNA and, therefore, that a multienzyme replication complex is not a necessary intermediate of replication. The data also imply that after the completion of chain elongation, the product strand is released from the replication complex and that the template and the replicase then dissociate.

Coliphages↗
PubMed1991

On the nature of spontaneous RNA synthesis by Q beta replicase.

Numerous RNA species of different length and nucleotide sequence grow spontaneously in vitro in Q beta replicase reactions where no RNA templates are added deliberately. Here, we show that this spontaneous RNA synthesis by Q beta replicase is template directed. The immediate source of template RNA can be the laboratory air, but there are ways to eliminate, or at least substantially reduce, the harmful effects of spontaneous synthesis. Solitary RNA molecules were detected in a thin layer of agarose gel containing Q beta replicase, where they grew to form colonies that became visible upon staining with ethidium bromide. This result provides a powerful tool for RNA cloning and selection in vitro. We also show that replicating RNAs similar to those growing spontaneously are incorporated into Q beta phage particles and can propagate in vivo for a number of phage generations. These RNAs are the smallest known molecular parasites, and in many aspects they resemble both the defective interfering genomes of animal and plant viruses and plant virus satellite RNAs.

Air↗
PubMed1989

Q beta replicase: mapping the functional domains of an RNA-dependent RNA polymerase.

We have localized a functional region of the RNA bacteriophage Q beta replicase following an extensive mutational analysis. Using the method of oligonucleotide linker-insertion mutagenesis, we specifically introduced mutations into a cloned DNA copy of the Q beta replicase gene so that the resulting replicase products would putatively contain small amino acid insertions. In a selective phenotypic assay, we screened mutant replicases for RNA-directed replication activity in vivo. Analysis of 37 different mutant clones indicated that Q beta replicase can accept amino acid substitutions and insertions at several sites at the amino and carboxy termini without abolishing functional activity in vivo or in vitro. However, disruption within the internal amino acid sequence resulted almost exclusively in nonfunctional enzyme. The results suggest that the central region of the replicase protein contains a rigid amino acid composition that is required for replicase function, whereas the amino and carboxy termini are much more receptive to small amino acid insertions and substitutions. These experiments should further enable us to analyze the coding function of the Q beta replicase gene independently of other phage RNA functions contained within this nucleotide region.

Amino Acids↗
PubMed1995

The mutant distribution of an RNA species replicated by Q beta replicase.

The mutant spectrum of an RNA species that is replicated by Q beta replicase, MNV-11, was investigated by retrotranscribing the RNA to DNA and cloning it into plasmids. The sequences of several cDNA clones of MNV-11 populations amplified by Q beta replicase under various conditions were determined and compared. A surprisingly broad mutant distribution was found: the consensus sequence never made up more than 40% of the total population and was accompanied by many mutants. Most mutants had several base exchanges, insertions and/or deletions; up to nine of the total 86 nucleotides were changed. The mutants found had replication rates comparable to that of the wild-type and were thus enriched in the population by selection forces. When the growth conditions were changed, the mutant distribution centre was shifted. The published consensus sequence of MNV-11 did not have the highest growth rate of the mutants, but was rather the best adapted to the various selection forces governing the growth phases the replicating RNA went through, i.e. it had found an optimal compromise between the rates of overall replication, enzyme binding and double strand formation.

Base Sequence↗
PubMed1995

Comparison of amplified Q beta replicase and PCR assays for detection of Mycobacterium tuberculosis.

Because of the long time required to isolate Mycobacterium tuberculosis in culture, there is an acute need for simple rapid methods for direct detection of M. tuberculosis from human sputum specimens. We have developed and characterized quantitative manual Q beta replicase and PCR assays for M. tuberculosis. The Q beta replicase assay was based on reversible target capture of M. tuberculosis 23S rRNA followed by amplification of a replicatable detector probe with Q beta replicase. For PCR assays, primers generating a 370-bp amplification product from the IS6110 insertion element were used in combination with a control plasmid containing an internal deletion in the IS6110 amplicon. Serial dilutions of M. tuberculosis were spiked into sputum and subjected to digestion and decontamination with N-acetyl-L-cysteine and NaOH. Assay conditions were optimized for hybridization and sample processing chemistries in order to maximize sample utilization. Following assay optimization, the sensitivities of the Q beta replicase and PCR assays of spiked sputum samples were 0.5 and 5.0 CFU per assay reaction, respectively. The effects of sputum matrix on each assay were examined by testing 20 patient sputum samples which had been cultured for M. tuberculosis. The culture-positive samples included smear-positive and smear-negative samples. The results of the Q beta replicase assay were not inhibited by sputum and were in 100% agreement with those of culture, including detection of 10 culture-positive specimens. However, using an internal control plasmid coamplified with each PCR as an indicator, we detected PCR inhibition in 9 of 20 samples tested. Decreasing the amount of sample assayed in the PCR 24-fold alleviated the inhibitory effects in all but two specimens, one of which was culture positive. The decreased sample utilization also resulted in a false-negative result with a third specimen which was culture positive for M. tuberculosis. Quantitative smear results and QB replicase assay estimates of the number of organisms present in these specimens were in close agreement. The QB replicase assay performed well in comparison with both culture and PCR and should offer a rapid means for detecting and controlling infection due to M. tuberculosis.

Bacteriological Techniques↗
PubMed1983

Localization of the Q beta replicase recognition site in MDV-1 RNA.

Fragments of MDV-1 RNA (a small, naturally occurring template for Q beta replicase) that were missing nucleotides at either their 5' end or their 3' end were still able to form a complex with Q beta replicase. By assaying the binding ability of fragments of different length, it was established that the binding site for Q beta replicase is determined by nucleotide sequences that are located near the middle of MDV-1 RNA. Fragments missing nucleotides at their 5' end were able to serve as templates for the synthesis of complementary strands, but fragments missing nucleotides at their 3' end were inactive, indicating that the 3'-terminal region of the template is required for the initiation of RNA synthesis. The nucleotide sequences of both the 3' terminus and the central binding region of MDV-1 (+) RNA are almost identical to sequences at the 3' terminus and at an internal region of Q beta (-) RNA.

Base Sequence↗
PubMed1991

Self-catalysed affinity labeling of Q beta replicase.

The spatial neighbourhood of the active center of Q beta replicase can be selectively modified by the method of self-catalysed affinity labeling. In the template-directed, mainly intramolecular enzymatic catalysis, the product [32P]GpG becomes specifically attached to the beta subunit. Using limited digestion of the radioactively labeled polypeptide by cyanogen bromide or N-chlorosuccinimide, we have mapped the attachment site to the region of subunit beta between Trp93 and Met130. Under our reaction conditions, Lys95 is the amino acid most likely to be modified, suggesting that Lys95 lies near the nucleotide binding site in the active center.

Affinity Labels↗
PubMed1996

Single molecule detection of RNA reporter probes by amplification with Q beta replicase.

The addition of target-specific probe sequences within MDV RNA, an otherwise efficient template for Q beta replicase, generally resulted in RNA molecules with inferior replication properties, including reduced replication rates and poor sensitivities. We have discovered that the replication characteristics of MDV RNA molecules with internally placed probe sequences are dramatically affected by short RNA sequences (spacer elements) at the 5' and 3' ends of the probe sequence. For a given probe sequence, the sequences of the flanking spacer elements effected replication sensitivity by six orders of magnitude and replication rate by three fold. By taking advantage of spacer elements, internal MDV probes were developed that permitted the reproducible, real time, fluorescence detection of a single RNA molecule in less than 25 min through amplification with Q beta replicase. RNA structural analysis of such probes suggested that the spacer elements functioned by allowing the RNA to fold in a way which substantially maintained the tertiary structure of the MDV domain. MDV reporter probes with suitable replication properties were obtained from libraries of RNA molecules in which the probe sequence was flanked by many different spacer elements (generated by random nucleotide synthesis). We demonstrated that this is a general method for developing RNA reporter molecules which are rapidly and reproducibly amplified by Q beta replicase, even from a single molecule.

Base Sequence↗
PubMed1996

RNA replication by Q beta replicase: a working model.

Two classes of RNA ligands that bound to separate, high affinity nucleic acid binding sites on Q beta replicase were previously identified. RNA ligands to the two sites, referred to as site I and site II, were used to investigate the molecular mechanism of RNA replication employed by the four-subunit replicase. Replication inhibition by site I- and site II-specific ligands defined two subsets of replicatable RNAs. When provided with appropriate 3' ends, ligands to either site served as replication templates. UV crosslinking experiments revealed that site I is associated with the S1 subunit, site II with elongation factor Tu, and polymerization with the viral subunit of the holoenzyme. These results provide the framework for a three site model describing template recognition and product strand initiation by Q beta replicase.

Base Sequence↗
PubMed1980

Q beta replicase containing wild type and mutant tufA and tufB gene.

The protein synthesis elongation factor EF-Tu, complexed with EF-Ts, forms part of Q beta RNA replicase. In an effort to determine its function in the RNA synthesis reaction, we have developed procedures which allow us to replace the endogenous EF-Tu in purified Q beta replicase with EF-Tu from a variety of sources. In this communication we report purification of EF-Tu from strains containing (a) a wild type tufA gene only, (b) a kirromycin-resistant mutant tufA gene only, and (c) a kirromycin-resistant mutant tufA gene and a mutant tufB gene which codes for EF-Tu that does not bind ribosomes. When each of these EF-Tu preparations is inserted in Q beta replicase, the wild type tufA gene product and and the tufB gene product function appearently normally, but the kirromycin-resistant tufA gene product causes the formation of an altered enzyme. The Q beta replicase containing kirromycin-resistant EF-Tu is unstable; it is rapidly inactivated in the reaction mixture, even at temperatures as low as 20 degrees C. This property results in an apparent increase in template specificity; while wild type Q beta replicase will transcribe poly(C) and other synthetic RNA species, the mutant enzyme will do so only in the presence of Mn2+, which reduces template specificity. The kirromycin-resistant Q beta replicase will also transcribe Q beta RNA. The results imply that EF-Tu is involved in maintenance of enzyme structure, which, in turn, is implicated in template specificity.

Anti-Bacterial Agents↗
PubMed1991

Efficient templates for Q beta replicase are formed by recombination from heterologous sequences.

A very efficient replicase template has been isolated from the products of spontaneous RNA synthesis in an in vitro Q beta replicase reaction that was incubated in the absence of added RNA. This template was named RQ135 RNA because it is 135 nucleotides in length. Its sequence consists entirely of segments that are homologous to ribosomal 23 S RNA and the phage lambda origin of replication. The sequence segments are unrelated to the sequence of Q beta bacteriophage genomic RNA. Nonetheless, this natural recombinant is replicated in vitro at a rate equal to the most efficient of the known Q beta RNA variants. Apparently, the structural properties that ensure recognition of an RNA template by Q beta replicase are not confined to viral RNA, but can appear as a result of recombination among other RNAs that usually occur in cells.

Base Sequence↗
PubMed1982

Q beta replicase containing altered forms of ribosomal protein S1.

Transcription of Q beta RNA replicase requires that the host-encoded Escherichia coli ribosomal protein S1 be present as a subunit of the replicase. To determine whether the activities of S1 in protein synthesis are operational in Q beta RNA transcription, we formed altered replicase enzymes by reconstituting replicase lacking S1 (R(-S1)) with modified S1 species whose properties in nucleic acid binding and protein synthesis had been previously established. S1 derivatized with N-ethylmaleimide reconstitutes a modified replicase that is 81% as active as replicase reconstituted with unmodified S1. S1 derivatized with N-ethylmaleimide and unmodified S1 bind with comparable affinity to R(-S1) (1 X 10(8) M-1). These results indicate that the helix-unwinding properties of S1, which are known to be inactivated by N-ethylmaleimide modification, are not required for Q beta RNA transcription and that the sulfhydryl-derivatized region of S1 is not utilized in replicase subunit contacts. In contrast with its established ability to replace E. coli S1 on the ribosome, Caulobacter crescentus S1 does not reconstitute Q beta RNA transcription activity when added to R(-S1). Our results suggest this inactivity may be due to poor binding to R(-S1).

Coliphages↗
PubMed1994

Identification of recognition elements on bacteriophage Q beta minus strand RNA that are essential for template activity with Q beta replicase.

In order to identify the structural elements important for the activity of the Q beta minus strand RNA as a template for Q beta replicase, a series of minus strand RNAs with internal or external deletions were prepared by in vitro transcription from suitable expression plasmids. The template activities of the deletion mutants were determined by single-round replication assays using purified replicase holoenzyme or core enzyme (lacking subunit alpha) in vitro. Two elements of RNA structure and/or sequence important for template activity were found. The first is a segment in the 5'-terminal region (map segment 4078 to 4132) containing a potential stem-loop structure, whose sequence was previously recognized to be highly conserved in the small variant MDV-1 RNA and suggested to be involved in its template recognition. The second element is defined by two partially complementary sequence segments in the 3'-terminal region (map positions 557 to 576 and 24 to 35), that appear to be engaged in long-range base-pairing and may form the stem of a large secondary structure domain, whose branches are not necessary for template recognition. The results obtained with replicase holoenzyme and core enzyme were identical within the accuracy of the method. They confirm the absence of any role of S1 protein in the interaction of replicase with minus strand RNA and further emphasize the profound difference in the interactions of replicase with the plus and minus strand.

Allolevivirus↗
PubMed1990

Amplified detection of viral nucleic acid at subattomole levels using Q beta replicase.

A sensitive, amplified assay for HIV-1 pol region RNA was developed using RNA probes which are replicated by the RNA-dependent RNA polymerase, Q beta replicase. A synthetic target RNA was hybridized in cell lysates prepared with guanidine thiocyanate with an RNA reporter probe and four deoxyoligonucleotide "capture" probes. The RNA reporter probe was a recombinant MDV RNA molecule generated by transcription from a cloned cDNA template. Capture probes are synthetic oligonucleotides that are complementary to the target nucleic acid and that bear 3' poly d(A) tails. The ternary hybrids (of target RNA with capture probe and reporter probe) were captured on oligo d(T)-derivatized paramagnetic particles by hybridization with the d(A) tails of the capture probes. Non-hybridized reporter probes were removed by washing and successively eluting and recapturing the ternary hybrids on fresh particles. After three cycles of elution and capture, the hybrids were eluted in a low ionic strength buffer and the MDV RNA reporter probes were amplified directly by Q beta replicase. Amplified product RNA was detected by fluorescence using propidium iodide. The assay detects one femtogram (600 molecules) of a synthetic target RNA containing the pol region of HIV-1. The complete assay takes about 2.5 hours.

Fluorescence↗
PubMed1993

Different mechanisms of recognition of bacteriophage Q beta plus and minus strand RNAs by Q beta replicase.

Our earlier work on the recognition of Q beta plus strand RNA by replicase had shown by RNase degradation and by electron microscopic techniques that specific binding interactions occurred at two internal sites, the S-site and the M-site, but not at the 3'-end, i.e. the site of initiation of synthesis. Using essentially similar methods, we have found now for binding complexes of replicase with the minus strand a completely different pattern, namely considerable terminal binding, whereas binding to internal sites was without detectable specificity. In the case of plus strand complexes, simultaneous binding at the two internal sites and at a terminal site could be demonstrated by electron microscopy after initiation of RNA synthesis in the presence of host factor, GTP and ATP. A variant plus strand RNA containing a 490 nucleotide duplication near the 5'-end resulted in similar double-looped complexes, however with an elongated free arm, showing that the protein-bound terminal site was the 3'-end of the RNA. Interestingly, the same two-looped structures were also found for complexes consisting of plus strand RNA and host factor without replicase. This suggests that the role of the host factor on the plus strand template is to bring the 3'-end into the proximity of the S-site/M-site domain, where replicase can initiate on it. In contrast, the 3'-end of the minus strand appears to be directly available to the enzyme.

Coliphages↗
PubMed1980

Modification of cytidines in a Q beta replicase template: analysis of conformation and localization of lethal nucleotide substitutions.

The solution conformation of MDV-1( + ) RNA, a small RNA template replicated autocatalytically in vitro by Q beta replicase, was investigated with sodium bisulfite, a reagent that selectively converts single-stranded cytidines to uridines. The reactivity of 45 of the 76 cytidines in MDV-1( + ) RNA was determined by nucleotide sequence analysis. Only 14 of these 45 cytidines were converted to uridine. Treatment of the RNA with methoxyamine, another single-strand-specific cytidine modification reagent, gave results in good agreement with the bisulfite data. The limited reactivity of MDV-1 ( + ) RNA with these reagents indicates that it is a highly structured molecule. A secondary structure consistent with the chemical modification data is proposed. Modification of MDV-1 ( + ) RNA by bisulfite renders it inactive as a template for RNA replication. This inactivation and the modification of the cytidines at the 3' end of the molecule occur at very similar rates. By using a short complementary RNA "mask" to protect just these cytidines, we demonstrated that the loss of activity resulted from their modification. This implies that one or more of the cytidines in the 3'-terminal sequence is required for template activity and that changes within this sequence can have lethal consequences. The effects of modification elsewhere in the sequence are discussed.

Base Sequence↗
PubMed1997

Detection of Mycobacterium tuberculosis directly from sputum by using a prototype automated Q-beta replicase assay.

We have adapted an assay for the direct detection of Mycobacterium tuberculosis using a prototype automated instrument platform in which probes are amplified with Q-beta replicase. The assay was based on amplification of specific detector probe following four cycles of background reduction (reversible target capture) in a closed disposable pack. The assay signal was the time required for fluorescence to exceed background levels (response time [RT]). RT was inversely related to the number of M. tuberculosis rRNA target molecules in the sample. Equivalent signals and noises were observed in assays containing either sputum or buffer. All mock samples containing > or = 10 CFU of M. tuberculosis responded in the assay (average RT, 13.91 min), while most (83%) samples containing as many as 10(7) CFU of Mycobacterium avium gave no response during a 25-min amplification reaction. The samples containing M. avium which did respond had an average RT of 17.04 min. Seventy-five percent (167 of 223) of samples containing no target gave no responses; the remaining 25% had an average RT of 15.53 min. Eighty-three frozen sputum samples were tested to develop a candidate cutoff RT for the assay prior to more extensive clinical testing. After resolution of discrepant results and with a 14-min RT cutoff, 30 of 38 M. tuberculosis-positive samples were positive by the assay; 1 of 45 negative samples responded within 14 min. Assay sensitivity, specificity, and positive and negatives predictive values in this pilot study were 79, 98, 97, and 85%, respectively.

DNA Probes↗