On producing more complexity than entropy in replication.
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RNA replication in the bacteriophage Q beta system can, in principle, transmit sequence complexity at a higher rate than it increases entropy. Expanding the variety of nucleotides, through novel base-pair interactions, would move the threshold at which synthesis produces more complexity than entropy away from near equilibrium while accelerating the system approach to equilibrium. A decrease in sequence complexity during polymerization, leading to a many-to-one monomer correspondence with template, cannot be reversed, owing to symmetry restrictions. In terms of the kinetic mechanism, uncertainty associated with the the path of depolymerization yields a path entropy which selectively prolongs the reverse reaction. Together with an elevation in thermodynamic entropy, therefore, there are two possible sources of irreversibility in a physical process. Some implications of kinetic irreversibility are considered in relation to the second law of thermodynamics and to the processing and translation of mRNA. (+info)
A study on the function of the glycine residue in the YGDD motif of the RNA-dependent RNA polymerase beta-subunit from RNA coliphage Q beta 1.
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Q beta replicases in which the Gly residue of the beta-subunit in the motif sequence, YGDD, was replaced with Ala, Ser, Pro, Met, or Val lost their replicase activity in vivo. In an in vitro Mg(2+)-dependent RNA-synthesizing system using poly(rC) or MDV-poly(+) RNA (a derivative of the naturally occurring small RNA that accumulates in the cells during Q beta phage infection) as templates, the lysates from the cells expressing such defective replicases exhibited only 2-6% of the enzyme activity of the lysate from those expressing wild-type replicase. However, the defective replicases, especially A357, with Ala substituted for the Gly, recovered enzyme activity when Mn2+ was added to the reaction mixture. Furthermore, the characteristics of the MDV-poly(+) RNA-dependent RNA synthesis by A357 replicase were similar to those by wild-type replicase in the presence of Mn2+. Gel retardation assay showed that all of the defective replicases could bind MDV-poly(+) RNA. These results suggest that the Gly residue in this motif of Q beta replicase is involved in Mg(2+)-catalyzed polymerization. In the Mn(2+)-catalyzed polymerization, A357 and S357 replicases can act as well as the wild-type replicase. (+info)
Viral Q beta RNA as a high expression vector for mRNA translation in a cell-free system.
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Dihydrofolate reductase (DHFR) mRNA was inserted into Q beta phage RNA instead of its coat protein cistron. Translation of this recombinant mRNA in the Escherichia coli cell-free system resulted in the synthesis of DHFR, which was two orders of magnitude higher than that in the case of translation of the control DHFR mRNA. Additionally, it resulted in a significantly enhanced synthesis of Q beta replicase as compared with its synthesis when the original Q beta RNA was used. (+info)
The expression of nifA in Azorhizobium caulinodans requires a gene product homologous to Escherichia coli HF-I, an RNA-binding protein involved in the replication of phage Q beta RNA.
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We report the characterization of a mutant of Azorhizobium caulinodans, isolated after ethyl methanesulfonate mutagenesis. This Nod+ Nif- Fix- mutant is unable to synthesize 10 of 15 polypeptides normally induced under conditions of nitrogen fixation. By using lacZ fusions it was shown that nifA and nifA-regulated genes were not expressed in this strain. The mutation was complemented by a constitutively expressed nifA gene or by a 1.1-kb DNA fragment from the wild-type strain, whose nucleotide sequence revealed a single open reading frame of 255 bp coding for an 85-amino acid polypeptide. The deduced amino acid sequence is similar to that of HF-I, an RNA-binding protein of Escherichia coli, which is required for replication of bacteriophage Q beta RNA. The similarity can be extended to the function since hfq, the structural gene for HF-I, complemented the A. caulinodans mutant. The corresponding gene in A. caulinodans was termed nrfA (for nif regulatory factor). Inactivation of nrfA in the mutant was due to a missense mutation resulting in the replacement of a cysteine residue by arginine. A null mutant, constructed by disruption of nrfA, exhibited the same phenotype as the missense mutant. Thus, an additional factor can be added to the already complex system of nifA regulation in A. caulinodans. (+info)
Regulation of the Escherichia coli hfq gene encoding the host factor for phage Q beta.
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The host factor (HF-I) for phage Q beta RNA replication is a small protein of 102 amino acid residues encoded by the hfq gene at 94.8 min on the Escherichia coli chromosome. The synthesis rate of HF-I at the exponential-growth phase is higher than at the stationary phase, and it increases concomitantly with the increase in cell growth rate. The intracellular level of HF-I is about 30,000 to 60,000 molecules per cell, the majority being associated with ribosomes as one of the salt wash proteins. Taken together, we suggest that HF-I is one of the growth-related proteins. (+info)
The RNA-binding protein HF-I, known as a host factor for phage Qbeta RNA replication, is essential for rpoS translation in Escherichia coli.
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The rpoS-encoded sigma(S) subunit of RNA polymerase in Escherichia coli is a global regulatory factor involved in several stress responses. Mainly because of increased rpoS translation and stabilization of sigma(S), which in nonstressed cells is a highly unstable protein, the cellular sigma(S) content increases during entry into stationary phase and in response to hyperosmolarity. Here, we identify the hfq-encoded RNA-binding protein HF-I, which has been known previously only as a host factor for the replication of phage Qbeta RNA, as an essential factor for rpoS translation. An hfq null mutant exhibits strongly reduced sigma(S) levels under all conditions tested and is deficient for growth phase-related and osmotic induction of sigma(S). Using a combination of gene fusion analysis and pulse-chase experiments, we demonstrate that the hfq mutant is specifically impaired in rpoS translation. We also present evidence that the H-NS protein, which has been shown to affect rpoS translation, acts in the same regulatory pathway as HF-I at a position upstream of HF-I or in conjunction with HF-I. In addition, we show that expression and heat induction of the heat shock sigma factor sigma(32) (encoded by rpoH) is not dependent on HF-I, although rpoH and rpoS are both subject to translational regulation probably mediated by changes in mRNA secondary structure. HF-I is the first factor known to be specifically involved in rpoS translation, and this role is the first cellular function to be identified for this abundant ribosome-associated RNA-binding protein in E. coli. (+info)
Photo-induced inactivation of viruses: adsorption of methylene blue, thionine, and thiopyronine on Qbeta bacteriophage.
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The adsorption of cationic organic dyes (methylene blue, thionine, and thiopyronine) on Qbeta bacteriophage was studied by UV-visible and fluorescence spectroscopy. The dyes have shown a strong affinity to the virus and some have been used as sensitizers for photo-induced inactivation of virus. In the methylene blue concentration range of 0.1-5 microM and at high ratios of dye to virus (greater than 1000 dye molecules per virion), the dyes bind as aggregates on the virus. Aggregation lowers the efficiency of photoinactivation because of self-quenching of the dye. At lower ratios of dye to virus (lower than 500 dye molecules per virion), the dye binds to the virus as a monomer. Fluorescence polarization and time-resolved studies of the fluorescence support the conclusions based on fluorescence quenching. Increasing the ionic strength (adding NaCl) dissociates bound dye aggregates on the virus and releases monomeric dye into the bulk solution. (+info)
MS2 coat protein mutants which bind Qbeta RNA.
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The coat proteins of the RNA phages MS2 and Qbetaare structurally homologous, yet they specifically bind different RNA structures. In an effort to identify the basis of RNA binding specificity we sought to isolate mutants that convert MS2 coat protein to the RNA binding specificity of Qbeta. A library of mutations was created which selectively substitutes amino acids within the RNA binding site. Genetic selection for the ability to repress translation from the Qbetatranslational operator led to the isolation of several MS2 mutants that acquired binding activity for QbetaRNA. Some of these also had reduced abilities to repress translation from the MS2 translational operator. These changes in RNA binding specificity were the results of substitutions of amino acid residues 87 and 89. Additional codon- directed mutagenesis experiments confirmed earlier results showing that the identity of Asn87 is important for specific binding of MS2 RNA. Glu89, on the other hand, is not required for recognition of MS2 RNA, but prevents binding of QbetaRNA. (+info)