Compositional complementarity between genomic RNA and coat proteins in positive-sense single-stranded RNA viruses

During packaging in positive-sense single-stranded RNA (+ssRNA) viruses, coat proteins (CPs) interact directly with multiple regions in genomic RNA (gRNA), but the underlying physicochemical principles remain unclear. Here we analyze the high-resolution cryo-EM structure of bacteriophage MS2 and show that the gRNA/CP binding sites, including the known packaging signal, overlap significantly with regions where gRNA nucleobase-density profiles match the corresponding CP nucleobase-affinity profiles. Moreover, we show that the MS2 packaging signal corresponds to the global minimum in gRNA/CP interaction energy in the unstructured state as derived using a linearly additive model and knowledge-based nucleobase/amino-acid affinities. Motivated by this, we predict gRNA/CP interaction sites for a comprehensive set of 1082 +ssRNA viruses. We validate our predictions by comparing them with site-resolved information on gRNA/CP interactions derived in SELEX and CLIP experiments for 10 different viruses. Finally, we show that in experimentally studied systems CPs frequently interact with autologous coding regions in gRNA, in agreement with both predicted interaction energies and a recent proposal that proteins in general tend to interact with own mRNAs, if unstructured. Our results define a self-consistent framework for understanding packaging in +ssRNA viruses and implicate interactions between unstructured gRNA and CPs in the process.     

Different base per unit length ratios exist in single-stranded RNA and single-stranded DNA.

A significant difference was found to exist in the number of bases per unit length of single-stranded RNA as compared to single-stranded DNA when single-stranded RNA or DNA molecules of known nucleotide sequence were measured by electron microscopy using a cytochrome spreading technique. Using this technique, single-stranded RNA was found to have 17.5% more bases per unit of length than single-stranded DNA. These ratios were verified using four different denaturing conditions for the RNA: 80% formamide, 80% formamide plus glyoxal, 80% formamide/4M urea and 80% formamide/4M urea plus glyoxal. Molecules ranging in size from 1541 to 5386 nucleotides were examined and the number of bases per unit length was found to vary inversely with micrometer was consistent when RNA and DNA molecules of the same length were compared.     

Semiconservative synthesis of single-stranded RNA by bacteriophage phi 6 RNA polymerase.

The RNA polymerase in the nucleocapsid of Pseudomonas phaseolicola bacteriophage phi 6 transcribed large, medium, and small single-stranded RNA from the viral double-stranded RNA genome by a semiconservative (displacement) mechanism. Approximately 23%, 63%, and 65% of the nucleocapsid particles in the assay mixture synthesized at least one round of large, medium, and small single-stranded RNA molecules, respectively. Some of these particles reinitiated synthesis such that an average of 1.5 large, 33 medium, and 24 small single-stranded RNAs were synthesized from each double-stranded RNA.     

RNA polymerase II subunit composition, stoichiometry, and phosphorylation.

RNA polymerase II subunit composition, stoichiometry, and phosphorylation were investigated in Saccharomyces cerevisiae by attaching an epitope coding sequence to a well-characterized RNA polymerase II subunit gene (RPB3) and by immunoprecipitating the product of this gene with its associated polypeptides. The immunopurified enzyme catalyzed alpha-amanitin-sensitive RNA synthesis in vitro. The 10 polypeptides that immunoprecipitated were identical in size and number to those previously described for RNA polymerase II purified by conventional column chromatography. The relative stoichiometry of the subunits was deduced from knowledge of the sequence of the subunits and from the extent of labeling with [35S]methionine. Immunoprecipitation from 32P-labeled cell extracts revealed that three of the subunits, RPB1, RPB2, and RPB6, are phosphorylated in vivo. Phosphorylated and unphosphorylated forms of RPB1 could be distinguished; approximately half of the RNA polymerase II molecules contained a phosphorylated RPB1 subunit. These results more precisely define the subunit composition and phosphorylation of a eucaryotic RNA polymerase II enzyme.     

ISG20, a new interferon-induced RNase specific for single-stranded RNA,defines an alternative antiviral pathway against RNA genomic viruses

Interferons (IFNs) encode a family of secreted proteins that provide the front-line defense against viral infections. Their diverse biological actions are thought to be mediated by the products of specific but usually overlapping sets of cellular genes induced in the target cells. We have recently isolated a new human IFN-induced gene that we have termed ISG20, which codes for a 3' to 5' exonuclease with specificity for single-stranded RNA and, to a lesser extent, for DNA. In this report, we demonstrate that ISG20 is involved in the antiviral functions of IFN. In the absence of IFN treatment, ISG20-overexpressing HeLa cells showed resistance to infections by vesicular stomatitis virus (VSV), influenza virus, and encephalomyocarditis virus (three RNA genomic viruses) but not to the DNA genomic adenovirus. ISG20 specifically interfered with VSV mRNA synthesis and protein production while leaving the expression of cellular control genes unaffected. No antiviral effect was observed in cells overexpressing a mutated ISG20 protein defective in exonuclease activity, demonstrating that the antiviral effects were due to the exonuclease activity of ISG20. In addition, the inactive mutant ISG20 protein, which is able to inhibit ISG20 exonuclease activity in vitro, significantly reduced the ability of IFN to block VSV development. Taken together, these data suggested that the antiviral activity of IFN against VSV is partly mediated by ISG20. We thus show that, besides RNase L, ISG20 has an antiviral activity, supporting the idea that it might represent a novel antiviral pathway in the mechanism of IFN action.     

RNA polymerase II as a control panel for multiple coactivator complexes.

1999 marks the 30th anniversary of the reported discovery of sigma factor and the bacterial RNA polymerase holoenzyme. In 1994, an RNA polymerase II complex was discovered in yeast - mammalian complexes were subsequently identified. Recent developments regarding the composition and function of RNA polymerase II complexes suggest, however, that the concept of the holoenzyme, as defined in bacteria, might not be relevant to eukaryotes.     

Specificity factor of yeast mitochondrial RNA polymerase. Purification and interaction with core RNA polymerase

We have identified a mitochondrial protein from Saccharomyces cerevisiae which confers the ability to recognize mitochondrial promoters onto a nonspecifically transcribing mitochondrial core RNA polymerase and we have purified this specificity factor 10,700-fold from a whole cell extract. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified fraction followed by elution and renaturation of protein activity shows that the specificity factor is a 43-kDa polypeptide which directs mitochondrial core RNA polymerase to promoters belonging to rRNA-, tRNA-, and protein-encoding genes, as well as to mitochondrial replication origins. Gel filtration and glycerol gradient sedimentation studies indicate that the specificity factor shows little association with core RNA polymerase in the absence of DNA, and that it behaves like a monomeric 43-kDa protein.     

RNA polymerase activity in purified virions of avian reticuloendotheliosis viruses.

An RNA polymerase activity that synthesizes a U-rich RNA hydrogen bonded to a large viral RNA molecule was found in the cores of virions of avian reticuloendotheliosis viruses (REV). The RNA polymerase activity was separable from the DNA polymerase activity of REV virions. The 5'-terminus of the newly synthesized RNA was A. In addition, a tRNA nucleotidyl transferase activity, which added -CpCpA ends to tRNA, appears to be present in the REV virions.     

In vivo genomic footprint of a yeast centromere.

We have used in vivo genomic footprinting to investigate the protein-DNA interactions within the conserved DNA elements (CDEI, CDEII, and CDEIII) in the centromere from chromosome III of the yeast Saccharomyces cerevisiae. The in vivo footprint pattern obtained from wild-type cells shows that some guanines within the centromere DNA are protected from methylation by dimethyl sulfate. These results are consistent with studies demonstrating that yeast cells contain sequence-specific centromere DNA-binding proteins. Our in vivo experiments on chromosomes with mutant centromeres show that some mutations which affect chromosome segregation also alter the footprint pattern caused by proteins bound to the centromere DNA. The results of this study provide the first fine-structure map of proteins bound to centromere DNA in living yeast cells and suggest a direct correlation between these protein-DNA interactions and centromere function.     

Both yeast W double-stranded RNA and its single-stranded form 20S RNA are linear.

Most yeast strains carry a cytoplasmic double-stranded RNA (dsRNA) molecule called W, of 2.5 kb in size. We have cloned and sequenced most of W genome (1), and we proposed that W (+) strands were identical to 20S RNA, a single-stranded RNA (ssRNA) species, whose copy number is highly induced under stress conditions. Recently it was proposed that 20S RNA was circular (2). In this paper, however, we demonstrate that both W dsRNA and 20S RNA are linear. Linearity of W dsRNA is shown by the stoichiometric labelling of both strands of W with 32P-pCp and T4 RNA ligase. The last 3' end nucleotide of both strands is about 70 to 80% C and 20 to 30% A. Linearity of 20S RNA is directly demonstrated by a site-specific cleavage of 20S RNA with RNase H, using an oligodeoxynucleotide complementary to an internal site of 20S RNA. The cleavage produced not one but two RNA fragments expected from the linearity of 20S RNA.