1H NMR study of the interaction of ATP with Escherichia coli RNA polymerase containing in vivo-incorporated Co(II)

The DNA-dependent RNA polymerase containing two intrinsic cobalt ions (Co2-RPase) instead of the naturally occurring zinc was purified from Escherichia coli cells grown in zinc-depleted, cobalt-enriched media. Longitudinal NMR relaxation rates of the H2 and H8 protons of ATP were measured in the absence and presence of up to 92 microM Co2-RPase. No enhancement of the proton relaxation rates was observed in the presence of cobalt-containing enzyme, suggesting that the ATP substrate does not undergo rapid exchange at a site close to either of the intrinsic cobalt ions. This result is in contrast to that previously observed when Co2+ was incorporated into RPase by an in vitro procedure involving partial urea denaturation of the protein.     

Antibodies against the folding domain of histone H5 cross-react with H1(0) but not with H1

Antibodies to the folding domain (residues 22-100) of histone H5 were elicited in rabbits. Analysis of the specificity of these antibodies by enzyme-linked immunoassay and by diazobenzyloxymethyl cellulose transfer techniques revealed that the antibody cross-reacts strongly with intact H5 and histones H1(0)a and H1(0)b purified from ox liver but not with the four core calf thymus, or with high mobility group proteins. We conclude that the globular region of H5 is serologically homologous to that of H1 degrees and suggest that possible functional similarities between the two proteins reside in this region.     

SPT5 stabilization of promoter-proximal RNA polymerase II

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Association of poly(A) polymerase with U1 RNA

Previous studies (Stetler, D. A., and Jacob, S. T. (1984) J. Biol. Chem. 259, 7239-7244) have shown that poly(A) polymerase from adult rat liver (liver-type) is structurally and immunologically distinct from the corresponding rat hepatoma (tumor-type) enzyme. When hepatoma 7777 (McA-RH 7777) cells were labeled with [32P]inorganic phosphate, followed by immunoprecipitation with anti-hepatoma poly(A) polymerase antibodies and analysis of the RNAs in the immunoprecipitate, only one labeled small nuclear RNA corresponding to U1 RNA was found. Preimmune sera did not form a complex with U1 RNA. Hepatoma poly(A) polymerase antisera did not immunoprecipitate U1 RNA or any other small nuclear RNA from a cell line (H4-11-EC3) which does not contain the tumor-type poly(A) polymerase. Immunoblot analysis of hepatoma 7777 nuclear extract or purified poly(A) polymerase with anti-ribonucleoprotein antisera did not show any cross-reactivity of the latter sera with poly(A) polymerase. The major RNA immunoprecipitated from the hepatoma nuclear extracts using trimethyl cap (m3G) antisera corresponded to the RNA immunoprecipitated with poly(A) polymerase antisera. These data indicate that U1 RNA is closely associated with poly(A) polymerase and suggest the potential involvement of this RNA in the cleavage/polyadenylation of mRNA precursor.     

RNA polymerase: structural similarities between bacterial RNA polymerase and eukaryotic RNA polymerase II

Bacterial RNA polymerase and eukaryotic RNA polymerase II exhibit striking structural similarities, including similarities in overall structure, relative positions of subunits, relative positions of functional determinants, and structures and folding topologies of subunits. These structural similarities are paralleled by similarities in mechanisms of interaction with DNA.     

Hepatitis C virus (HCV) NS5A binds RNA-dependent RNA polymerase (RdRP) NS5B and modulates RNA-dependent RNA polymerase activity.

Hepatitis C virus (HCV) NS5B is RNA-dependent RNA polymerase (RdRP), the essential catalytic enzyme for HCV replication. Recently, NS5A has been reported to be important for the establishment of HCV replication in vitro by the adaptive mutations, although its role in viral replication remains uncertain. Here we report that purified bacterial recombinant NS5A and NS5B directly interact with each other in vitro, detected by glutathione S-transferase (GST) pull-down assay. Furthermore, complex formation of these proteins transiently coexpressed in mammalian cells was detected by coprecipitation. Using terminally and internally truncated NS5A, two discontinuous regions of NS5A (amino acids 105-162 and 277-334) outside of the adaptive mutations were identified to be independently essential for the binding both in vivo and in vitro (Yamashita, T., Kaneko, S., Shirota, Y., Qin, W., Nomura, T., Kobayashi, K., and Mkyrakami, S. (1998) J. Biol. Chem. 273, 15479-15486). We previously examined the effect of His-NS5A on RdRP activity of the soluble recombinant NS5Bt in vitro (see Yamashita et al. above). Wild NS5A weakly stimulated at first (when less than 0.1 molar ratio to NS5B) and then inhibited the NS5Bt RdRP activity in a dose-dependent manner. The internal deletion mutants defective in NS5B binding exhibited no inhibitory effect, indicating that the NS5B binding is necessary for the inhibition. Taken together, our results support the idea that NS5A modulates HCV replication as a component of replication complex.     

Higher-level phylogeny of Foraminifera inferred from the RNA polymerase II (RPB1) gene

Macroevolutionary relations among main lineages of Foraminifera have traditionally been inferred from the small subunit ribosomal genes (SSU rDNA). However, important discrepancies in the rates of SSU rDNA evolution between major lineages led to difficulties in accurate interpretation of SSU-based phylogenetic reconstructions. Recently, actin and beta-tubulin sequences have been used as alternative markers of foraminiferal phylogeny and their analyses globally confirm results obtained with SSU rDNA. In order to test new protein markers, we sequenced a fragment of the largest subunit of the RNA polymerase II (RPB1), a nuclear encoded single copy gene, for 8 foraminiferal species representing major orders of Foraminifera. Analyses of our data robustly confirm previous SSU rDNA and actin phylogenies and show (i) the paraphyly and ancestral position of monothalamid Foraminifera; (ii) the independent origin of miliolids; (iii) the monophyly of rotaliids, including buliminids and globigerinids; and (iv) the polyphyly of planktonic families Globigerinidae and Candeinidae. Additionally, the RPB1 phylogeny suggests Allogromiidae as the most ancestral foraminiferal lineage. In the light of our study, RPB1 appears as a valuable phylogenetic marker, particularly useful for groups of protists showing extreme variations of evolutionary rates in ribosomal genes.     

Primer-independent abortive initiation by wheat-germ RNA polymerase B (II)

Highly purified RNA polymerase B (II) from wheat germ catalyses the formation of dinucleoside tetraphosphates from ribonucleoside triphosphates in the absence of an oligonucleotide primer or additional protein factors. The reaction requires bivalent cations such as Mn2+ or Mg2+ and proceeds linearly for several hours. It is strongly inhibited by 1 microgram/ml alpha-amanitin or 2 micrograms/ml heparin. The reaction strictly depends on the addition of a specific linear or circular DNA template, such as the plasmid pSmaF or a DNA fragment containing the gene for nopaline dehydrogenase. Bacteriophage T7 D111 DNA has almost no template activity. The start sites for dinucleotide synthesis on the template are limited. With the DNA fragment containing the gene for nopaline dehydrogenase only pppApA and pppApU are synthesised substantially whereas pppUpU is formed only in trace amounts. No significant dinucleotide synthesis is observed with other ribonucleoside triphosphates either singly or in a combination of two. The various regions of the DNA fragment differ distinctly in template activity.     

The carboxyl-terminal domain of murine H1(0). Immunochemical and partial amino acid sequence comparisons with other H1(0)/H1/H5 histones

The carboxyl-terminal domain of murine H1(0) histone was compared with that of human H1(0), bovine H1(0) and other H1 and H5 histones. Two sets of antibodies were induced by murine H1(0). One set reacted with only the carboxyl-terminal domain of murine H1(0) and preferred the murine over the bovine and human proteins. The second set of antibodies reacted with the globular domain of murine H1(0) and did not distinguish among murine, bovine and human H1(0) species. There were five positions in the first 60 residues of the carboxyl-terminal domain in which the murine H1(0) differed from the human H1(0). In this region, the murine H1(0) had no more than 49% overall homology with other H1 and H5 histones; however, short sequences in the domain were very similar to short sequences that occur in rabbit H1.3, trout H1 and goose or chicken H5. In comparisons based on these and other published data, the carboxyl-terminal domain of H1(0) is found to be more variable among species than is the globular domain; the first two-thirds of the H1(0) carboxyl-terminal domain is largely unique and does not show great overall homology with H1 or H5, whereas the last third is again more conserved. As the first two-thirds of the domain is the only portion where the homology with H5 is less than 50%, it may be responsible for functional differences between H1(0) and H5.     

Histones H1 and H4 of surface-spread meiotic chromosomes

The chromatin conformation of somatic and meiotic chromosomes is, at least in part, a function of electrostatic nucleosome interactions that are mediated by transient acetylation of the histone H4 N-terminal domain and phosphorylation of histone H1. The distribution of those histones in the chromatin of meiotic chromosomes is reported here. Antibodies to testis-specific histone 1, H1t, detect H1t in the chromatin of mouse meiotic prophase chromosomes only after synapsis and synaptonemal complex (SC) assembly is completed and before core separation is initiated. The H1t protein is evenly distributed over euchromatin, heterochromatin and the SC. Antibodies to acetylated lysine residues 5, 12 or 16 of histone H4, indicate that the euchromatin is more acetylated than the centromeric heterochromatin. The pattern is most pronounced for acetylated residue 5 and least for 16. Antibodies to phosphorylated H1 epitopes do not react with chromatin but, instead, recognize the chromosome cores and SCs. Possibly these are not phosphorylated histone H1 epitopes, but SC proteins with similar potentially phosphorylatable sequences such as KTPTK of the synaptic protein Syn1.