The nature of initiation sites on DNA for the core RNA polymerase

Summary The biological significance of the low level of symmetric and non-specific RNA synthesis catalyzed by the core RNA polymerase devoid of the sigma factor has been analyzed. Shearing of DNA's including T4 DNA markedly increased the template activities with the core enzyme but not with the holoenzyme. This finding suggests that RNA synthesis by the core enzyme increases concomittantly with the production of termini in DNA. Double-stranded circular DNA's such as dv and fd-RFI were found to be inactive as templates for the core enzyme, but were made active by introduction of single-strand nicks with deoxyribonuclease. In contrast, single-stranded circular DNA (X 174) served as a good template for RNA synthesis by the core RNA polymerase. These findings suggest that the sigma factor may activate double-stranded DNA at the promotor sites by creating proper initiation points for RNA synthesis. Partial separation of duplex DNA into single-stranded forms at the promotor sites could be one of the processes in the reaction catalyzed by the holoenzyme containing the sigma factor.     

Elucidating the Role of Microprocessor Protein DGCR8 in Bending RNA Structures

Although conformational dynamics of RNA molecules are potentially important in microRNA (miRNA) processing, the role of the protein binding partners in facilitating the requisite structural changes is not well understood. In previous work, we and others have demonstrated that nonduplex structural elements and the conformational flexibility they support are necessary for efficient RNA binding and cleavage by the proteins associated with the two major stages of miRNA processing. However, recent studies showed that the protein DGCR8 binds primary miRNA and duplex RNA with similar affinities. Here, we study RNA binding by a small recombinant construct of the DGCR8 protein and the RNA conformation changes that result. This construct, the DGCR8 core, contains two double-stranded RNA-binding domains (dsRBDs) and a C-terminal tail. To assess conformational changes resulting from binding, we applied small-angle x-ray scattering with contrast variation to detect conformational changes of primary-miR-16-1 in complex with the DGCR8 core. This method reports only on the RNA conformation within the complex and suggests that the protein bends the RNA upon binding. Supporting work using smFRET to study the conformation of RNA duplexes bound to the core also shows bending. Together, these studies elucidate the role of DGCR8 in interacting with RNA during the early stages of miRNA processing.     

Polyadenylic acid synthesis activity of purified DNA-dependent RNA polymerase from Caulobacter

Characterization of purified DNA-dependent RNA polymerase (EC 2.7.7.6) of Caulobacter crescentus, strain CB15 has led to the conclusion that this enzyme catalyzes poly(A) synthesis in the absence of template. Poly(A) synthetase activity co-purifies with both holoenzyme and core polymerase on DNA-cellulose columns, and core polymerase purified to 98% homogeneity by glycerol gradient centrifugation is still capable of catalyzing poly(A) polymerization. Both RNA synthesis and poly(A) polymerization activities are sensitive to rifampicin. In addition, RNA polymerase purified from partially rifampicin-sensitive mutants exhibits the same partial sensitivity in vitro to the drug in the synthesis of RNA and poly(A). The enzyme used in these studies was prepared by a simple method which allows a high yield of pure RNA polymerase from large batches of exponential cells. The procedure includes high speed centrifugation of cell extracts, DEAE-cellulose column, DNA-affinity chromatography, and low salt glycerol gradient centrifugation. Holoenzyme can be resolved into core and sigma subunit by either DNA-cellulose chromatography or glycerol gradient centrifugation, and the latter step allows recovery of pure sigma factor.     

Sigma factor is not released during transcription in Bacillus subtilis.

The relationship between sigma (sigma) and delta (delta) factors of Bacillus subtilis RNA polymerase has been analyzed during initiation of RNA synthesis. When core enzyme (E) containing delta factor (E delta) binds to DNA, the delta factor is released with the formation of an E-DNA complex. The addition of sigma to the E-DNA complex results in the formation of a stable E sigma-DNA complex which can synthesize RNA upon addition of nucleoside triphosphates. Sigma factor, significantly, is not released from the core during RNA synthesis. These results suggest that delta and sigma factors can act sequentially during initiation of RNA synthesis with delta acting as a DNA recognition factor and sigma acting as an initiation factor. The results do not preclude the possibility that E sigma can initiate RNA synthesis correctly since E sigma alone can bind to DNA and initiate RNA synthesis.     

A noncycling activity assay for the omega subunit of Escherichia coli RNA polymerase.

We describe a new method for quantitatively assaying the omega subunit of Escherichia coli RNA polymerase. The assay is based on the ability of RNA polymerase holoenzyme to catalyze the continuous synthesis of the dinucleotide pApU on a poly[d(A-T)] . poly[d(A-T)] template when supplied with AMP and UTP as substrates. Core enzyme, lacking omega subunit, catalyzed this reaction at a rate less than 1% that of holoenzyme. The omega subunit was not released from the enzyme/DNA complex during dinucleotide synthesis. Using this assay, a titration of a fixed concentration of core enzyme was observed with increasing concentrations of added omega subunit. Below a 1:1 omega:core ratio the measured activity increased linearly with omega concentration, whereas above a 1:1 ratio the activity remained constant. An immediate application of the assay is in determining the concentration of active omega, or equivalently of active holoenzyme, in any RNA polymerase preparation.     

Comparison of the Virion Polymerase of Reovirus with the Enzyme Purified from Reovirus-Infected Cells

Reovirus has in its protein coat an enzyme which catalyzes the net synthesis of the three size classes of virus-specific, single-stranded ribonucleic acid (RNA). For synthesis of 24, 19, and 14S single-stranded RNA, Mn(++) was the preferred divalent cation, and ammonium sulfate at an optimal concentration of 4.2% of saturation was an absolute requirement. During synthesis, the parental double-stranded RNA was conserved in the viral core and the newly synthesized completed RNA chains were released as free RNA. The viral cores synthesizing RNA had properties consistent with the presence of nascent RNA on their outer surface. The enzyme-template complex from the infected cells described in an earlier paper was comprised of viral cores already active in the in vivo synthesis of single-stranded RNA. This pool of viral cores was newly made during infection, and exponential increase in the number of particles in this pool, as detected by the increase in enzymatic activity, occurred 2 hr earlier than that in mature virus.     

Crystal Structure of the Human Primase

DNA replication in bacteria and eukaryotes requires the activity of DNA primase, a DNA-dependent RNA polymerase that lays short RNA primers for DNA polymerases. Eukaryotic and archaeal primases are heterodimers consisting of small catalytic and large accessory subunits, both of which are necessary for RNA primer synthesis. Understanding of RNA synthesis priming in eukaryotes is currently limited due to the lack of crystal structures of the full-length primase and its complexes with substrates in initiation and elongation states. Here we report the crystal structure of the full-length human primase, revealing the precise overall organization of the enzyme, the relative positions of its functional domains, and the mode of its interaction with modeled DNA and RNA. The structure indicates that the dramatic conformational changes in primase are necessary to accomplish the initiation and then elongation of RNA synthesis. The presence of a long linker between the N- and C-terminal domains of p58 provides the structural basis for the bulk of enzyme''s conformational flexibility. Deletion of most of this linker affected the initiation and elongation steps of the primer synthesis.     

In Vitro Activities of the Multifunctional RNA Silencing Polymerase QDE-1 of Neurospora crassa

QDE-1 is an RNA- and DNA-dependent RNA polymerase that has functions in the RNA silencing and DNA repair pathways of the filamentous fungus Neurospora crassa. The crystal structure of the dimeric enzyme has been solved, and the fold of its catalytic core is related closely to that of eukaryotic DNA-dependent RNA polymerases. However, the specific activities of this multifunctional enzyme are still largely unknown. In this study, we characterized the in vitro activities of the N-terminally truncated QDE-1ΔN utilizing structure-based mutagenesis. Our results indicate that QDE-1 displays five distinct catalytic activities, which can be dissected by mutating critical amino acids or by altering reaction conditions. Our data also suggest that the RNA- and DNA-dependent activities have different modes for the initiation of RNA synthesis, which may reflect the mechanism that enables the polymerase to discriminate between template nucleic acids. Moreover, we show that QDE-1 is a highly potent terminal nucleotidyltransferase. Our results suggest that QDE-1 is able to regulate its activity mode depending on the template nucleic acid. This work extends our understanding of the biochemical properties of the QDE-1 enzyme and related RNA polymerases.