Primer-independent RNA sequencing with bacteriophage φ6 RNA polymerase and chain terminators

Transcription termination by bacteriophage T7 RNA polymerase at rho-independent terminators.

We have investigated the mechanism of transcription termination by T7 RNA polymerase using templates encoding variants of the transcription-termination structure (attenuator) of the regulatory region of the threonine (thr) operon of Escherichia coli. The thr attenuator comprises the following two distinct structural elements: a G + C-rich inverted repeat, which encodes an RNA hairpin structure,...

Poly(A)- and primer-independent RNA polymerase of Norovirus.

Replication of positive-strand caliciviruses is mediated by a virus-encoded RNA-dependent RNA polymerase (RdRp). To study the replication of Norovirus (NV), a member of the family Caliciviridae, we used a recombinant baculovirus system to express an enzymatically active RdRp protein from the 3D region of the NV genome and defined conditions for optimum enzymatic activity. Using an RNA template ...

Enhancement of RNA Interference Effect in P19 EC Cells by an RNA-dependent RNA Polymerase

Background: RNA interference (RNAi) is a phenomenon uses double-stranded RNA (dsRNA) to specifically inhibit gene expression. The non-specific silencing caused by interferon response to dsRNA in mammalian cells limits the potential of utilizing RNAi to study gene function. Duplexes of 21-nucleotide short interfering dsRNA (siRNA) inhibit gene expression by RNAi. In some organisms, siRNA can als...

Primer-dependent synthesis by poliovirus RNA-dependent RNA polymerase (3Dpol)

Properties of poliovirus RNA-dependent RNA polymerase (3D(pol)) including optimal conditions for primer extension, processivity and the rate of dissociation from primer-template (k(off)) were examined in the presence and absence of viral protein 3AB. Primer-dependent polymerization was examined on templates of 407 or 1499 nt primed such that fully extended products would be 296 or 1388 nt, resp...

Enzymatic RNA Synthesis using Bacteriophage T7 RNA Polymerase