Misidentifying Helicobacter cinadei.

An RNA polymerase identified in the genome of Pseudomonas phage Njord offers a promising tool for the synthesis of mRNA and other therapeutic nucleic acids. Originating from a marine microbial ecosystem, Njord RNAP transcribes RNA at high yield even under low temperature conditions. Key properties of the enzyme relevant to mRNA synthesis are presented including transcriptional fidelity, promoter specificity, incorporation of modified nucleotides, and the impurity profile of the RNA. Specific attention is given to the formation of contaminating double-stranded RNA (dsRNA) species. Analysis of transcription reactions shows that DNA-templated promoter-independent transcription is a major source of detectable dsRNA impurities and that Njord RNAP displays a minimal level of this activity. Consistent with the known inflammatory role of dsRNA in synthetic mRNA, transcriptomic analysis of cell culture and a live animal study demonstrates that mRNA synthesized with Njord RNAP elicits only a minimal immune response. This natural enzyme enables efficient mRNA synthesis at ambient temperature and produces transcripts essentially free of dsRNA, offering significant potential to streamline mRNA manufacturing processes.

DNA-Directed RNA Polymerases↗

PubMed2003

The L-L oligomerization domain resides at the very N-terminus of the sendai virus L RNA polymerase protein.

The Sendai virus RNA-dependent RNA polymerase is composed of the L and P proteins. We previously showed that the L protein gives intragenic complementation and forms an oligomer where the L-L interaction site mapped to the N-terminal half of the protein (S. Smallwood et al., 2002, Virology, 00, 000-000). We now show that L oligomerization does not depend on P protein and progressively smaller N-terminal fragments of L from amino acids (aa) 1-1146 through aa 1-174 all bind wild-type L. C-terminal truncations up to aa 424, which bind L, can complement the transcription defect in an L mutant altered at aa 379, although these L truncation mutants do not bind P. The fragment of L comprising aa 1-895, furthermore, acts as a dominant-negative mutant to inhibit transcription of wild-type L. N-terminal deletions of aa 1-189 and aa 1-734 have lost the ability to form the L-L complex as well as the L-P complex, although they still bind C protein. These data are consistent with the L-L interaction site residing in aa 1-174. Site-directed mutations in the N-terminal 347 aa, of L which abolish P binding, do not affect L-L complex formation, so while the L and P binding sites on L are overlapping they are mediated by different amino acids. The N-terminal portions of L with aa 1-424, aa 1-381, and to a lesser extent aa 1-174, can complement the transcription defect in an L mutant altered at aa 77-81, showing their L-L interaction is functional.

DNA-Directed RNA Polymerases↗

PubMed2003

Essential steps in the ppGpp-dependent regulation of bacterial ribosomal RNA promoters can be explained by substrate competition.

Transcription of stable RNA genes is known to be dramatically reduced in the presence of guanosine tetraphosphate (ppGpp), the mediator of the stringent response. Using in vitro transcription systems with ribosomal RNA P1 promoters, we have analyzed which step of the initiation cycle is inhibited by the effector ppGpp. We show that formation of the ternary transcription initiation complex consisting of RNA polymerase holoenzyme, the promoter DNA, and the first initiating nucleotide triphosphate is the major step at which ppGpp exerts its regulation. Neither primary binding of RNA polymerase to the promoter nor isomerization to the open binary complexes or the subsequent promoter clearance steps contributes notably to the observed inhibition. The effect of ppGpp-dependent inhibition in the formation of the ternary transcription initiation complex could be mimicked by nucleotide derivatives known to bind to the RNA polymerase active center. Using these model compounds, almost identical inhibition characteristics were observed as seen with ppGpp. The results support the previously published model, which suggests that ppGpp-dependent inhibition is based on competition between the inhibitor molecules and NTP substrates for access to the active center of RNA polymerase.

DNA-Directed RNA Polymerases↗