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3' end additions by T7 RNA polymerase are RNA self-templated, distributive and diverse in character-RNA-Seq analyses.

ABSTRACT: Synthetic RNA is widely used in basic science, nanotechnology and therapeutics research. The vast majority of this RNA is synthesized in vitro by T7 RNA polymerase or one of its close family members. However, the desired RNA is generally contaminated with products longer and shorter than the DNA-encoded product. To better understand these undesired byproducts and the processes that generate them, we analyze in vitro transcription reactions using RNA-Seq as a tool. The results unambiguously confirm that product RNA rebinds to the polymerase and self-primes (in cis) generation of a hairpin duplex, a process that favorably competes with promoter driven synthesis under high yield reaction conditions. While certain priming modes can be favored, the process is heterogeneous, both in initial priming and in the extent of priming, and already extended products can rebind for further extension, in a distributive process. Furthermore, addition of one or a few nucleotides, previously termed 'nontemplated addition,' also occurs via templated primer extension. At last, this work demonstrates the utility of RNA-Seq as a tool for in vitro mechanistic studies, providing information far beyond that provided by traditional gel electrophoresis.

SUBMITTER: Gholamalipour Y

PROVIDER: S-EPMC6182178 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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3' end additions by T7 RNA polymerase are RNA self-templated, distributive and diverse in character-RNA-Seq analyses.

Gholamalipour Yasaman Y Karunanayake Mudiyanselage Aruni A Martin Craig T CT

Nucleic acids research 20181001 18

Synthetic RNA is widely used in basic science, nanotechnology and therapeutics research. The vast majority of this RNA is synthesized in vitro by T7 RNA polymerase or one of its close family members. However, the desired RNA is generally contaminated with products longer and shorter than the DNA-encoded product. To better understand these undesired byproducts and the processes that generate them, we analyze in vitro transcription reactions using RNA-Seq as a tool. The results unambiguously confi ...[more]

PMID: 30219859

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Project description:BACKGROUND:T7 lysozyme (T7L), also known as N-acetylmuramoyl-L-alanine amidase, is a T7 bacteriophage gene product. It involves two functions: It can cut amide bonds in the bacterial cell wall and interacts with T7 RNA polymerase (T7RNAP) as a part of transcription inhibition. In this study, with the help of molecular dynamics (MD) calculations and computational interaction studies, we investigated the effect of varying pH conditions on conformational flexibilities of T7L and their influence on T7RNAP -T7L interactions. RESULTS:From the MD studies of the T7L at three different pH strengths viz. 5, neutral and 7.9 it was observed that T7L structure at pH 5 exhibited less stable nature with more residue level fluctuations, decrease of secondary structural elements and less compactness as compared to its counterparts: neutral pH and pH 7.9. The T-pad analysis of the MD trajectories identified local fluctuations in few residues that influenced the conformational differences in three pH strengths. From the docking of the minimum energy representative structures of T7L at different pH strengths (obtained from the free energy landscape analysis) with T7RNAP structures at same pH strengths, we saw strong interaction patterns at pH 7.9 and pH 5. The MD analysis of these complexes also confirmed the observations of docking study. From the combined in silico studies, it was observed that there are conformational changes in N-terminal and near helix 1 of T7L at different pH strengths, which are involved in the T7RNAP interaction, thereby varying the interaction pattern. CONCLUSION:Since T7L has been used for developing novel therapeutics and T7RNAP one of the most biologically useful protein in both in-vitro and in vivo experiments, this in silico study of pH dependent conformational differences in T7L and the differential interaction with T7RNAP at different pH can provide a significant insight into the structural investigations on T7L and T7RNAP in varying pH environments.

Dataset Information

3' end additions by T7 RNA polymerase are RNA self-templated, distributive and diverse in character-RNA-Seq analyses.

Publications

3' end additions by T7 RNA polymerase are RNA self-templated, distributive and diverse in character-RNA-Seq analyses.

Similar Datasets

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