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A specific, promoter-independent activity of T7 RNA polymerase suggests a general model for DNA/RNA editing in single subunit RNA Polymerases.

ABSTRACT: Insertional RNA editing has been observed and characterized in mitochondria of myxomycetes. The single subunit mitochondrial RNA polymerase adds nontemplated nucleotides co-transcriptionally to produce functional tRNA, rRNA and mRNAs with full genetic information. Addition of nontemplated nucleotides to the 3' ends of RNAs have been observed in polymerases related to the mitochondrial RNA polymerase. This activity has been observed with T7 RNA polymerase (T7 RNAP), the well characterized prototype of the single subunit polymerases, as a nonspecific addition of nucleotides to the 3' end of T7 RNAP transcripts in vitro. Here we show that this novel activity is an editing activity that can add specific ribonucleotides to 3' ends of RNA or DNA when oligonucleotides, able to form intramolecular or intermolecular hairpin loops with recessed 3' ends, are added to T7 RNA polymerase in the presence of at least one ribonucleotide triphosphate. Specific ribonucleotides are added to the recessed 3' ends through Watson-Crick base pairing with the non-base paired nucleotide adjacent to the 3' end. Optimization of this activity is obtained through alteration of the lengths of the 5'-extension, hairpin loop, and hairpin duplex. These properties define a T7 RNAP activity different from either transcriptional elongation or initiation.

SUBMITTER: Sarcar SN

PROVIDER: S-EPMC6141538 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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A specific, promoter-independent activity of T7 RNA polymerase suggests a general model for DNA/RNA editing in single subunit RNA Polymerases.

Sarcar Subha Narayan SN Miller Dennis L DL

Scientific reports 20180917 1

Insertional RNA editing has been observed and characterized in mitochondria of myxomycetes. The single subunit mitochondrial RNA polymerase adds nontemplated nucleotides co-transcriptionally to produce functional tRNA, rRNA and mRNAs with full genetic information. Addition of nontemplated nucleotides to the 3' ends of RNAs have been observed in polymerases related to the mitochondrial RNA polymerase. This activity has been observed with T7 RNA polymerase (T7 RNAP), the well characterized prototy ...[more]

PMID: 30224735

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Project description:Key messageWe identified sequence motifs, which enhance or reduce the ability of the Arabidopsis phage-type RNA polymerases RPOTm (mitochondrial RNAP), RPOTp (plastidial RNAP), and RPOTmp (active in both organelles) to recognize their promoters in vitro with help of a 'specificity loop'. The importance of this data for the evolution and function of the organellar RNA polymerases is discussed. The single-subunit RNA polymerase (RNAP) of bacteriophage T7 is able to perform all steps of transcription without additional transcription factors. Dicotyledonous plants possess three phage-type RNAPs, RPOTm-the mitochondrial RNAP, RPOTp-the plastidial RNAP, and RPOTmp-an RNAP active in both organelles. RPOTm and RPOTp, like the T7 polymerase, are able to recognize promoters, while RPOTmp displays no significant promoter specificity in vitro. To find out which promoter motifs are crucial for recognition by the polymerases we performed in vitro transcription assays with recombinant Arabidopsis RPOTm and RPOTp enzymes. By comparing different truncated and mutagenized promoter constructs, we observed the same minimal promoter sequence supposed to be needed in vivo for transcription initiation. Moreover, we identified elements of core and flanking sequences, which are of critical importance for promoter recognition and activity in vitro. We further intended to reveal why RPOTmp does not efficiently recognize promoters in vitro and if promoter recognition is based on a structurally defined specificity loop of the plant enzymes as described for the yeast and T7 RNAPs. Interestingly, the exchange of only three amino acids within the putative specificity loop of RPOTmp enabled the enzyme for specific promoter transcription in vitro. Thus, also in plant phage-type RNAPs the specificity loop is engaged in promoter recognition. The results are discussed with respect to their relevance for transcription in organello and to the evolution of RPOT enzymes including the divergence of their functions.

Dataset Information

A specific, promoter-independent activity of T7 RNA polymerase suggests a general model for DNA/RNA editing in single subunit RNA Polymerases.

Publications

A specific, promoter-independent activity of T7 RNA polymerase suggests a general model for DNA/RNA editing in single subunit RNA Polymerases.

Similar Datasets

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