RNA capping by mitochondrial and multi-subunit RNA polymerases

Abstract

Recently, it was found that bacterial and eukaryotic transcripts are capped with cellular cofactors installed by their respective RNA polymerases (RNAPs) during transcription initiation. We now show that mitochondrial RNAP efficiently caps transcripts with ADP - containing cofactors. However, a functional role of universal RNAP - catalysed capping is not yet clear.

Keywords: FAD; NAD+; RNA capping; RNA polymerase; UDP-GlcNAc; dephospho coenzymeA; mitochondrial RNA polymerase; non-canonical capping; transcription.

Figure 1.

A. A list of 6 heavily NAD⁺ modified RNA species found by Cahova et. al., [1] with half-lives reported in [22]. B. Cellular concentrations of nucleotides and analogs in E. coli cell reported by Bennett et. al., [9], and the K_m for their usage as a substrates in transcription initiation [8]. C. Regions of RNAP shown to influence capping efficiency. PDB 5D4D structure of Thermus thermophilus RNAP open complex with NADpC was used, NAD is shown in cyan. Part of rifampicin-binding pocket corresponding to cluster I of Rif region of β subunit is in magenta, region 3.2 of σ subunit is in green, template DNA is grey, Mg²⁺ ions are in ruby.

Figure 2.

Human mitochondrial RNAP (hmRNAP) incorporates ADP analogs in vitro. A partial sequence of the light strand promoter (LSP) is shown, with the initially transcribed sequence underlined. For the assay, 50 nM TFAM, 50 nM hmRNAP, 50 nM TFB2M (purified as described in [23]) were combined with 50 nM of linear DNA fragment containing LSP promoter (positions -70 to +50) in 10 µl of transcription buffer (40 mM Tris pH 8.0, 10 mM MgCl₂, 10 mM DTT), then ATP or ADP analogs were added to the final concentration of 1mM. Transcription was initiated by the addition of 10 mM MgCl₂, 50 µM ATP, 300 µM GTP, 10µM [α³²P]-UTP, 25 Ci/mmol (Hartmann Analytic). After 30 min incubation at 37°C, 500 nM NudC was added to half of the reactions and incubated for additional 15 minutes at 37°C. Transcripts modified with NAD⁺, NADH and DP-CoA (but not ATP or FAD) were susceptible to NudC (lanes 5,9,11) judging from increased mobility of the products. Reactions were stopped by the addition of formamide-containing loading buffer. Products were separated on denaturing polyacrylamide gels (20% acrylamide, 3% bis-acrylamide, 6M urea, 1xTBE), revealed by PhosphorImaging (GE Healthcare), and analysed using ImageQuant software (GE Healthcare).