1. Biochemistry and Chemical Biology
  2. Chromosomes and Gene Expression

Highly efficient 5' capping of mitochondrial RNA with NAD+ and NADH by yeast and human mitochondrial RNA polymerase

  1. Jeremy G Bird
  2. Urmimala Basu
  3. David Kuster
  4. Aparna Ramachandran
  5. Ewa Grudzien-Nogalska
  6. Atif Towheed
  7. Douglas C Wallace
  8. Megerditch Kiledjian
  9. Dmitry Temiakov
  10. Smita S Patel  Is a corresponding author
  11. Richard H Ebright  Is a corresponding author
  12. Bryce E Nickels  Is a corresponding author
  1. Rutgers University, United States
  2. Heidelberg University, Germany
  3. The Children’s Hospital of Philadelphia, United States
  4. The Children's Hospital of Philadelphia, Perelman School of Medicine, United States
  5. Thomas Jefferson University, United States
https://doi.org/10.7554/eLife.42179
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Cite this article
  1. Jeremy G Bird
  2. Urmimala Basu
  3. David Kuster
  4. Aparna Ramachandran
  5. Ewa Grudzien-Nogalska
  6. Atif Towheed
  7. Douglas C Wallace
  8. Megerditch Kiledjian
  9. Dmitry Temiakov
  10. Smita S Patel
  11. Richard H Ebright
  12. Bryce E Nickels
(2018)
Highly efficient 5' capping of mitochondrial RNA with NAD+ and NADH by yeast and human mitochondrial RNA polymerase
eLife 7:e42179.
https://doi.org/10.7554/eLife.42179
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  3. Download .RIS
5 figures, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement
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S. cerevisiae and human mtRNAPs cap RNA with NAD+ and NADH in vitro.

(A) Structures of ATP, NAD+, and NADH. Grey, identical atoms; black, distinct atoms. (B) Processing of RNA 5' ends by RppH and NudC. A, adenosine; N+, NAD+ nicotinamide; N, NADH nicotinamide; p, phosphate. (C and D) NCIN capping with NAD+ and NADH by S. cerevisiae mtRNAP (C) and human mtRNAP (D). Top, promoter derivatives. Middle, initial RNA products of in vitro transcription reactions with ATP, NAD+, or NADH as initiating nucleotide and [α32P]-GTP as extending nucleotide. Bottom, full-length RNA products of in vitro transcription reactions with ATP, NAD+, or NADH as initiating nucleotide and [α32P]-GTP, ATP, UTP, and 3'-deoxy-CTP (C), or [α32P]-GTP, ATP, and UTP (D) as extending nucleotides. Products were treated with RppH or NudC as indicated. Grey box and arrow, transcription start site (TSS);+31 and+17/18, position of last NTP incorporated into full-length RNA products; M, 10-nt marker.

https://doi.org/10.7554/eLife.42179.002
Figure 1—source data 1

Source data for Figure 1C,D.

https://doi.org/10.7554/eLife.42179.004
Figure 1—source data 2

Data for Figure 1—figure supplement 1.

https://doi.org/10.7554/eLife.42179.005
Figure 1—figure supplement 1
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S. cerevisiae and human mtRNAPs cap RNA with NAD+ in vitro: additional data.

(A) Processing of radiolabeled RNA 5'-ends by alkaline phosphatase (AP) and NudC. A, adenosine; N+, NAD+ nicotinamide; p, phosphate. *, radiolabeled phosphate. (B and C). NCIN capping with NAD+ by S. cerevisiae mtRNAP (B) and human mtRNAP (C). Top, promoter derivatives. Bottom, full-length RNA products of in vitro transcription reactions with [γ32P]-ATP or [α32P]-NAD+ as initiating nucleotide and GTP, ATP, UTP, and 3'-deoxy-CTP (C), or GTP, ATP, and UTP (D) as extending nucleotides. Products were treated with NudC alone, AP alone, or NudC and AP, as indicated. Grey box and arrow, TSS; +31 and +17/18, position of last NTP incorporated into RNA; M, 10-nt marker.

https://doi.org/10.7554/eLife.42179.003
Figure 2 with 2 supplements
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S. cerevisiae and human mtRNAPs cap RNA with NAD+ and NADH more efficiently than bacterial and nuclear RNAPs.

(A and B) Dependence of NCIN-mediated capping with NAD+ and NADH on [NCIN] / [ATP] ratio for S. cerevisiae mtRNAP (A) and human mtRNAP (B) (mean ± SD; n = 3). DNA templates and representative data are shown in Figure 2—figure supplement 1. (C) Dependence of NCIN-mediated capping with NAD+ and NADH on [NCIN] / [ATP] ratio for mtRNAPs vs. E. coli RNAP and S. cerevisiae RNAP II. Top, tailed template. Grey box and arrow indicate TSS. Bottom, dependence of NCIN-mediated capping with NAD+ and NADH on [NCIN] / [ATP] ratio for S. cerevisiae mtRNAP (Sce mtRNAP), human mtRNAP, E. coli RNAP (Eco RNAP) and S. cerevisiae RNAP II (Sce RNAP II) (mean ± SD; n = 3).

https://doi.org/10.7554/eLife.42179.006
Figure 2—source data 1

Data for Figure 2A,B.

https://doi.org/10.7554/eLife.42179.009
Figure 2—source data 2

Data for Figure 2C,D, and Figure 2—figure supplement 1.

https://doi.org/10.7554/eLife.42179.010
Figure 2—figure supplement 1
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Dependence of NCIN-mediated capping with NAD+ and NADH on [NCIN] / [ATP] ratio for mtRNAPs: representative data.

(A and B) Panels show DNA templates and full-length RNA products of in vitro transcription reactions performed with S. cerevisiae mtRNAP (A) and human mtRNAP (B) with the indicated [NCIN] / [ATP] ratio. Grey box and arrow, TSS; +31, +17/18, +15/16, position of last NTP incorporated into RNA; M, 10-nt marker.

https://doi.org/10.7554/eLife.42179.007
Figure 2—figure supplement 2
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S. cerevisiae and human mtRNAPs cap RNA with NAD+ and NADH at least as efficiently as bacteriophage T7 RNAP.

Dependence of NCIN-mediated capping with NAD+ and NADH on [NCIN] / [ATP] ratio for mtRNAPs vs. T7 RNAP. Top, tailed template. Grey box and arrow indicate TSS. Bottom, Dependence of NCIN-mediated capping with NAD+ and NADH on [NCIN] / [ATP] ratio for S. cerevisiae mtRNAP (Sce mtRNAP), human mtRNAP, and T7 RNAP (mean ± SD; n = 3).

https://doi.org/10.7554/eLife.42179.008
Figure 3 with 1 supplement
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Promoter sequence determines efficiency of RNA capping with NAD+ by mtRNAP.

(A) S. cerevisiae mitochondrial 21S promoter DNA depicted in the context of the mtRNAP-promoter open complex. DNA nontemplate strand (NT) on top; DNA template strand (T) on bottom; Unwound, non-base-paired DNA region, ‘transcription bubble,’ indicated by raised and lowered nucleotides; +1 and grey boxes, bases at the TSS; −1, bases immediately upstream of the TSS (the 21S promoter is a −1Y promoter). (B) Products of transcription reactions with NAD+ as initiating nucleotide and [α32P]-CTP as extending nucleotide for templates having complementary or non-complementary nucleotides at position +1. (C) Dependence of NAD+ capping on [NAD+] / [ATP] ratio for homoduplex templates having A:T, G:C, T:A, or C:G at position −1 relative to TSS (mean ± SD; n = 3). Red, −1R promoters; black, −1Y promoters. (D) Dependence of NAD+ capping on [ATP] / [NAD+] ratio for heteroduplex templates having an abasic site (*) on the DNA nontemplate strand (mean ± SD; n = 3). Red, promoters with a template-strand Y; black, promoters with a template-strand R. (E) Sequence preferences at position −1 for S. cerevisiae mtRNAP, E. coli RNAP, and T7 RNAP. Graphs show normalized values of (kcat/KM)NAD+ / (kcat/KM)ATP determined for homoduplex templates having A:T, G:C, T:A, or C:G at position −1 (mean ± SD; n = 3). Normalized values were calculated by dividing the value for each individual promoter by the average value measured for −1R promoters. Data for S. cerevisiae mtRNAP are from panel C, data for E. coli RNAP are from (Vvedenskaya et al., 2018), and data for T7 RNAP are from Figure 3—figure supplement 1.

https://doi.org/10.7554/eLife.42179.011
Figure 3—source data 1

Data for Figure 3B.

https://doi.org/10.7554/eLife.42179.013
Figure 3—source data 2

Data for Figure 3C,D,E, and Figure 3—figure supplement 1.

https://doi.org/10.7554/eLife.42179.014
Figure 3—figure supplement 1
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Promoter sequence determines efficiency of RNA capping with NAD+: bacteriophage T7 RNAP.

(A) Bacteriophage T7 RNAP-dependent promoter derivatives analyzed. Red, −1R promoters; black, −1Y promoters; +1 and grey box, bases at the TSS; −1, bases immediately upstream of the TSS. (B) Dependence of NAD+ capping on [NAD+] / [ATP] ratio for homoduplex templates having A:T, G:C, T:A, or C:G at position −1 relative to TSS (mean ± SD; n = 3).

https://doi.org/10.7554/eLife.42179.012
Figure 4
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Detection and quantitation of NAD+- and NADH-capped mitochondrial RNA in vivo: boronate affinity electrophoresis with DNAzyme-cleaved cellular RNA and DNAzyme-cleaved synthetic NCIN-capped RNA standards.

(A) Use of DNAzyme (DZ) to process RNA to yield a defined, short 5'-end-containing subfragment, in parallel in vivo (red) and in vitro (blue). Uncapped, 5'-triphosphate (ppp) end generated using ATP-mediated initiation; 5'-NAD+, NAD+-capped end generated using NAD+-mediated initiation; 5'-NADH, NADH-capped end generated using NADH-mediated initiation. (B) Use of boronate affinity electrophoresis to resolve 5'-uncapped, 5'-NAD+, and 5'-NADH containing RNAs. Grey, structure of phenylboronic acid (PB) polyacrylamide gel. (C) PB-polyacrylamide gel (left) and polyacrylamide gel (right) analysis of DNAzyme-generated 5'-end-containing subfragments of S. cerevisiae mitochondrial RNA COX2. Red, observed 5'-end-containing RNA subfragments resolved by PB-polyacylamide-gel (left) or not resolved by polyacrylamide gel (right); identities of these subfragments are defined in Panel D. (D) Comparison of electrophoretic mobilities of observed 5'-end-containing subfragments of COX2 RNA generated in vivo to 5'-end-containing subfragments of synthetic RNA standards generated in vitro. a, NAD+-capped RNA; b, NADH-capped RNA; c, uncapped RNA (mean ± SD; n = 3).

https://doi.org/10.7554/eLife.42179.015
Figure 4—source data 1

Data for Figure 4D.

https://doi.org/10.7554/eLife.42179.016
Figure 5
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Detection and quantitation of NAD+- and NADH-capped mitochondrial RNA in vivo: effects of intracellular NAD+ and NADH levels in S. cerevisiae and human cells.

(A) Changes in intracellular NAD+/NADH ratios result in changes in levels of NAD+- and NADH-capped mitochondrial RNA (mean ± SD; n = 3). Gel images show representative data for S. cerevisiae COX2 RNA (left) and 21S RNA (right). Blue annotations as in Figure 4. (B) Changes in intracellular NAD(H) levels result in changes in levels of NAD+- and NADH-capped mitochondrial RNA (mean ± SD; n = 3). Gel images show representative data for LSP-derived RNAs. Red, NAD(H) biosynthesis inhibitor FK866; NAMPT, Nicotinamide phosphoribosyltransferase; NMNAT, Nicotinamide mononucleotide adenylyltransferase.

https://doi.org/10.7554/eLife.42179.017
Figure 5—source data 1

Data for Figure 5 (gels).

https://doi.org/10.7554/eLife.42179.018
Figure 5—source data 2

Data for Figure 5 (values of NCIN capping).

https://doi.org/10.7554/eLife.42179.019

Tables

Key resources table
Reagent type
(species) or
resource		DesignationSource or
reference		IdentifiersAdditional information
Strain, strain
background
(E. coli)		BL21(DE3) bacteriaNEBC2527H
Strain, strain
background
(E. coli)		NiCo21(DE3) bacteriaNEBC2529H
Strain, strain
background
(E. coli)		Artic Express (DE3)
bacteria		Fisher ScientificNC9444283
Strain, strain
background
(S. cerevisiae)		246.1.1
(MATa ura3 trp1 leu2 his4)		Gift of A. Vershon
Cell line
(human)		HEK293T
(human embryonic
kidney cells)		ATCCCRL-3216
Recombinant
DNA reagent		pIA900Gift of I.
Artsimovitch
Recombinant
DNA reagent		pET NudC-His(Bird et al., 2016)
Recombinant
DNA reagent		pJJ1399gift of J. Jaehning
Recombinant
DNA reagent		pTrcHisC-Mtf1gift of J. Jaehning
Recombinant
DNA reagent		pPROEXHTb-POLRMT
(43–1230)−6xHis		(Ramachandran et al., 2017)
Recombinant
DNA reagent		pPROEXHTb-TFAM
(43-245)−6xHis		(Ramachandran et al., 2017)
Recombinant
DNA reagent		pT7TEV-HMBP4(Yakubovskaya et al., 2014)
Recombinant
DNA reagent		pAR1219(Jia et al., 1996)
Sequence-based
reagent		DK64Integrated DNA
Technologies (IDT)		tailed template with
PEG6 linker		GGCTCGCCTCGGCTCG/iSp18/
CGAGCCGAGGCGAGCGTCACCAA
Sequence-based
reagent		JB459IDThuman LSP DNA template
 + 1 AGU variant nontemplate
strand		GTGTTAGTTGGGGGGTGACTGTT
AAAAGTGCATACCGCCAAAGTATA
AAATTTGTGGGCC
Sequence-based
reagent		JB460IDThuman LSP DNA template
+ 1 AGU variant template
strand		GGCCCACAAATTTTATACTTTGGC
GGTATGCACTTTTAACAGTCACCC
CCCAACTAACAC
Sequence-based
reagent		JB469IDTT7φ2.5–35 n nontemplate
strand (−1T)		CAGTAATACGACTCACTATTAGCGAA
GCGGGCATGCGGCCAGCCATAGC
CGATCA
Sequence-based
reagent		JB470IDTT7φ2.5–35 n template
strand (−1A)		TGATCGGCTATGGCTGGCCGCATGCC
CGCTTCGCTAATAGTGAGTCGTA
TTACTG
Sequence-based
reagent		JB471IDTT7φ2.5–35 n nontemplate
strand (−1A)		CAGTAATACGACTCACTATAAGCGAAGC
GGGCATGCGGCCAGCCATAG
CCGATCA
Sequence-based
reagent		JB472IDTT7φ2.5–35 n template
strand (−1T)		TGATCGGCTATGGCTGGCCGCATGCCC
GCTTCGCTTATAGTGAGTCGTATTACTG
Sequence-based
reagent		JB473IDTT7φ2.5–35 n nontemplate
strand (−1G)		CAGTAATACGACTCACTATGAGCGAAG
CGGGCATGCGGCCAGCCATAG
CCGATCA
Sequence based
reagent		JB474IDTT7φ2.5 35 n template
strand (−1C)		TGATCGGCTATGGCTGGCCGCATGCC
CGCTTCGCTCATAGTGAGTCGTATTACTG
Sequence-based
reagent		JB475IDTT7φ2.5–35 n nontemplate
strand (−1C)		CAGTAATACGACTCACTATCAGCGAA
GCGGGCATGCGGCCAGCCA
TAGCCGATCA
Sequence-based
reagent		JB476IDTT7φ2.5–35 n template
strand (−1G)		TGATCGGCTATGGCTGGCCGCATGC
CCGCTTCGCTGATAGTG
AGTCGTATTACTG
Sequence-based
reagent		JB515IDTprobe for human LSP-generated
RNA (complementary to
positions + 2 to+31)		CACCAGCCTAACCAGATTTCAA
ATTTTATC
Sequence-based
reagent		JB525IDTprobe for S. cerevisiae
21S RNA (complementary
to positions + 9 to+42)		CTATATAATAAATATTTCAAATC
TATTATTCTAC
Sequence-based
reagent		JB526IDTS. cerevisiae 21S RNA
DNAzyme; cleaves transcript
at position + 53		ACTCCATGATTAGGCTAGCTACAA
CGACTCTTTAAATCT
Sequence-based
reagent		JB555IDTprobe for S. cerevisiae
COX2 RNA (complementary
to positions + 8 to+46)		ATCTTAACCTTTAGACTCTTTTGTC
TATTTATAATATGT
Sequence-based
reagent		JB557IDTS. cerevisiae COX2 DNAzyme;
cleaves at position + 57		TCTTAATAAATCTAAGGCTAGCTACA
ACGAATTTTAATAAATCTT
Sequence-based
reagent		JB559IDThuman LSP-generated RNA
DNAzyme; cleaves at
position + 67		GCACTTAAACAGGCTAGCTACAA
CGAATCTCTGCCA
Sequence-based
reagent		JB560IDTS. cerevisiae COX2 −40
to + 125 nontemplate strand
oligo (for generation of
in vitro transcription template)		TATATAATAATAAATTATAAATAAATTTT
AATTAAAAGTAGTATTAACATATTATAAA
TAGACAAAAGAGTCTAAAGGTTAAGATT
TATTAAAATGTTAGATTTATTAAGATTAC
AATTAACAAC
Sequence-based
reagent		JB561IDTS. cerevisiae COX2 −40 to + 3
forward primer (for generation
of in vitro transcription template)		TATATAATAATAAATTATAAATAAATTTT
AATTAAAAGTAGT
Sequence-based
reagent		JB562IDTS. cerevisiae COX2 + 83 to+125
reverse primer (for generation
of in vitro transcription template)		GTTGTTAATTGTAATCTTAATAAATCTAA
CATTTTAATAAATC
Sequence-based
reagent		UB1IDThuman LSP DNA template (−43
to + 19) nontemplate strand		ATGTGTTAGTTGGGGGGTGACTGTTAA
AAGTGCATACCGCCAAAAGATAAAATT
TGAAATCTG
Sequence-based
reagent		UB2IDThuman LSP DNA template
(−43 to + 19) template
strand		CAGATTTCAAATTTTATCTTTTGGCGGT
ATGCACTTTTAACAGTCACCCCCCAAC
TAACACAT
Sequence-based
reagent		UB3IDThuman HSP1 DNA template
(−43 to + 20) nontemplate
strand		ACACACCGCTGCTAACCCCATACCCCGA
ACCAACCAAACCCCAAAGACACCCGCC
ACAGTTTA
Sequence-based
reagent		UB4IDThuman HSP1 DNA template
(−43 to + 20) template
strand		TAAACTGTGGCGGGTGTCTTTGGGGT
TTGGTTGGTTCGGGGTATGGGGTTA
GCAGCGGTGTGT
Sequence-based
reagent		UB5IDTS. cerevisiae 15S DNA
template (−25 to + 1;
C-less cassette) nontemplate
strand		ATAATTTATTTATTATTATATAAGTAAT
AAATAATTGTTTTATATAATAAGAA
TTCTCCTTC
Sequence-based
reagent		UB6IDTS. cerevisiae 15S DNA template
(−25 to + 1; C-less cassette)
template strand		GAAGGAGAATTCTTATTATATAAAACA
ATTATTTATTACTTATATAATAATAA
ATAAATTAT
Sequence-based
reagent		UB7IDTS. cerevisiae 21S DNA
template (−25 to + 1;
C-less cassette) nontemplate
strand		TATTATTATTATTATATATATAAGTAG
TAAAAAGTAGAATAATAGATTT
GAAATACC
Sequence-based
reagent		UB8IDTS. cerevisiae 21S DNA
template (−25 to + 1;
C-less cassette) template
strand		GAAGGAGACCAACCACAAACACACA
ACAACCACCAACTACTTATATAATAA
TAAATAAATTAT
Sequence-based
reagent		UB9IDTS. cerevisiae 21S DNA
template (−25 to + 1; C-less
and A-less cassette) nontemplate
strand		ATAATTTATTTATTATTATATAAGTAG
TTGGTGGTTGTTGTGTGTTTGTG
GTTGGTCTCCTTC
Sequence-based
reagent		UB10IDTS. cerevisiae 21S DNA
template (−25to + 1;
C-less and A-less cassette)
template strand		GAAGGAGACCAACCACAAACACAC
AACAACCACCAACTACTTATATAA
TAATAAATAAATTAT
Sequence-based
reagent		UB11IDTS. cerevisiae 21S
nontemplate strand
(−1A)		ATAATTTATTTATTATTATATAAGAA
GTTGGTGGTTGTTGTGTGTTTGTG
GTTGGTCTCCTTC
Sequence-based
reagent		UB12IDTS. cerevisiae 21S template
strand (−1T)		GAAGGAGACCAACCACAAACACA
CAACAACCACCAACTTCTTATATA
ATAATAAATAAATTAT
Sequence-based
reagent		UB13IDTS. cerevisiae 21S
nontemplate strand
(−1G)		ATAATTTATTTATTATTATATAAGG
AGTTGGTGGTTGTTGTGTGTTTG
TGGTTGGTCTCCTTC
Sequence-based
reagent		UB14IDTS. cerevisiae 21S
template strand
(−1C)		GAAGGAGACCAACCACAAACACA
CAACAACCACCAACTCCTTATAT
AATAATAAATAAATTAT
Sequence-based
reagent		UB15IDTS. cerevisiae 21S
nontemplate strand
(−1C)		ATAATTTATTTATTATTATATAAG
CAGTTGGTGGTTGTTGTGTGT
TTGTGGTTGGTCTCCTTC
Sequence-based
reagent		UB16IDTS. cerevisiae 21S
template strand
(−1G)		GAAGGAGACCAACCACAAACACA
CAACAACCACCAACTGCTTATATA
ATAATAAATAAATTAT
Sequence-based
reagent		UB17IDTS. cerevisiae 21S
nontemplate strand
(+1C)		ATAATTTATTTATTATTATATAAGTC
GTTGGTGGTTGTTGTGTGTTTGT
GGTTGGTCTCCTTC
Sequence-based
reagent		UB18IDTS. cerevisiae 21S
template strand
(+1G)		GAAGGAGACCAACCACAAACACAC
AACAACCACCAACGACTTATATAA
TAATAAATAAATTAT
Sequence-based
reagent		JB527IDTS. cerevisiae 21S
nontemplate strand
(−1 abasic)		ATAATTTATTTATTATTATATAAG/
idSp/AGTTGGTGGTTGTTGTGTGT
TTGTGGTTGGTCTCCTTC
Peptide, recombinant
protein (S. cerevisiae)		Rpo41 (mtRNAP)(Tang et al., 2009)
Peptide, recombinant
protein (S. cerevisiae)		Mtf1(Paratkar and Patel, 2010)
Peptide, recombinant
protein (Human)		POLRMT (mtRNAP)(Ramachandran et al., 2017)
Peptide, recombinant
protein (Human)		TFAM(Ramachandran et al., 2017)
Peptide, recombinant
protein (Human)		TFB2(Yakubovskaya et al., 2014)
Peptide, recombinant
protein (S. cerevisiae)		RNA
polymerase II		Gift of C. Kaplan
Peptide, recombinant
protein (E. coli)		RNA polymerase
core (β'−6xHis)		(Artsimovitch et al., 2003)
Peptide, recombinant
protein		T7 RNA polymerase(Jia et al., 1996)
Peptide, recombinant
protein (E. coli)		NudC(Cahová et al., 2015)
Peptide, recombinant
protein		Phusion Flash HF
master mix		ThermoFisherF-548L
Peptide, recombinant
protein		T4 Polynucleotide
Kinase		NEBM0201L
Peptide, recombinant
protein		RNA 5'
pyrophosphohydrolase
(RppH)		NEBM0356S
Peptide, recombinant
protein		FastAP Alkaline
Phosphatase		Thermo FisherEF0651
Commercial assay
or kit		Monarch PCR and DNA
clean up kit		NEBT1030S
Chemical compound,
drug		Nuclease-free water
(not DEPC-treated)		ThermoFisherAM9932
Chemical compound,
drug		Bacto agarVWR90000–760
Chemical compound,
drug		Bacto tryptoneVWR90000–286
Chemical compound,
drug		Bacto yeast extractVWR90000–726
Chemical compound,
drug		D-Glucose
monhydrate		Amresco0643–1 kg
Chemical compound,
drug		GlycerolEMD Millipore55069521
Chemical compound,
drug		DMEM mediumThermo Fisher11965–092
Chemical compound,
drug		Fetal Bovine SerumAtlanta BiologicalS11150H
Chemical compound,
drug		dNTP solution mix,
10 mM of each NTP		NEBN0447S
Chemical compound,
drug		NTP set (ultra-pure),
100 mM solutions		GE Healthcare27-2025-01
Chemical compound,
drug		NAD+Roche
(Sigma-Aldrich)		10127965001
Chemical compound,
drug		NADHRoche
(Sigma-Aldrich)		10107735001
Chemical compound,
drug		Tris base (Amresco)VWR97061–800
Chemical compound,
drug		Boric Acid (ACS grade)VWR97061–980
Chemical compound,
drug		EDTA disodium salt
dyhydrate		VWR97061–018
Chemical compound,
drug		0.5 M EDTA pH 8ThermoFisherAM9260G
Chemical compound,
drug		Dibasic Sodium
phosphate		EMD MilliporeSX0715-1
Chemical compound,
drug		Sodium ChlorideEMD MilliporeSX0420-3
Chemical compound,
drug		Potassium ChlorideEMD Millipore7300–500 GM
Chemical compound,
drug		Sodium CitrateEMD Millipore7810–1 KG
Chemical compound,
drug		Sodium Acetate,
trihydrate		VWRMK736406
Chemical compound,
drug		Ficoll 400VWRAAB22095-18
Chemical compound,
drug		PolyvinylpyrrolidoneEMD Millipore7220–1 KG
Chemical compound,
drug		Diethyl Pyrocarbonate
(DEPC)		VWRAAB22753-14
Chemical compound,
drug		Formamide,
deionized		VWREM-4610
Chemical compound,
drug		Sodium
dodecylsulfate (SDS)		VWR97064–470
Chemical compound,
drug		Magnesium chloride
hexahydrate		VWREM-5980
Chemical compound,
drug		Magnesium sulfate
heptahydrate		VWREM-MX0070-1
Chemical compound,
drug		Glycerol (ACS grade)VWREMGX0185-5
Chemical compound,
drug		Bovine Serum Albumin
(BSA) fraction V		VWR101174–932
Chemical compound,
drug		Bromophenol BlueVWREM-BX1410-7
Chemical compound,
drug		Xylene CyanolSigma-AldrichX4126-10G
Chemical compound,
drug		Amaranth DyeVWR200030–400
Chemical compound,
drug		Temed (JT Baker)VWRJT4098-1
Chemical compound,
drug		Ammonium PersulfateVWR97064–594
Chemical compound,
drug		Dithiothreitol (DTT)Gold BioDTT50
Chemical compound,
drug		Glycogen from Oyster
(type II)		Sigma-AldrichG8751
Chemical compound,
drug		Hydrochloric Acid
(ACS plus)		Fisher ScientificA144-212
Chemical compound,
drug		Ethyl AlcoholPharmco-AAPER111000200
Chemical compound,
drug		GeneMate LE Quick
Dissolve agarose		BioExpressE-3119–500
Chemical compound,
drug		SequaGel sequencing
system		National
Diagnostics		EC833
Chemical compound,
drug		Nytran SuPerCharge
Nylon Membrane		VWR10416296
Chemical compound,
drug		SigmaSpin G25 cleanup
columns		Sigma-AldrichS5059
Chemical compound,
drug		32P NAD+ 250 uCiPerkin ElmerBLU023X250UC
Chemical compound,
drug		γ-32P ATP Easy
Tide 1 mCi		Perkin ElmerBLU502Z001MC
Chemical compound,
drug		α-32P CTP Easy
Tide 250 uCi		Perkin ElmerBLU508H250UC
Chemical compound,
drug		α-32P GTP Easy
Tide 250 uCi		Perkin ElmerBLU506H250UC
Chemical compound,
drug		α-32P UTP Easy
Tide 250 uCi		Perkin ElmerBLU507H250UC
Chemical compound,
drug		Decade MarkerThermo FisherAM7778
Chemical compound,
drug		TRI ReagentMolecular
Research Center		TR118
Chemical compound,
drug		Acid phenol:chloroform
(CHCl3) pH 4.5		ThermoFisherAM9720
Chemical compound,
drug		FK866 hydrochloride
hyrate		Sigma-AldrichF8557
Software, algorithmExcelMicrosoft365
Software, algorithmImageQuantGE HealthcareTL 5.1, TL v8.2
Software, algorithmSigmaPlotSystat Software Inc.Version 10
Software, algorithmPymolSchrodinger, LLChttp://www.pymol.org
Software, algorithmIllustratorAdobeVersion CS6
OtherTyphoon RBG ImagerGE Healthcare
OtherNanoDrop 2000C
spectrophotometer		Thermo Fisher
OtherUV CrosslinkerFisher ScientificFB-UVXL-1000
OtherHybridization oven 5420VWR97005–252
OtherSequi-Gen GT sequencing
systems (21 × 50) (38 × 30)		Bio-Rad1653871 and 1653873

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https://doi.org/10.7554/eLife.42179.020

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  1. Jeremy G Bird
  2. Urmimala Basu
  3. David Kuster
  4. Aparna Ramachandran
  5. Ewa Grudzien-Nogalska
  6. Atif Towheed
  7. Douglas C Wallace
  8. Megerditch Kiledjian
  9. Dmitry Temiakov
  10. Smita S Patel
  11. Richard H Ebright
  12. Bryce E Nickels
(2018)
Highly efficient 5' capping of mitochondrial RNA with NAD+ and NADH by yeast and human mitochondrial RNA polymerase
eLife 7:e42179.
https://doi.org/10.7554/eLife.42179