Identification of multiple rate-limiting steps during the human mitochondrial transcription cycle in vitro

Abstract

We have reconstituted human mitochondrial transcription in vitro on DNA oligonucleotide templates representing the light strand and heavy strand-1 promoters using protein components (RNA polymerase and transcription factors A and B2) isolated from Escherichia coli. We show that 1 eq of each transcription factor and polymerase relative to the promoter is required to assemble a functional initiation complex. The light strand promoter is at least 2-fold more efficient than the heavy strand-1 promoter, but this difference cannot be explained solely by the differences in the interaction of the transcription machinery with the different promoters. In both cases, the rate-limiting step for production of the first phosphodiester bond is open complex formation. Open complex formation requires both transcription factors; however, steps immediately thereafter only require transcription factor B2. The concentration of nucleotide required for production of the first dinucleotide product is substantially higher than that required for subsequent cycles of nucleotide addition. In vitro, promoter-specific differences in post-initiation control of transcription exist, as well as a second rate-limiting step that controls conversion of the transcription initiation complex into a transcription elongation complex. Rate-limiting steps of the biochemical pathways are often those that are targeted for regulation. Like the more complex multisubunit transcription systems, multiple steps may exist for control of transcription in human mitochondria. The tools and mechanistic framework presented here will facilitate not only the discovery of mechanisms regulating human mitochondrial transcription but also interrogation of the structure, function, and mechanism of the complexes that are regulated during human mitochondrial transcription.

FIGURE 1.

Human mitochondrial transcription machinery produced in E. coli supports promoter-dependent transcription on established plasmid-based templates in vitro. a, recombinant human mitochondrial transcription proteins used in this study. Proteins (5 μg) were resolved by SDS-PAGE on a 10% gel and visualized by Coomassie staining. b, plasmid-based templates for in vitro transcription. Schematic of pUC18-LSP, pUC18-HSP1, and pUC18-LSP-HSP1 plasmids used in this study (21). The position of unique restriction sites and the sizes (in nucleotides, nt) of the corresponding run-off transcripts are indicated. A 120-nt transcript will be produced from LSP regardless of the restriction enzyme used because of premature termination caused by the conserved sequence block II (CSBII). c, transcription assays. Reactions were performed by combining h-mtTFA (100 nm) and h-mtTFB2 (20 nm) with linearized plasmid DNA (4 nm) in reaction buffer containing NTP mix (400 μm ATP, 150 μm CTP, 150 μm GTP, 10 μm UTP, and 0.2 μCi/μl [α-³²P]UTP) at 32 °C, initiated by addition of h-mtRNAP (20 nm), and quenched after 30 min by addition of stop buffer. Products were resolved by denaturing PAGE on 5% gels and visualized by phosphorimaging. The size of run-off transcripts produced from the different linearized plasmid templates is indicated. The size of selected bands from a 10-bp DNA ladder (M) is indicated as a reference.

FIGURE 2.

Formation of an initiation complex that is resistant to heparin is dependent on ATP concentration. a, experimental design. Reactions were performed by combining h-mtTFA (100 nm), h-mtTFB2 (20 nm), and h-mtRNAP (20 nm) to linearized plasmid DNA (4 nm) in reaction buffer at 32 °C for 5 min. Some reactions were pulsed with ATP (400 μm) for 30 s prior to initiation of transcription with all four NTPs (400 μm ATP, 150 μm CTP, 150 μm GTP, 10 μm UTP, and 0.2 μCi/μl [α-³²P]UTP) in the absence or presence of heparin (1 μm). Reactions were quenched after a 30-min incubation. b, ATP is required to form heparin-resistant complexes. Products from reactions on LSP (left) or HSP1 (right) were resolved by denaturing PAGE on 5% gels and visualized by phosphorimaging. In the absence of heparin, products were formed independent of an ATP pulse on LSP (lanes 1 and 2) and HSP1 (lanes 5 and 6). In the presence of heparin, products were formed on LSP and HSP1 when pulsed with ATP (lanes 2 and 7) but not in the absence of the ATP pulse (lanes 4 and 8). The size of selected bands from a 10-bp DNA ladder (M) is indicated as a reference. c, ATP concentration dependence for transcription in the absence and presence of heparin. The reaction was performed in the absence (lanes 1–6) or presence (lanes 7–12) of heparin as described in b; however, the concentration of ATP used in the pulse was varied from 0.01 to 1 mm. d, formation of heparin-resistant (initiation) complexes requires high concentration of ATP. The RNA product data (volume reported by ImageQuant software) were plotted as a function of ATP concentration. Transcription efficiency reaches a plateau at 1 mm ATP only in the absence of heparin, suggesting that concentrations in excess of 1 mm are required for initiation. When the data are fit to a hyperbola, K_0.5(ATP) values for ATP of 0.2 and 2 mm are obtained in the absence and presence of heparin, respectively.

FIGURE 3.

DNA oligonucleotide-based templates for the study of human mitochondrial transcription in vitro. a, double-stranded DNA oligonucleotides used in this study. DNA oligonucleotides (90 bp) for LSP and HSP1 contained 50 bp upstream of the TSS (+1) and a 40-bp templating region. The reported h-mtTFA DNA-binding sites (h-mtTFA-BS) are highlighted (38). The top strand is the nontemplating strand (NTS); the bottom strand is the templating strand (TS). b, utilization of the LSP and HSP1 oligo templates by the polymerase requires h-mtTFA and h-mtTFB2. Reactions were performed by combining one or more of the following: h-mtTFA (0.5 μm), h-mtTFB2 (0.5 μm), and h-mtRNAP (0.5 μm) with LSP or HSP1 oligo (0.5 μm) in reaction buffer at 32 °C for 5 min prior to initiating transcription by addition of NTP mix (400 μm ATP, 150 μm CTP, 150 μm GTP, 10 μm UTP, and 0.2 μCi/μl [α-³²P]UTP). Reaction was quenched after 10 min. Products were resolved by denaturing PAGE on 15% gels and visualized by phosphorimaging. c, direct observation of early (abortive) initiation products. Reactions were performed by combining h-mtTFA (2.5 μm), h-mtTFB2 (2.5 μm), and h-mtRNAP (2.5 μm) with DNA oligonucleotide duplex (2.5 μm) in reaction buffer at 32 °C for 5 min prior to initiating transcription by addition of NTPs (500 μm each) containing 0.2 μCi/μl [α-³²P]ATP. Reactions were quenched at various times by addition of stop buffer. Product analysis was as described in b; however a 23% gel was used. d, production of full-length RNA is inefficient. The yield of 2-, 3-, and 40-mer RNA was determined on LSP and HSP1 oligos as described under “Experimental Procedures.”

FIGURE 4.

DNase I footprinting provides evidence for assembly of initiation complexes on LSP and HSP1 oligo templates. DNase I footprinting was performed using LSP and HSP1 DNA oligonucleotide templates. Proteins were assembled on the indicated ³²P-labeled DNA oligo (2.5 μm) by combining one or more of the following components: h-mtTFA (2.5 μm), h-mtTFB2 (2.5 μm), and h-mtRNAP (2.5 μm), as indicated, in reaction buffer at 32 °C. DNA cleavage was initiated by addition of RQ1 DNase (0.002 unit/μl) and CaCl₂ (1 mm) and quenched after 2 min by addition of stop/trap buffer. Products were resolved by denaturing PAGE on 8% gels and visualized by phosphorimaging. The locations/orientations of the h-mtTFA-binding site and transcription start site are indicated for the LSP and HSP1 promoters. A 10-bp ladder was used as a size marker (M). a, footprinting was performed on LSP and HSP1 containing ³²P in the 5′ end of the templating strand. b, quantification of selected lanes of the gels shown in a. The intensities were normalized to the −40 position of the control (lane 2 of LSP or HSP1 in a). Initiation complex refers to lane 6 of LSP or HSP1 in a. Asterisk refers to the location of the ³²P label.

FIGURE 5.

Initiation of RNA synthesis by using a dinucleotide RNA primer does not increase transcription efficiency relative to initiation de novo on LSP or HSP1. a, experimental design. Reactions were performed by combining h-mtTFA (2.5 μm), h-mtTFB2 (2.5 μm), and h-mtRNAP (2.5 μm) with DNA oligonucleotide duplex (2.5 μm) and ³²P-labeled dinucleotide (*pApA) RNA (10 μm) in reaction buffer at 32 °C for 5 min. The reaction was initiated by addition of NTPs (500 μm each) in the absence or presence of heparin (10 μm) and then quenched at various times by addition of stop buffer. Reaction products were resolved by denaturing PAGE on 25% gels and visualized by phosphorimaging. b, representative gel of an experiment using LSP as template. c, representative gel of an experiment using HSP1 as template. d, comparison of the average amount of the indicated RNA product formed at 120 s on LSP and HSP1 in the absence of heparin. e, comparison of the average amount of the indicated RNA product formed at 120 s on LSP or HSP1 in the presence of heparin.

FIGURE 6.

RNA synthesis initiated using a dinucleotide RNA primer requires lower concentrations of ATP for maximal transcriptional efficiency than RNA synthesis initiated de novo on LSP or HSP1. Reactions were performed by combining h-mtTFA (0.5 μm), h-mtTFB2 (0.5 μm), and h-mtRNAP (0.5 μm) with LSP or HSP1 oligo templates (0.5 μm) in the absence (−pApA) or presence (+pApA) of dinucleotide primer (10 μm) in reaction buffer at 32 °C for 5 min. Transcription was initiated by adding NTPs (150 μm GTP, 150 μm CTP, 50 μm UTP, and 0.2 μCi/μl [α-³²P]UTP) with the concentration of ATP ranging from 50 to 1000 μm. Reactions were quenched after 2 min by addition of stop buffer. Products were resolved by denaturing PAGE on 25% gels and visualized by phosphorimaging. a, representative gel of an experiment using LSP as template. b, representative gel of an experiment using HSP1 as template. c, RNA product data obtained on LSP in absence (−pApA, ■) or presence (+pApA, ●) of dinucleotide primer were expressed as a fraction of that observed at 1 mm ATP and plotted as a function of ATP concentration. The data were fit to a hyperbola, yielding K_0.5(ATP) values for ATP of 500 ± 90 μm and 70 ± 10 μm in the absence or presence of dinucleotide, respectively. d, RNA product data obtained on HSP1 in absence (−pApA, ■) or presence (+pApA, ●) of dinucleotide primer were expressed as a fraction of that observed at 1 mm ATP and plotted as a function of ATP concentration. The data were fit to a hyperbola, yielding K_0.5(ATP) values for ATP of 400 ± 60 and 60 ± 10 μm in the absence or presence of dinucleotide, respectively.

FIGURE 7.

Open complex formation is a rate-limiting step for transcription initiation on LSP and HSP1 revealed by using bubble templates. Reactions were performed by combining h-mtTFA (2.5 μm), h-mtTFB2 (2.5 μm), and h-mtRNAP (2.5 μm) with LSP or HSP1 bubble templates (2.5 μm) and ³²P-labeled dinucleotide (*pApA, 10 μm) in reaction buffer at 32 °C for 5 min. The reaction was initiated by addition of NTPs (500 μm each) in the presence or absence of heparin (10 μm) and then quenched at various times by addition of stop buffer. For the pretrap experiment, heparin (10 μm) was included during the assembly of proteins on DNA. a, representative gel of an experiment using LSP bubble template. b, representative gel of an experiment using HSP1 bubble template. c, comparison of the average amount of the indicated RNA product formed at 120 s on LSP and HSP1 bubble templates in the presence of heparin. d, comparison of the average amount of the indicated RNA product formed at 120 s on LSP and HSP1 in the absence of heparin.

FIGURE 8.

Unique factor dependence of LSP relative to HSP1 when bubble templates are used. Proteins were assembled on the LSP1 or HSP1 bubble template (2.5 μm) by combining one or more of the following components: h-mtTFA (2.5 μm), h-mtTFB2 (2.5 μm), and h-mtRNAP (2.5 μm), as indicated, and ³²P-labeled dinucleotide primer (*pApA, 10 μm) in reaction buffer at 32 °C for 5 min. The reaction was initiated by addition of NTPs (500 μm each) in the presence or absence of heparin (10 μm) and then quenched at various times by addition of stop buffer. Products were resolved by denaturing PAGE on 25% gels and visualized by phosphorimaging. a, representative gel of an experiment using LSP bubble template. b, representative gel of an experiment using HSP1 bubble template.

FIGURE 9.

Hypothetical model for the human mitochondrial transcription cycle. The images used of the proteins and DNA should be considered schematics but have been drawn to scale. Interactions of h-mtTFA with h-mtTFB2 and h-mtTFB2 with h-mtRNAP occur through carboxyl- or amino-terminal tails of h-mtTFA or h-mtTFB2, respectively, and are not shown explicitly (27, 29). The data presented in this study are consistent with the following model. Transcription factors and polymerase assemble on DNA to form an initiation complex by some unknown mechanism. Once formed, the initiation complex is quite stable. Open complexes do not accumulate based on potassium permanganate reactivity, creating a significant barrier to transcription initiation that can be bypassed by using bubble templates. Formation of the first phosphodiester bond requires high concentrations of ATP; therefore, the efficiency of this step will be controlled by the availability of ATP. Once a dinucleotide is formed, the dependence of the reaction on the concentration of ATP is reduced by a factor of 10. The transition into an elongation complex occurs at some point after formation of the trinucleotide, occurs inefficiently, and occurs differently on the two promoters.