G-quadruplex structures in RNA stimulate mitochondrial transcription termination and primer formation

Abstract

The human mitochondrial transcription machinery generates the primers required for initiation of leading-strand DNA replication. According to one model, the 3' end of the primer is defined by transcription termination at conserved sequence block II (CSB II) in the mitochondrial DNA control region. We here demonstrate that this site-specific termination event is caused by G-quadruplex structures formed in nascent RNA upon transcription of CSB II. We also demonstrate that a poly-dT stretch downstream of CSB II has a modest stimulatory effect on the termination efficiency. The mechanism is reminiscent of Rho-independent transcription termination in prokaryotes, with the exception that a G-quadruplex structure replaces the hairpin loop formed in bacterial mRNA during transcription of terminator sequences.

Fig. 1.

Premature termination of transcription is reduced in 7-deaza-GTP. (A) Partial sequence of the mtDNA control region. The LSP is denoted by a bent arrow. Positions of premature transcription termination (nucleotides 282–300) are shown in red. Conserved sequence blocks I–III are marked with lines above the sequence, and the guanines with potential to contribute to one of the predicted G-quadruplexes at CSB II are underlined. (B) Products of in vitro transcription on WT and mutant 318–288 CSB II template with GTP or 7-deaza-GTP (7-dzGTP) were separated on a 6% PAGE gel. RO, full-length runoff transcript; PT, preterminated transcript; asterisk denotes a minor product that was unaffected in 7-deaza-GTP and acts as an internal control.

Fig. 2.

The effect of ionic conditions and template topology on premature transcription termination. (A) Wt (lanes 1–6) and mutant (lanes 7–12) templates were transcribed in the presence of GTP (lanes 1–3, 7–9) or 7-deaza-GTP (lanes 4–6, 10–12) and 35 mM of added NaCl, KCl, or LiCl. PT(%), percentage of pretermination; M, marker lane. (B) In vitro transcription on SspI-linearized (lanes 1–3 and 7–9) or supercoiled (lanes 4–6, 10–12) template. A final concentration of 35 mM of the indicated ion was added, and reactions contained either GTP or 7-deaza-GTP. PT, preterminated transcript; RO lin, runoff transcript from linearized template (∼240 nts); RO sc, runoff transcripts from supercoiled template. M, marker lane.

Fig. 3.

Native gel analysis of oligonucleotides containing wild-type or mutated CSB II. (A) Wt or G → A mutant RNA oligonucleotides (5 pmol total, of which 1.4 pmol labeled) were incubated overnight at 37 °C and separated on a 12% native gel. DNA size marker in the right-hand lane. bi-G4, bimolecular quadruplex; uni-G4, unimolecular quadruplex; asterisk denotes the more compact uni-G4* species. (B) RNA oligos (WT and mutant) were incubated in increasing concentrations of NaCl, LiCl, or KCl (0, 50, 150, or 1,000 mM) overnight at 37 °C. Reactions were separated on a 12% native gel. bi-G4, bimolecular quadruplex; uni-G4, unimolecular quadruplex; asterisk denotes a more compact G-quadruplex conformation. The arrow indicates the linear form of oligo, which is seen only with the mutant lacking the Gs required for quadruplex formation.

Fig. 4.

Mutations in the G stretches of CSB II reduce pretermination. (Upper) In vitro transcription using a series of mutant templates with a G-to-A mutation at the indicated position. Transcription reactions contained GTP and 50 mM KCl. RO, full-length runoff transcript; PT, preterminated transcript; PT(%), average percentage of pretermination in three independent experiments. (Lower) The percentage of prematurely terminated transcript in transcription assays carried out in 50 mM KCl with either GTP (black bars) or 7-deaza-GTP (white bars) on mutant or wild-type template. The nucleotide position of the G → A mutation is indicated. N = 3 and error bars represent standard error of the mean.

Fig. 5.

CSBII mutants exhibiting altered G-quadruplex formation affect transcription pretermination accordingly. (A) Wild-type or mutant CSBII RNA oligonucleotides (5 pmol per reaction, of which 0,4 pmol labeled) with GG → AA alterations at nucleotide positions 296–295, 306–305, and 313–312 were incubated overnight at 37 °C and separated on a 12 % native gel. M, DNA marker. (B) DNA templates with GG → AA mutations corresponding to the ones in A were used in the transcription assay in parallel with wild-type and mutant 318–218 templates in the presence of 50 mM KCl and either GTP or 7-deaza-GTP. (C) Templates with mutations encompassing the poly-T (nt positions 291–286), poly-A (294–292), or both (294–286) runs downstream of CSB II were used in transcription assays in 50 mM KCl and either GTP or 7-deaza-GTP. RO, runoff transcript; PT, preterminated transcript; PT(%), average percentage of pretermination in three independent experiments; M, marker lane.