The GAA*TTC triplet repeat expanded in Friedreich's ataxia impedes transcription elongation by T7 RNA polymerase in a length and supercoil dependent manner - PubMed (original) (raw)
The GAA*TTC triplet repeat expanded in Friedreich's ataxia impedes transcription elongation by T7 RNA polymerase in a length and supercoil dependent manner
E Grabczyk et al. Nucleic Acids Res. 2000.
Abstract
Large expansions of the trinucleotide repeat GAA*TTC within the first intron of the X25 (frataxin) gene cause Friedreich's ataxia, the most common inherited ataxia. Expansion leads to reduced levels of frataxin mRNA in affected individuals. Here we show that GAA*TTC tracts, in the absence of any other frataxin gene sequences, can reduce the amount of GAA-containing transcript produced in a defined in vitro transcription system. This effect is due to an impediment to elongation that forms in the GAA*TTC tract during transcription, a phenomenon that is exacerbated by both superhelical stress and increased tract length. On supercoiled templates the major truncations of the GAA-containing transcripts occur in the distal (3') end of the GAA repeat. To account for these observations we present a model in which an RNA polymerase advancing within a long GAA*TTC tract initiates the transient formation of an R*R*Y intramolecular DNA triplex. The non-template (GAA) strand folds back creating a loop in the template strand, and the polymerase is paused at the distal triplex-duplex junction.
Figures
Figure 1
GAA•TTC tracts inhibit transcript production, but not initiation. (A) Templates, digested with the restriction enzyme _Ssp_I which cuts 512 bp 3′ of the repeats, were transcribed as described in Materials and Methods. The site of T7 initiation is 55 bp 5′ of the repeat. After phenol extraction, precipitation with ethanol and resuspension in loading buffer the material was resolved on an agarose gel and then stained with ethidium bromide. The faint band above each template is an artifact of sample preparation. The number of GAA triplets in the transcripts is indicated above the lanes. Lane M is the 1 kb DNA ladder (Life Technologies). (B) Autoradiograph showing the products of abortive transcription reactions performed on the same templates used in (A). Only GTP and ATP were present (500 µM each) with [γ-32P]GTP (25 µCi per reaction) to label the 5′ end of the reaction products. Samples were taken after 2 and 20 min. The number of repeats in each template is indicated above the lane. The products were separated on a 23% denaturing polyacrylamide gel. The arrow indicates the position of the hexamer products. The GAA•TTC repeat tracts begin ∼50 bp beyond the termination of the abortive reactions and were not transcribed. The lane marked with a minus sign contains the product of a 20 min reaction with no template.
Figure 2
Exogenous RNA has no effect on the transcription of (GAA•TTC)88 templates. Exogenous control (GAA)0 RNA was produced from a template digested with _Ssp_I (568 base transcript) and exogenous (GAA)88 RNA was prepared from an _Xho_I digested template (380 base transcript). The amounts added approximated the usual transcript yield obtained in this type of reaction for the (GAA•TTC)88 template (1×) and for the control (4×). Reactions were stopped with 2 vol formamide loading buffer, then heated to 65°C before loading to denature secondary structures. (A) Ethidium bromide stained agarose gel displays the transcripts (black arrow, 834 base transcript) produced from a (GAA•TTC)88 template linearized with _Ssp_I and transcribed in the presence of exogenous control RNA (lanes 2 and 3) and (GAA)88 RNA (lanes 4 and 5). (B) Ethidium bromide stained agarose gel displays the transcripts (black arrow, 773 base transcript) produced from a control template with no repeats linearized with _Xmn_I and transcribed in the presence of exogenous control RNA (lanes 2 and 3) and (GAA)88 RNA (lanes 4 and 5).
Figure 3
Transcription through a long GAA•TTC tract reduces transcription of a second template in trans. Transcription reactions were performed in the presence of [γ-32P]GTP (10 µCi per reaction) on aliquots of _Ssp_I linearized templates with no repeat (lanes 1–3), (GAA•TTC)88 (lanes 4–6) or both templates together (lanes 7–9). Arrows indicate the locations of the two transcripts in this denaturing gel. Serial 5-fold dilutions of T7 RNAP (10, 2 and 0.4 U per 20 µl reaction) were used for each set of three (indicated by a triangle). The concentration of each template was kept constant, in the mixed reactions the total template DNA concentration was therefore doubled. The smearing below the transcript with 88 repeats comes from deletions within the repeat in the template used for this experiment, and is not due to RNase activity.
Figure 4
Negative supercoils exacerbate transcription inhibition by a (GAA•TTC)88 tract. (A) The templates used in these experiments contain the sequence for a self-cleaving ribozyme that cuts the transcript ∼270 bases 3′ to the end of the repeat tract, so the size of the full-length cleaved transcript (590 bases) is the same for both linear (L) and supercoiled (SC) templates. Linear templates were opened with restriction enzyme _Ssp_I and the primary transcript was 2698 bases. The templates produced RNA containing either (CUG)88 or (GAA)88 as indicated above the lanes. Templates were transcribed in the presence of [γ-32P]GTP (10 µCi per reaction). Bands immediately below the full-length transcripts extending to a length of ∼350 bases are due to deletions within the repeats in the templates. The numbers to the left indicate the size in bases of selected bands of the _Msp_I digest of pBR322 used as a marker. (B) A scan of lane 4 aligned to the gel highlights the distribution of truncation products within the (GAA)88 tract. The bracket to the right of the scan labeled repeat tract indicates the location of the 88 triplets within the 5′ end-labeled transcripts in both the scan in (B) and the gel in (A).
Figure 5
A pH dependent inhibition of (UUC)n but not (GAA)n synthesis. (A) Line drawings of intramolecular R•R•Y and Y•R•Y triplexes. The purine (R) strand is black, the pyrimidine (Y) strand is gray. The single black dots indicate normal Watson–Crick base pairs and the smaller double dots indicate alternative hydrogen bonding interactions that are pH independent. Hoogsteen base pairs involving a protonated cytosine are indicated with a plus sign. (B) and (C) Ethidium bromide stained agarose gels display the products of transcription containing the indicated number of GAA or UUC triplets. Aliquots of the _Ssp_I linearized templates were transcribed in reactions buffered at pH 8.0 (left half of the gel) or pH 7.0 (right half). The reactions were stopped by adding an equal volume of loading buffer (25 mM EDTA, 200 mM Tris pH 8.0, 10% v/v glycerol) and loaded directly on a 1% agarose gel with TAE pH 8.0 as the running buffer. (B) GAA-containing transcript accumulation is similar at both pH 8.0 and pH 7.0. Transcribed templates directing GAA 44 and 88 transcription exhibit smearing at both pHs when loaded directly from the transcription reaction, but the same amounts were used in the reactions as the controls (compare to Fig.1). (C) UUC-containing transcript accumulation, from templates in which the orientation of the GAA•TTC repeat relative to the T7 promoter had been reversed, transcribed at pH 8.0 and pH 7.0. Templates producing 44 and 88 UUC triplets exhibit smearing after transcription at pH 7, and transcript production is reduced compared to transcription at pH 8. Lane M is the 1 kb DNA ladder
Figure 6
DEPC reactivity of an oligonucleotide containing 5′-(GAA)22-(TTC)11-3′. (A) An autoradiograph of a denaturing 20% polyacrylamide gel showing the DEPC-specific piperidine cleavage of an end-labeled oligonucleotide containing 5′-(GAA)22-(TTC)11-3′ in the absence (lane 1) or presence (lane 2) of Mg2+ (6 mM). The numbers to the left of the gel indicate the GAA triplets from the 5′ end of the oligonucleotide. A line drawing to the right shows the general correspondence between bands on the gel and the linear oligonucleotide. The black portion indicates the (GAA)22 tract and the gray part corresponds to (TTC)11. Full-length, uncleaved material forms a dark band at the top of the gel in both lanes. The gray arrow indicates a region of Mg2+ independent DEPC hyper-reactivity; the black arrow indicates a region of Mg2+ reactivity. (B) and (C) Diagrammatic representation of the structure of the oligonucleotide in the absence (B) and presence (C) of Mg2+. The single black dots indicate Watson–Crick bonds and the smaller double dots indicate alternative hydrogen bonding interactions between different regions of the oligonucleotide. The gray arrow indicates the junction between the two tracts that is hyper-reactive with DEPC under these conditions. The black arrow in (C) indicates the DEPC reactive region seen in the middle of the GAA tract in the presence of Mg2+.
Figure 7
An intramolecular R•R•Y triplex as a structural impediment to transcription through the GAA•TTC repeat tract. Ribbon diagram showing the model for transcription dependent triplex formation leading to a pause at the promoter distal end of the structure. The GAA (R) strand is shown as black, the TTC (Y) strand is shown as white, and the flanking DNA is gray. (A) A standing wave of negative supercoiling follows RNA polymerase as it enters the GAA•TTC repeat tract. Underwound DNA is shown as a widened helix, the direction of the rotation imparted by the motion of the polymerase is shown by the curved arrow. (B) The non-template (GAA) strand is available to fold back in an R•R•Y interaction; the template strand is covered by RNAP. (C) Relaxation of negative supercoils by rotation of the helix (curved arrow shows direction) as it winds in the third strand aids in the formation of the triplex. (D) RNA polymerase is paused at the triplex/duplex junction in the distal end of the GAA•TTC tract (black arrow).
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