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Effects of Friedreich's ataxia (GAA)n*(TTC)n repeats on RNA synthesis and stability.

Krasilnikova MM, Kireeva ML, Petrovic V, Knijnikova N, Kashlev M, Mirkin SM - Nucleic Acids Res. (2007)

Bottom Line: To follow the effects of (GAA)n*(TTC)n repeats on gene expression, we have chosen E. coli as a convenient model system. (GAA)n*(TTC)n repeats were cloned into bacterial plasmids in both orientations relative to a promoter, and their effects on transcription and RNA stability were evaluated both in vitro and in vivo.Expanded (GAA)n repeats in the sense strand for transcription caused a significant decrease in the mRNA levels in vitro and in vivo.This decrease was likely due to the tardiness of the RNA polymerase within expanded (GAA)n runs but was not accompanied by the enzyme's dissociation and premature transcription termination.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, IL 60607, USA.

ABSTRACT
Expansions of (GAA)n repeats within the first intron of the frataxin gene reduce its expression, resulting in a hereditary neurodegenerative disorder, Friedreich's ataxia. While it is generally believed that expanded (GAA)n repeats block transcription elongation, fine mechanisms responsible for gene repression are not fully understood. To follow the effects of (GAA)n*(TTC)n repeats on gene expression, we have chosen E. coli as a convenient model system. (GAA)n*(TTC)n repeats were cloned into bacterial plasmids in both orientations relative to a promoter, and their effects on transcription and RNA stability were evaluated both in vitro and in vivo. Expanded (GAA)n repeats in the sense strand for transcription caused a significant decrease in the mRNA levels in vitro and in vivo. This decrease was likely due to the tardiness of the RNA polymerase within expanded (GAA)n runs but was not accompanied by the enzyme's dissociation and premature transcription termination. Unexpectedly, positioning of normal- and carrier-size (TTC)n repeats into the sense strand for transcription led to the appearance of RNA transcripts that were truncated within those repetitive runs in vivo. We have determined that these RNA truncations are consistent with cleavage of the full-sized mRNAs at (UUC)n runs by the E. coli degradosome.

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Related in: MedlinePlus

Schematic representation of the transcription experiment in vitro. Immobilized RNA polymerase binds to the RNA primer (arrow) annealed to a single-strand DNA oligonucleotide. A complementary non-template DNA is then added to the complex, creating a 3′ overhang for the repeat-containing fragment ligation. The repeat-containing fragment is excised from the plasmid and ligated to the elongation complex. After addition of ATP, CTP and GTP, the elongation complex is stalled upstream from the repeat. Three scenarios are possible after this stalled complex is released in the presence of all four NTPs: (i) successful transcription through the repeat; (ii) transcription arrest within the repeat; and (iii) transcription termination within the repeat.
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Figure 3: Schematic representation of the transcription experiment in vitro. Immobilized RNA polymerase binds to the RNA primer (arrow) annealed to a single-strand DNA oligonucleotide. A complementary non-template DNA is then added to the complex, creating a 3′ overhang for the repeat-containing fragment ligation. The repeat-containing fragment is excised from the plasmid and ligated to the elongation complex. After addition of ATP, CTP and GTP, the elongation complex is stalled upstream from the repeat. Three scenarios are possible after this stalled complex is released in the presence of all four NTPs: (i) successful transcription through the repeat; (ii) transcription arrest within the repeat; and (iii) transcription termination within the repeat.

Mentions: For these studies, we used the promoter-independent initiation with the immobilized transcription system (23). Briefly, the elongation complexes assembled on a synthetic DNA template with a 9-nt RNA primer, labeled at its 5′ end, were ligated in vitro to the DNA fragments containing (GAA)n·(TTC)n or a control sequence of a comparable length. The RNA polymerase was then ‘walked’ to the first UTP in position +46 in the presence of ATP, GTP and CTP. The resulting elongation complex was stalled just a few bases upstream from the repeat. Addition of all four NTPs allowed transcription run-off with three possible outcomes: (i) productive transcription through the repeat; (ii) transcription arrest within the repeat without RNA polymerase dissociation; or (iii) premature transcription termination inside the repeat (Figure 3). To access the transcription efficiency though the repeat, the amount of the 45-nt RNA (before chase) was compared with the amount of the run-off RNA from the complexes that successfully transcribed through the repeat.Figure 3.


Effects of Friedreich's ataxia (GAA)n*(TTC)n repeats on RNA synthesis and stability.

Krasilnikova MM, Kireeva ML, Petrovic V, Knijnikova N, Kashlev M, Mirkin SM - Nucleic Acids Res. (2007)

Schematic representation of the transcription experiment in vitro. Immobilized RNA polymerase binds to the RNA primer (arrow) annealed to a single-strand DNA oligonucleotide. A complementary non-template DNA is then added to the complex, creating a 3′ overhang for the repeat-containing fragment ligation. The repeat-containing fragment is excised from the plasmid and ligated to the elongation complex. After addition of ATP, CTP and GTP, the elongation complex is stalled upstream from the repeat. Three scenarios are possible after this stalled complex is released in the presence of all four NTPs: (i) successful transcription through the repeat; (ii) transcription arrest within the repeat; and (iii) transcription termination within the repeat.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1851639&req=5

Figure 3: Schematic representation of the transcription experiment in vitro. Immobilized RNA polymerase binds to the RNA primer (arrow) annealed to a single-strand DNA oligonucleotide. A complementary non-template DNA is then added to the complex, creating a 3′ overhang for the repeat-containing fragment ligation. The repeat-containing fragment is excised from the plasmid and ligated to the elongation complex. After addition of ATP, CTP and GTP, the elongation complex is stalled upstream from the repeat. Three scenarios are possible after this stalled complex is released in the presence of all four NTPs: (i) successful transcription through the repeat; (ii) transcription arrest within the repeat; and (iii) transcription termination within the repeat.
Mentions: For these studies, we used the promoter-independent initiation with the immobilized transcription system (23). Briefly, the elongation complexes assembled on a synthetic DNA template with a 9-nt RNA primer, labeled at its 5′ end, were ligated in vitro to the DNA fragments containing (GAA)n·(TTC)n or a control sequence of a comparable length. The RNA polymerase was then ‘walked’ to the first UTP in position +46 in the presence of ATP, GTP and CTP. The resulting elongation complex was stalled just a few bases upstream from the repeat. Addition of all four NTPs allowed transcription run-off with three possible outcomes: (i) productive transcription through the repeat; (ii) transcription arrest within the repeat without RNA polymerase dissociation; or (iii) premature transcription termination inside the repeat (Figure 3). To access the transcription efficiency though the repeat, the amount of the 45-nt RNA (before chase) was compared with the amount of the run-off RNA from the complexes that successfully transcribed through the repeat.Figure 3.

Bottom Line: To follow the effects of (GAA)n*(TTC)n repeats on gene expression, we have chosen E. coli as a convenient model system. (GAA)n*(TTC)n repeats were cloned into bacterial plasmids in both orientations relative to a promoter, and their effects on transcription and RNA stability were evaluated both in vitro and in vivo.Expanded (GAA)n repeats in the sense strand for transcription caused a significant decrease in the mRNA levels in vitro and in vivo.This decrease was likely due to the tardiness of the RNA polymerase within expanded (GAA)n runs but was not accompanied by the enzyme's dissociation and premature transcription termination.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, IL 60607, USA.

ABSTRACT
Expansions of (GAA)n repeats within the first intron of the frataxin gene reduce its expression, resulting in a hereditary neurodegenerative disorder, Friedreich's ataxia. While it is generally believed that expanded (GAA)n repeats block transcription elongation, fine mechanisms responsible for gene repression are not fully understood. To follow the effects of (GAA)n*(TTC)n repeats on gene expression, we have chosen E. coli as a convenient model system. (GAA)n*(TTC)n repeats were cloned into bacterial plasmids in both orientations relative to a promoter, and their effects on transcription and RNA stability were evaluated both in vitro and in vivo. Expanded (GAA)n repeats in the sense strand for transcription caused a significant decrease in the mRNA levels in vitro and in vivo. This decrease was likely due to the tardiness of the RNA polymerase within expanded (GAA)n runs but was not accompanied by the enzyme's dissociation and premature transcription termination. Unexpectedly, positioning of normal- and carrier-size (TTC)n repeats into the sense strand for transcription led to the appearance of RNA transcripts that were truncated within those repetitive runs in vivo. We have determined that these RNA truncations are consistent with cleavage of the full-sized mRNAs at (UUC)n runs by the E. coli degradosome.

Show MeSH
Related in: MedlinePlus