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Mitochondrial Transcription Factor A (TFAM) Binds to RNA Containing 4-Way Junctions and Mitochondrial tRNA.

Brown TA, Tkachuk AN, Clayton DA - PLoS ONE (2015)

Bottom Line: Kinetic binding assays and RNase-insensitive TFAM distribution indicate that DNA remains the preferred substrate within the nucleoid.The amount of each immunoprecipitated tRNA is not well correlated with tRNA celluar abundance, indicating unequal TFAM binding preferences.TFAM-mt-tRNA interaction suggests potentially new functions for this protein.

View Article: PubMed Central - PubMed

Affiliation: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, United States of America.

ABSTRACT
Mitochondrial DNA (mtDNA) is maintained within nucleoprotein complexes known as nucleoids. These structures are highly condensed by the DNA packaging protein, mitochondrial Transcription Factor A (TFAM). Nucleoids also include RNA, RNA:DNA hybrids, and are associated with proteins involved with RNA processing and mitochondrial ribosome biogenesis. Here we characterize the ability of TFAM to bind various RNA containing substrates in order to determine their role in TFAM distribution and function within the nucleoid. We find that TFAM binds to RNA-containing 4-way junctions but does not bind appreciably to RNA hairpins, internal loops, or linear RNA:DNA hybrids. Therefore the RNA within nucleoids largely excludes TFAM, and its distribution is not grossly altered with removal of RNA. Within the cell, TFAM binds to mitochondrial tRNAs, consistent with our RNA 4-way junction data. Kinetic binding assays and RNase-insensitive TFAM distribution indicate that DNA remains the preferred substrate within the nucleoid. However, TFAM binds to tRNA with nanomolar affinity and these complexes are not rare. TFAM-immunoprecipitated tRNAs have processed ends, suggesting that binding is not specific to RNA precursors. The amount of each immunoprecipitated tRNA is not well correlated with tRNA celluar abundance, indicating unequal TFAM binding preferences. TFAM-mt-tRNA interaction suggests potentially new functions for this protein.

No MeSH data available.


TFAM binding of complex DNA and RNA substrates by EMSA.Varying amounts of TFAM were bound to 20 fM of each biotinylated substrate with as follows; (A) Stem-loop RNA, 2.4–0.0375 μM TFAM with two-fold serial dilutions, (B) dsRNA with internal 8 nucleotide mismatch loop, TFAM dilutions as in (A), apparent Kd of 2.04 μM, (C) Alternating, four arm RNA:DNA 4-way junction, TFAM dilutions as in (A), apparent Kd of 299 nM, (D) RNA 4-way junction, 600–9.375 nM TFAM with two-fold serial dilutions, apparent Kd of 270 nM, (E) Mixed pairing RNA and DNA 4-way junction, TFAM dilutions as in (A), apparent Kd of 63 nM, (F) DNA 4-way junction, TFAM dilutions as in (D), apparent Kd of 63 nM. Left lane in each panel is free template without TFAM. Substrate diagrams appear to the right of each panel with RNA depicted in red and DNA in blue.
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pone.0142436.g002: TFAM binding of complex DNA and RNA substrates by EMSA.Varying amounts of TFAM were bound to 20 fM of each biotinylated substrate with as follows; (A) Stem-loop RNA, 2.4–0.0375 μM TFAM with two-fold serial dilutions, (B) dsRNA with internal 8 nucleotide mismatch loop, TFAM dilutions as in (A), apparent Kd of 2.04 μM, (C) Alternating, four arm RNA:DNA 4-way junction, TFAM dilutions as in (A), apparent Kd of 299 nM, (D) RNA 4-way junction, 600–9.375 nM TFAM with two-fold serial dilutions, apparent Kd of 270 nM, (E) Mixed pairing RNA and DNA 4-way junction, TFAM dilutions as in (A), apparent Kd of 63 nM, (F) DNA 4-way junction, TFAM dilutions as in (D), apparent Kd of 63 nM. Left lane in each panel is free template without TFAM. Substrate diagrams appear to the right of each panel with RNA depicted in red and DNA in blue.

Mentions: RNA most often forms structures that are not simply linear. We therefore assayed the capacity of TFAM to bind more complex substrates. EMSA data show that TFAM is unable to bind to a dsRNA hairpin loop, which is a common secondary structure in RNA (Fig 2A). However, TFAM is able to bind another common dsRNA structure that contains an internal loop (Fig 2B). This binding is weak and occurs with an apparent Kd of 2.04 μM, which is over 300-fold higher than the apparent Kd for the LSP dsDNA. TFAM has also been shown to bind to DNA 4-way junctions, which are typical of HMG-box proteins [14]. We have confirmed this and estimate TFAM binding with an apparent Kd of 63 nM. We then looked for the ability of TFAM to bind to RNA- and RNA:DNA-containing 4-way junction substrates and were able to qualitatively demonstrate binding. Several of the 4-way junction substrates yielded two binding isoforms that fill sequentially with increasing TFAM. The Hill coefficients for the 4-way substrates were 1.6–3, indicating cooperative binding of TFAM. This is consistent with previous studies in which TFAM binding to specific and nonspecific substrates yielded Hill coefficients of 1.5–2. [19]. We have estimated the equilibrium dissociation constants for these substrates by combining the bound forms and referring to the cumulative binding as the apparent Kd. Binding affinity estimates indicate that the RNA-only 4-way junction has an apparent Kd of 270 nM (Fig 2D). The apparent affinity of TFAM for RNA:DNA hybrid 4-way junctions depended partially on the arrangement of the hybrid strands. The 4-way junction that is all RNA:DNA hybrid has an apparent Kd that is similar (~299 nM) to the all-RNA 4-way junction. However, when the 4-way structure contains some RNA:DNA, some RNA:RNA, and some DNA:DNA paired structure, the apparent TFAM affinity is greater (Kd ~63 nM), and is identical to the all-DNA 4-way junction (Kd ~63 nM). This likely indicates that TFAM is preferentially binding to the dsDNA arm of this substrate when given a mixed arrangement around the junction.


Mitochondrial Transcription Factor A (TFAM) Binds to RNA Containing 4-Way Junctions and Mitochondrial tRNA.

Brown TA, Tkachuk AN, Clayton DA - PLoS ONE (2015)

TFAM binding of complex DNA and RNA substrates by EMSA.Varying amounts of TFAM were bound to 20 fM of each biotinylated substrate with as follows; (A) Stem-loop RNA, 2.4–0.0375 μM TFAM with two-fold serial dilutions, (B) dsRNA with internal 8 nucleotide mismatch loop, TFAM dilutions as in (A), apparent Kd of 2.04 μM, (C) Alternating, four arm RNA:DNA 4-way junction, TFAM dilutions as in (A), apparent Kd of 299 nM, (D) RNA 4-way junction, 600–9.375 nM TFAM with two-fold serial dilutions, apparent Kd of 270 nM, (E) Mixed pairing RNA and DNA 4-way junction, TFAM dilutions as in (A), apparent Kd of 63 nM, (F) DNA 4-way junction, TFAM dilutions as in (D), apparent Kd of 63 nM. Left lane in each panel is free template without TFAM. Substrate diagrams appear to the right of each panel with RNA depicted in red and DNA in blue.
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pone.0142436.g002: TFAM binding of complex DNA and RNA substrates by EMSA.Varying amounts of TFAM were bound to 20 fM of each biotinylated substrate with as follows; (A) Stem-loop RNA, 2.4–0.0375 μM TFAM with two-fold serial dilutions, (B) dsRNA with internal 8 nucleotide mismatch loop, TFAM dilutions as in (A), apparent Kd of 2.04 μM, (C) Alternating, four arm RNA:DNA 4-way junction, TFAM dilutions as in (A), apparent Kd of 299 nM, (D) RNA 4-way junction, 600–9.375 nM TFAM with two-fold serial dilutions, apparent Kd of 270 nM, (E) Mixed pairing RNA and DNA 4-way junction, TFAM dilutions as in (A), apparent Kd of 63 nM, (F) DNA 4-way junction, TFAM dilutions as in (D), apparent Kd of 63 nM. Left lane in each panel is free template without TFAM. Substrate diagrams appear to the right of each panel with RNA depicted in red and DNA in blue.
Mentions: RNA most often forms structures that are not simply linear. We therefore assayed the capacity of TFAM to bind more complex substrates. EMSA data show that TFAM is unable to bind to a dsRNA hairpin loop, which is a common secondary structure in RNA (Fig 2A). However, TFAM is able to bind another common dsRNA structure that contains an internal loop (Fig 2B). This binding is weak and occurs with an apparent Kd of 2.04 μM, which is over 300-fold higher than the apparent Kd for the LSP dsDNA. TFAM has also been shown to bind to DNA 4-way junctions, which are typical of HMG-box proteins [14]. We have confirmed this and estimate TFAM binding with an apparent Kd of 63 nM. We then looked for the ability of TFAM to bind to RNA- and RNA:DNA-containing 4-way junction substrates and were able to qualitatively demonstrate binding. Several of the 4-way junction substrates yielded two binding isoforms that fill sequentially with increasing TFAM. The Hill coefficients for the 4-way substrates were 1.6–3, indicating cooperative binding of TFAM. This is consistent with previous studies in which TFAM binding to specific and nonspecific substrates yielded Hill coefficients of 1.5–2. [19]. We have estimated the equilibrium dissociation constants for these substrates by combining the bound forms and referring to the cumulative binding as the apparent Kd. Binding affinity estimates indicate that the RNA-only 4-way junction has an apparent Kd of 270 nM (Fig 2D). The apparent affinity of TFAM for RNA:DNA hybrid 4-way junctions depended partially on the arrangement of the hybrid strands. The 4-way junction that is all RNA:DNA hybrid has an apparent Kd that is similar (~299 nM) to the all-RNA 4-way junction. However, when the 4-way structure contains some RNA:DNA, some RNA:RNA, and some DNA:DNA paired structure, the apparent TFAM affinity is greater (Kd ~63 nM), and is identical to the all-DNA 4-way junction (Kd ~63 nM). This likely indicates that TFAM is preferentially binding to the dsDNA arm of this substrate when given a mixed arrangement around the junction.

Bottom Line: Kinetic binding assays and RNase-insensitive TFAM distribution indicate that DNA remains the preferred substrate within the nucleoid.The amount of each immunoprecipitated tRNA is not well correlated with tRNA celluar abundance, indicating unequal TFAM binding preferences.TFAM-mt-tRNA interaction suggests potentially new functions for this protein.

View Article: PubMed Central - PubMed

Affiliation: Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, United States of America.

ABSTRACT
Mitochondrial DNA (mtDNA) is maintained within nucleoprotein complexes known as nucleoids. These structures are highly condensed by the DNA packaging protein, mitochondrial Transcription Factor A (TFAM). Nucleoids also include RNA, RNA:DNA hybrids, and are associated with proteins involved with RNA processing and mitochondrial ribosome biogenesis. Here we characterize the ability of TFAM to bind various RNA containing substrates in order to determine their role in TFAM distribution and function within the nucleoid. We find that TFAM binds to RNA-containing 4-way junctions but does not bind appreciably to RNA hairpins, internal loops, or linear RNA:DNA hybrids. Therefore the RNA within nucleoids largely excludes TFAM, and its distribution is not grossly altered with removal of RNA. Within the cell, TFAM binds to mitochondrial tRNAs, consistent with our RNA 4-way junction data. Kinetic binding assays and RNase-insensitive TFAM distribution indicate that DNA remains the preferred substrate within the nucleoid. However, TFAM binds to tRNA with nanomolar affinity and these complexes are not rare. TFAM-immunoprecipitated tRNAs have processed ends, suggesting that binding is not specific to RNA precursors. The amount of each immunoprecipitated tRNA is not well correlated with tRNA celluar abundance, indicating unequal TFAM binding preferences. TFAM-mt-tRNA interaction suggests potentially new functions for this protein.

No MeSH data available.