The Selenocysteine tRNA Gene in Leishmania major Is Transcribed by both RNA Polymerase II and RNA Polymerase III

Padilla-Mejía, Norma E.; Florencio-Martínez, Luis E.; Moreno-Campos, Rodrigo; Vizuet-de-Rueda, Juan Carlos; Cevallos, Ana Marı́a; Hernández‐Rivas, Rosaura; Manning‐Cela, Rebeca; Martínez‐Calvillo, Santiago

doi:10.1128/ec.00239-14

Cited by 12 publications

(7 citation statements)

References 51 publications

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“…The three eukaryotic RNA polymerases are specific for their own promoters, though some exceptions have been found where more than one polymerase can use the same promoter (James Faresse et al 2012; Duttke 2014; Padilla-Mejia et al 2015). Using specific enzyme inhibitors in human cells and extracts (Seidl et al 2013), we previously found evidence of coligo transcription exclusively by Pol III.…”

Section: Resultsmentioning

confidence: 99%

RNA polymerase III initiation on coligo DNA templates containing loops of variable sequence, size and nucleotide chemistry

Patel

Lama

Hoffmann

et al. 2017

Gene

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Circularized oligonucleotides, or coligos, were previously found to serve as RNA polymerase III (Pol III) templates in vitro and in human tissue culture cells. Here we randomized the 12-nucleotide larger loop (L-loop) of a well characterized coligo and found unexpectedly that in vitro transcription by FLAG-Pol III was not significantly affected. This observation allowed us to test the variable of coligo L-loop size separately from the variable of its sequence. Transcription efficiency increased with L-loop size from 3 to 12 nucleotides of randomized sequence, and the smallest loop forced initiation to move into the stem region. To test further the need for any specific sequence we compared seven nucleotide L-loops composed of random, abasic and abasic-acyclic nucleotides, and all supported transcription by Pol III. Transcription of a series of coligos containing twelve contiguous randomized nucleotides placed at different locations within the coligo structure provided further evidence that the stem-loop junction structure is important for precise initiation. Nearly the same transcript pattern was formed in vitro by Pol III from yeast and human cells. Overall, these experiments support structure, rather than L-loop sequence, as the major determinant of coligo transcription initiation by Pol III.

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Section: Resultsmentioning

confidence: 99%

RNA polymerase III initiation on coligo DNA templates containing loops of variable sequence, size and nucleotide chemistry

Patel

Lama

Hoffmann

et al. 2017

Gene

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“…These assays were performed as described before [30, 36] with isolated nuclei from 2 × 10 8 mid-log promastigotes. Labeled nascent RNA was hybridized to Hybond-N+ membranes (GE Healthcare) containing dots of 2 μ g of plasmid DNA.…”

Section: Methodsmentioning

confidence: 99%

TFIIIB Subunit Bdp1 Participates in RNA Polymerase III Transcription in the Protozoan Parasite Leishmania major

Román‐Carraro

Florencio-Martínez

Romero-Meza

et al. 2019

BioMed Research International

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Leishmania major, a protozoan parasite that diverged early from the main eukaryotic lineage, exhibits unusual mechanisms of gene expression. Little is known in this organism about the transcription factors involved in the synthesis of tRNA, 5S rRNA, and snRNAs, transcribed by RNA Polymerase III (Pol III). Here we identify and characterize the TFIIIB subunit Bdp1 in L. major (LmBdp1). Bdp1 plays key roles in Pol III transcription initiation in other organisms, as it participates in Pol III recruitment and promoter opening. In silico analysis showed that LmBdp1 contains the typical extended SANT domain as well as other Bdp1 conserved regions. Nevertheless, LmBdp1 also displays distinctive features, including the presence of only one aromatic residue in the N-linker region. We were not able to produce null mutants of LmBdp1 by homologous recombination, as the obtained double replacement cell line contained an extra copy of LmBdp1, indicating that LmBdp1 is essential for the viability of L. major promastigotes. Notably, the mutant cell line showed reduced levels of the LmBdp1 protein, and its growth was significantly decreased in relation to wild-type cells. Nuclear run-on assays demonstrated that Pol III transcription was affected in the mutant cell line, and ChIP experiments showed that LmBdp1 binds to 5S rRNA, tRNA, and snRNA genes. Thus, our results indicate that LmBdp1 is an essential protein required for Pol III transcription in L. major.

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“…These experiments were performed as described elsewhere (Martinez-Calvillo et al 2003;Padilla-Mejia et al 2015), with nuclei isolated from 2 × 10 8 mid-log cells incubated for 48 h in the presence of doxycycline. Labelled nascent RNA was hybridized to Hybond filters (Amersham) containing dots of 2 µg of plasmid DNA.…”

Section: Nuclear Run-on Experimentsmentioning

confidence: 99%

BRF1, a subunit of RNA polymerase III transcription factor TFIIIB, is essential for cell growth ofTrypanosoma brucei

et al. 2015

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RNA polymerase III (Pol III) synthesizes small RNA molecules that are essential for cell viability. Accurate initiation of transcription by Pol III requires general transcription factor TFIIIB, which is composed of three subunits: TFIIB-related factor BRF1, TATA-binding protein and BDP1. Here we report the molecular characterization of BRF1 in Trypanosoma brucei (TbBRF1), a parasitic protozoa that shows distinctive transcription characteristics. In silico analysis allowed the detection in TbBRF1 of the three conserved domains located in the N-terminal region of all BRF1 orthologues, namely a zinc ribbon motif and two cyclin repeats. Homology modelling suggested that, similarly to other BRF1 and TFIIB proteins, the TbBRF1 cyclin repeats show the characteristic structure of five α-helices per repeat, connected by a short random-coiled linker. As expected for a transcription factor, TbBRF1 was localized in the nucleus. Knock-down of TbBRF1 by RNA interference (RNAi) showed that this protein is essential for the viability of procyclic forms of T. brucei, since ablation of TbBRF1 led to growth arrest of the parasites. Nuclear run-on and quantitative real-time PCR analyses demonstrated that transcription of all the Pol III-dependent genes analysed was reduced, at different levels, after RNAi induction.

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The Selenocysteine tRNA Gene in Leishmania major Is Transcribed by both RNA Polymerase II and RNA Polymerase III

Cited by 12 publications

References 51 publications

RNA polymerase III initiation on coligo DNA templates containing loops of variable sequence, size and nucleotide chemistry

RNA polymerase III initiation on coligo DNA templates containing loops of variable sequence, size and nucleotide chemistry

TFIIIB Subunit Bdp1 Participates in RNA Polymerase III Transcription in the Protozoan Parasite Leishmania major

BRF1, a subunit of RNA polymerase III transcription factor TFIIIB, is essential for cell growth ofTrypanosoma brucei

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