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A novel sigma factor reveals a unique regulon controlling cell-specific recombination in Mycoplasma genitalium.

Torres-Puig S, Broto A, Querol E, Piñol J, Pich OQ - Nucleic Acids Res. (2015)

Bottom Line: Herein, we found that MG428 activates transcription of recA, ruvA and ruvB as well as several genes with unknown function.Additionally, MG428 was found to interact with the RNAP core enzyme, reinforcing the predicted role of this protein as an alternative sigma factor.Finally, our results indicate that MG428 contributes to the generation of genetic diversity in this model organism.

View Article: PubMed Central - PubMed

Affiliation: Institut de Biotecnologia i Biomedicina and Departament de Bioquímica i Biologia Molecular. Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain.

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Identification of the TSSs of several MG428-regulated genes. Primer extension analysis of the MG_220, recA, MG_RS02200 and MG_414 genes in the WT strain and the Tn::recA-2 mutant. All electropherograms were generated with Peak Scanner v1.0 (Applied Biosystems) analysis software. Red peaks represent ROX size standards while blue peaks correspond to the primer extension products. Schematic representations of the genome regions analyzed are shown and the presence of the identified promoters indicated with blue arrows. The approximate location of the primers used in these analyses is also indicated by arrows.
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Figure 6: Identification of the TSSs of several MG428-regulated genes. Primer extension analysis of the MG_220, recA, MG_RS02200 and MG_414 genes in the WT strain and the Tn::recA-2 mutant. All electropherograms were generated with Peak Scanner v1.0 (Applied Biosystems) analysis software. Red peaks represent ROX size standards while blue peaks correspond to the primer extension products. Schematic representations of the genome regions analyzed are shown and the presence of the identified promoters indicated with blue arrows. The approximate location of the primers used in these analyses is also indicated by arrows.

Mentions: A putative promoter sequence with sigma-70 architecture was identified within the upstream region (UR) of the genes or operons up-regulated by the MG428 protein. This putative promoter was composed of two conserved elements of six residues separated by 18 or 19 nucleotides and showed the consensus 5′-TTGTCA-N18/19-ATTWAT-3′ (Figure 5A). Of note, a conserved sequence with sigma-70 promoter architecture was also recognized immediately upstream of all of the M. pneumoniae genes homologous to the members of the M. genitalium MG428-regulon. The conserved sequence identified in M. pneumoniae showed the consensus 5′-TTGGCR-N18/19-ATTYAT-3′ (Figure 5B). We conducted primer extension analyses of the recA, ruvA and MG_220 genes in the WT strain and the Tn::recA-2 mutant (Figure 6). In repeated experiments, we did not detect transcription initiation within the URs of these genes using RNA from the WT strain. In contrast, single, unequivocal transcriptional start sites (TSS) were identified using RNA from the Tn::recA-2 mutant. As expected, all the identified TSSs were located immediately downstream of the anticipated promoter sequences (Figure 5A). For the ruvA gene, we found that transcription initiated in the promoter region of the MG_RS02200 ORF, which precedes the ruvAB genes. This specific TSS was confirmed with a second primer complementary of the MG_RS02200 ORF region (Supplementary Figure S5). Therefore, MG428-dependent activation of the ruvAB genes is driven from the MG_RS02200 promoter.


A novel sigma factor reveals a unique regulon controlling cell-specific recombination in Mycoplasma genitalium.

Torres-Puig S, Broto A, Querol E, Piñol J, Pich OQ - Nucleic Acids Res. (2015)

Identification of the TSSs of several MG428-regulated genes. Primer extension analysis of the MG_220, recA, MG_RS02200 and MG_414 genes in the WT strain and the Tn::recA-2 mutant. All electropherograms were generated with Peak Scanner v1.0 (Applied Biosystems) analysis software. Red peaks represent ROX size standards while blue peaks correspond to the primer extension products. Schematic representations of the genome regions analyzed are shown and the presence of the identified promoters indicated with blue arrows. The approximate location of the primers used in these analyses is also indicated by arrows.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4446450&req=5

Figure 6: Identification of the TSSs of several MG428-regulated genes. Primer extension analysis of the MG_220, recA, MG_RS02200 and MG_414 genes in the WT strain and the Tn::recA-2 mutant. All electropherograms were generated with Peak Scanner v1.0 (Applied Biosystems) analysis software. Red peaks represent ROX size standards while blue peaks correspond to the primer extension products. Schematic representations of the genome regions analyzed are shown and the presence of the identified promoters indicated with blue arrows. The approximate location of the primers used in these analyses is also indicated by arrows.
Mentions: A putative promoter sequence with sigma-70 architecture was identified within the upstream region (UR) of the genes or operons up-regulated by the MG428 protein. This putative promoter was composed of two conserved elements of six residues separated by 18 or 19 nucleotides and showed the consensus 5′-TTGTCA-N18/19-ATTWAT-3′ (Figure 5A). Of note, a conserved sequence with sigma-70 promoter architecture was also recognized immediately upstream of all of the M. pneumoniae genes homologous to the members of the M. genitalium MG428-regulon. The conserved sequence identified in M. pneumoniae showed the consensus 5′-TTGGCR-N18/19-ATTYAT-3′ (Figure 5B). We conducted primer extension analyses of the recA, ruvA and MG_220 genes in the WT strain and the Tn::recA-2 mutant (Figure 6). In repeated experiments, we did not detect transcription initiation within the URs of these genes using RNA from the WT strain. In contrast, single, unequivocal transcriptional start sites (TSS) were identified using RNA from the Tn::recA-2 mutant. As expected, all the identified TSSs were located immediately downstream of the anticipated promoter sequences (Figure 5A). For the ruvA gene, we found that transcription initiated in the promoter region of the MG_RS02200 ORF, which precedes the ruvAB genes. This specific TSS was confirmed with a second primer complementary of the MG_RS02200 ORF region (Supplementary Figure S5). Therefore, MG428-dependent activation of the ruvAB genes is driven from the MG_RS02200 promoter.

Bottom Line: Herein, we found that MG428 activates transcription of recA, ruvA and ruvB as well as several genes with unknown function.Additionally, MG428 was found to interact with the RNAP core enzyme, reinforcing the predicted role of this protein as an alternative sigma factor.Finally, our results indicate that MG428 contributes to the generation of genetic diversity in this model organism.

View Article: PubMed Central - PubMed

Affiliation: Institut de Biotecnologia i Biomedicina and Departament de Bioquímica i Biologia Molecular. Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain.

Show MeSH
Related in: MedlinePlus