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Transcriptional and posttranscriptional regulation of Bacillus sp. CDB3 arsenic-resistance operon ars1.

Yu X, Zheng W, Bhat S, Aquilina JA, Zhang R - PeerJ (2015)

Bottom Line: Compared to other ars gene clusters, regulation of the Bacillus sp.CDB3 ars1 operon is more complex.It represents another example of specific mRNA degradation in the transporter gene region and possibly the first case of attenuator-mediated regulation of ars operons.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Biological Sciences, University of Wollongong , Wollongong, NSW , Australia.

ABSTRACT
Bacillus sp. CDB3 possesses a novel eight-gene ars cluster (ars1, arsRYCDATorf7orf8) with some unusual features in regard to expression regulation. This study demonstrated that the cluster is a single operon but can also produce a short three-gene arsRYC transcript. A hairpin structure formed by internal inverted repeats between arsC and arsD was shown to diminish the expression of the full operon, thereby probably acting as a transcription attenuator. A degradation product of the arsRYC transcript was also identified. Electrophoretic mobility shift analysis demonstrated that ArsR interacts with the ars1 promoter forming a protein-DNA complex that could be impaired by arsenite. However, no interaction was detected between ArsD and the ars1 promoter, suggesting that the CDB3 ArsD protein may not play a regulatory role. Compared to other ars gene clusters, regulation of the Bacillus sp. CDB3 ars1 operon is more complex. It represents another example of specific mRNA degradation in the transporter gene region and possibly the first case of attenuator-mediated regulation of ars operons.

No MeSH data available.


Mapping of RNA degradation product.(A) Electropherogram showing the result of primer extension assay. The extension product is indicated by arrowhead and size standards are labelled below. (B) The first 50 nucleotides sequence of CDB3 arsY RNA coding region indicating an inverted repeat (underlined) and 5’-end of 1.5-kb RNA (pointed by diamond).
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fig-3: Mapping of RNA degradation product.(A) Electropherogram showing the result of primer extension assay. The extension product is indicated by arrowhead and size standards are labelled below. (B) The first 50 nucleotides sequence of CDB3 arsY RNA coding region indicating an inverted repeat (underlined) and 5’-end of 1.5-kb RNA (pointed by diamond).

Mentions: To confirm the identity of the 1.5-kb RNA, a primer extension assay was conducted to map the 5’-end. Fluorescent labeled primer PED-R (29-bp) was annealed from 295 bp downstream of the arsY coding region. The electropherogram of extension products showed a predominant peak corresponding to a 261-bp cDNA fragment (Fig. 3A), demonstrating that the 5’-end of 1.5-kb RNA was the 35th bp of arsY. This nucleotide (uridine) is immediately downstream of an inverted repeat region in Y RNA sequence (Fig. 3B). This further supports the assumption that the smaller RNA resulted from degradation.


Transcriptional and posttranscriptional regulation of Bacillus sp. CDB3 arsenic-resistance operon ars1.

Yu X, Zheng W, Bhat S, Aquilina JA, Zhang R - PeerJ (2015)

Mapping of RNA degradation product.(A) Electropherogram showing the result of primer extension assay. The extension product is indicated by arrowhead and size standards are labelled below. (B) The first 50 nucleotides sequence of CDB3 arsY RNA coding region indicating an inverted repeat (underlined) and 5’-end of 1.5-kb RNA (pointed by diamond).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4562236&req=5

fig-3: Mapping of RNA degradation product.(A) Electropherogram showing the result of primer extension assay. The extension product is indicated by arrowhead and size standards are labelled below. (B) The first 50 nucleotides sequence of CDB3 arsY RNA coding region indicating an inverted repeat (underlined) and 5’-end of 1.5-kb RNA (pointed by diamond).
Mentions: To confirm the identity of the 1.5-kb RNA, a primer extension assay was conducted to map the 5’-end. Fluorescent labeled primer PED-R (29-bp) was annealed from 295 bp downstream of the arsY coding region. The electropherogram of extension products showed a predominant peak corresponding to a 261-bp cDNA fragment (Fig. 3A), demonstrating that the 5’-end of 1.5-kb RNA was the 35th bp of arsY. This nucleotide (uridine) is immediately downstream of an inverted repeat region in Y RNA sequence (Fig. 3B). This further supports the assumption that the smaller RNA resulted from degradation.

Bottom Line: Compared to other ars gene clusters, regulation of the Bacillus sp.CDB3 ars1 operon is more complex.It represents another example of specific mRNA degradation in the transporter gene region and possibly the first case of attenuator-mediated regulation of ars operons.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Biological Sciences, University of Wollongong , Wollongong, NSW , Australia.

ABSTRACT
Bacillus sp. CDB3 possesses a novel eight-gene ars cluster (ars1, arsRYCDATorf7orf8) with some unusual features in regard to expression regulation. This study demonstrated that the cluster is a single operon but can also produce a short three-gene arsRYC transcript. A hairpin structure formed by internal inverted repeats between arsC and arsD was shown to diminish the expression of the full operon, thereby probably acting as a transcription attenuator. A degradation product of the arsRYC transcript was also identified. Electrophoretic mobility shift analysis demonstrated that ArsR interacts with the ars1 promoter forming a protein-DNA complex that could be impaired by arsenite. However, no interaction was detected between ArsD and the ars1 promoter, suggesting that the CDB3 ArsD protein may not play a regulatory role. Compared to other ars gene clusters, regulation of the Bacillus sp. CDB3 ars1 operon is more complex. It represents another example of specific mRNA degradation in the transporter gene region and possibly the first case of attenuator-mediated regulation of ars operons.

No MeSH data available.