Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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5'-RACE-Seq analysis of Salmonella hilD and leuO promoters' response to adjacent transcription

ABSTRACT: Gene silencing by nucleoid-associated protein H-NS is sensitive to neighbouring transcription. This effect does not require neighbouring transcription to elongate into the silenced region. Here we measured how activating transcription of a tetRA cassette positioned upstream of either hilD or leuO promoters of Salmonella affected the activity of these promoters. The tetRA cassette included a strong Rho-independent transcription terminator at the 3' end of tetA. Transcription of the cassette was activated with anhydrotetracycline (AHTc). The activities of hilD and leuO promoters were measured upon performing template-switching reverse transcription follwed by semi-quantitative PCR and Illumina high-throughput sequencing.

INSTRUMENT(S): NextSeq 500

ORGANISM(S): Salmonella enterica subsp. enterica

SUBMITTER: Lionello Bossi

PROVIDER: E-MTAB-13482 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Pervasive Transcription originates from spurious promoters present in both orientations within coding sequences. Spurious transcripts are typically untranslated and thus for the most part are terminated by transcription termination factor Rho. They can be studied by impairing Rho activity, which in this study was achieved by depleting cells for Rho co-factor NusG. To this end, strains in which the only chromosomal copy of the nusG gene is transcribed from a promoter that can be repressed by arabinose (ARA) were used. This study was aimed at determining how pervasive transcription affect H-NS mediated gene silencing in Salmonella Pathogenicity Island 1 (SPI-1). In particular, the analysis concentrated on the region surrounding the master regulator gene HilD. The activities of promoters in this region were assessed performing semiquantitative 5’RACE-Seq. RNA extracted from cells exposed to the indicated treatments was reverse-transcribed with gene-specific primers in the presence of a template-switching oligonucleotide (TSO). The latter carries three 3’-terminal riboguanosines that hybridize to the polycytosine overhang (typically 3 or 4 Cs) added by the RT enzyme when it reaches the 5’ end of the RNA.This allows the RT enzyme to switch template and polymerise the TSO. Subsequent PCR amplification, with a primer complementary to the TSO and another annealing inside the reverse transcribed region, followed by sequence analysis, allows identifying the 5’ end of the RNA as the sequence lying immediately adjacent to the poly-G track. Performing the PCR step semiquantitatively provides information as to relative RNA levels. Here, the technique was adapted to high throughput sequencing by using PCR primers carrying Illumina adapters at their 5’ ends. Three strains were used, all three carrying the ARA-repressible nusG allele, MA12996, MA14302 and MA14358. This last strain carries a deleted version of SPI-1 with the Anhydrotetracycline (AHTc)-inducible Ptet promoter positioned at edge of the H-NS patch covering hilD, which made it possible to study the effects of NusG depletion in the presence or absence of overlapping transcription.

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5'-RACE-Seq analysis of Salmonella hilD and leuO promoters' response to adjacent transcription

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