Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of E. coli expressing small RNA MgrR

ABSTRACT: MgrR is a newly characterized Hfq dependent small RNA RNA. The expression of MgrR is regulated by Two component system, PhoPQ regulon, which senses low Mg2+ in environment. It has been reported that Hfq-binding sRNAs base pair with target RNAs, frequently leading to rapid degradation of target messages or, less frequently, to stabilization, both of which can be assayed by using microarrays. In order to search for the target genes of MgrR, we therefore examined the consequences of MgrR expression on mRNA abundance under two conditions. In condition 1, the chromosomal copy of mgrR was deleted and MgrR was expressed for 15â from an induced plac-mgrR plasmid and compared to cells carrying a vector induced for the same period. In condition 2, the expression of mRNAs was compared in wild-type cells (mgrR+) and the mgrR deletion strain, both grown in LB; because MgrR levels are fairly high under our normal growth conditions, this allowed analysis of both the direct and indirect (long-term) effects of MgrR. Experiment Overall Design: The strains used are either wild type which is derivatives of MG1655 or mgrR::kn knockout to restrict MgrR expression to the inducible pBR Plac-mgrR vector. The mgrR knockout mutants carried either pBR Plac-mgrR or the control vector pBR Plac. A Fresh colony of bacteria was inoculated in LB media with ampicilline at final concentration of 100ug/ml at 37Â°C with agitation. To induce MgrR expression, cultures carrying the pBR Plac-mgrR construct were grown to an O.D.600 of 0.5 and IPTG was added to the culture at a final concentration of 100uM. Both cultures for wild type and mgrR deletion mutant were grown to O.D. 600 of 0.5 and cells were collected. Total RNA was extracted from cells at the indicated time using the hot phenol procedure.

ORGANISM(S): Escherichia coli K-12

SUBMITTER: Kyung Moon

PROVIDER: E-GEOD-18935 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:RyhB is a non-coding RNA regulated by the Fur repressor. It has previously been shown to cause the rapid degradation of a number of mRNAs that encode proteins that utilize iron. Here we examine the effect of ectopic RyhB production on global gene expression by microarray analysis. Many of the previously identified targets were found, as well as other mRNAs encoding iron-binding proteins, bringing the total number of regulated operons to at least 18, encoding 56 genes. The two major operons involved in Fe-S cluster assembly showed different behavior; the isc operon appears to be a direct target of RyhB action, while the suf operon does not. This is consistent with previous findings suggesting that the suf genes but not the isc genes are important for Fe-S cluster synthesis under iron-limiting conditions, presumably for essential iron-binding proteins. In addition, we observed repression of Fur-regulated genes upon RyhB expression, interpreted as due to intracellular iron-sparing resulting from reduced synthesis of iron-binding proteins. Our results demonstrate the broad effects of a single non-coding RNA on iron homeostasis. Experiment Overall Design: Derivatives of MG1655 carrying either pBAD-ryhB or the control vector pNM12 were used in all experiments. Experiment Overall Design: The strains used contain the (delta)ara714 allele to prevent catabolism of arabinose and the (delta)ryhB::cat allele to restrict RyhB expression to the inducible pBAD-ryhB vector. Experiment Overall Design: Overnight bacterial cultures were incubated in LB media with ampicillin at a final concentration of 50 ug/mL at 37oC and diluted 1000-fold into 50 mL of fresh LB-ampicillin media at 37oC with agitation. To induce RyhB expression, cultures carrying the pBAD-ryhB construct were grown to an O.D.600 of 0.5 and arabinose was added to the culture at a final concentration of 0.1%. In some experiments, 50 uM FeSO4 was added to the new culture after dilution from overnight culture. Total RNA was extracted from cells at the indicated time using the hot phenol procedure.

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Transcription profiling of E. coli expressing small RNA MgrR

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