Dataset Information

Genetic evidence for a novel interaction between transcriptional activator SoxS and region 4 of the ?(70) subunit of RNA polymerase at class II SoxS-dependent promoters in Escherichia coli.

ABSTRACT: Escherichia coli SoxS activates transcription of the genes of the soxRS regulon, which provide the cell's defense against oxidative stress. In response to this stress, SoxS is synthesized de novo. Because the DNA binding site of SoxS is highly degenerate, SoxS efficiently activates transcription by the mechanism of prerecruitment. In prerecruitment, newly synthesized SoxS first forms binary complexes with RNA polymerase. These complexes then scan the chromosome for class I and II SoxS-dependent promoters, using the specific DNA-recognition properties of SoxS and ?(70) to distinguish SoxS-dependent promoters from the vast excess of sequence-equivalent soxboxes that do not reside in promoters. Previously, we determined that SoxS interacts with RNA polymerase in two ways: by making protein-protein interactions with the DNA-binding determinant of the ? subunit and by interacting with ?(70) region 4 (?(70) R4) both "on-DNA" and "off-DNA." Here, we address the question of how SoxS and ?(70) R4 coexist at class II promoters, where the binding site for SoxS either partially or completely overlaps the -35 region of the promoter, which is usually bound by ?(70) R4. To do so, we created a tri-alanine scanning library that covers all of ?(70) R4. We determined that interactions between ?(70) R4 and the DNA in the promoter's -35 region are required for activation of class I promoters, where the binding site lies upstream of the -35 hexamer, but they are not required at class II promoters. In contrast, specific three-amino-acid stretches are required for activation of class I (lac) and class II (galP1) cyclic AMP receptor protein-dependent promoters. We conclude from these data that SoxS and ?(70) R4 interact with each other in a novel way at class II SoxS-dependent promoters such that the two proteins do not accommodate one another in the -35 region but instead SoxS binding there occludes the binding of ?(70) R4.

SUBMITTER: Zafar MA

PROVIDER: S-EPMC3070153 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Genetic evidence for a novel interaction between transcriptional activator SoxS and region 4 of the σ(70) subunit of RNA polymerase at class II SoxS-dependent promoters in Escherichia coli.

Zafar M Ammar MA Sanchez-Alberola Neus N Wolf Richard E RE

Journal of molecular biology 20101231 3

Escherichia coli SoxS activates transcription of the genes of the soxRS regulon, which provide the cell's defense against oxidative stress. In response to this stress, SoxS is synthesized de novo. Because the DNA binding site of SoxS is highly degenerate, SoxS efficiently activates transcription by the mechanism of prerecruitment. In prerecruitment, newly synthesized SoxS first forms binary complexes with RNA polymerase. These complexes then scan the chromosome for class I and II SoxS-dependent ...[more]

PMID: 21195716

Similar Datasets

Project description:BACKGROUND:The genome-integrated T7 expression system offers significant advantages, in terms of productivity and product quality, even when expressing the gene of interest (GOI) from a single copy. Compared to plasmid-based expression systems, this system does not incur a plasmid-mediated metabolic load, and it does not vary the dosage of the GOI during the production process. However, long-term production with T7 expression system leads to a rapidly growing non-producing population, because the T7 RNA polymerase (RNAP) is prone to mutations. The present study aimed to investigate whether two ?70 promoters, which were recognized by the Escherichia coli host RNAP, might be suitable in genome-integrated expression systems. We applied a promoter engineering strategy that allowed control of expressing the model protein, GFP, by introducing lac operators (lacO) into the constitutive T5 and A1 promoter sequences. RESULTS:We showed that, in genome-integrated E. coli expression systems that used ?70 promoters, the number of lacO sites must be well balanced. Promoters containing three and two lacO sites exhibited low basal expression, but resulted in a complete stop in recombinant protein production in partially induced cultures. In contrast, expression systems regulated by a single lacO site and the lac repressor element, lacIQ, on the same chromosome caused very low basal expression, were highly efficient in recombinant protein production, and enables fine-tuning of gene expression levels on a cellular level. CONCLUSIONS:Based on our results, we hypothesized that this phenomenon was associated with the autoregulation of the lac repressor protein, LacI. We reasoned that the affinity of LacI for the lacO sites of the GOI must be lower than the affinity of LacI to the lacO sites of the endogenous lac operon; otherwise, LacI autoregulation could not take place, and the lack of LacI autoregulation would lead to a disturbance in lac repressor-mediated regulation of transcription. By exploiting the mechanism of LacI autoregulation, we created a novel E. coli expression system for use in recombinant protein production, synthetic biology, and metabolic engineering applications.

Dataset Information

Genetic evidence for a novel interaction between transcriptional activator SoxS and region 4 of the ?(70) subunit of RNA polymerase at class II SoxS-dependent promoters in Escherichia coli.

Publications

Genetic evidence for a novel interaction between transcriptional activator SoxS and region 4 of the σ(70) subunit of RNA polymerase at class II SoxS-dependent promoters in Escherichia coli.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure