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Promoter Recognition by Extracytoplasmic Function ? Factors: Analyzing DNA and Protein Interaction Motifs.

ABSTRACT: Extracytoplasmic function (ECF) ? factors are the largest and the most diverse group of alternative ? factors, but their mechanisms of transcription are poorly studied. This subfamily is considered to exhibit a rigid promoter structure and an absence of mixing and matching; both -35 and -10 elements are considered necessary for initiating transcription. This paradigm, however, is based on very limited data, which bias the analysis of diverse ECF ? subgroups. Here we investigate DNA and protein recognition motifs involved in ECF ? factor transcription by a computational analysis of canonical ECF subfamily members, much less studied ECF ? subgroups, and the group outliers, obtained from recently sequenced bacteriophages. The analysis identifies an extended -10 element in promoters for phage ECF ? factors; a comparison with bacterial ? factors points to a putative 6-amino-acid motif just C-terminal of domain ?2, which is responsible for the interaction with the identified extension of the -10 element. Interestingly, a similar protein motif is found C-terminal of domain ?2 in canonical ECF ? factors, at a position where it is expected to interact with a conserved motif further upstream of the -10 element. Moreover, the phiEco32 ECF ? factor lacks a recognizable -35 element and ?4 domain, which we identify in a homologous phage, 7-11, indicating that the extended -10 element can compensate for the lack of -35 element interactions. Overall, the results reveal greater flexibility in promoter recognition by ECF ? factors than previously recognized and raise the possibility that mixing and matching also apply to this group, a notion that remains to be biochemically tested.ECF ? factors are the most numerous group of alternative ? factors but have been little studied. Their promoter recognition mechanisms are obscured by the large diversity within the ECF ? factor group and the limited similarity with the well-studied housekeeping ? factors. Here we extensively compare bacterial and bacteriophage ECF ? factors and their promoters in order to infer DNA and protein recognition motifs involved in transcription initiation. We predict a more flexible promoter structure than is recognized by the current paradigm, which assumes rigidness, and propose that ECF ? promoter elements may complement (mix and match with) each other's strengths. These results warrant the refocusing of research efforts from the well-studied housekeeping ? factors toward the physiologically highly important, but insufficiently understood, alternative ? factors.

SUBMITTER: Guzina J

PROVIDER: S-EPMC4936102 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

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Promoter Recognition by Extracytoplasmic Function σ Factors: Analyzing DNA and Protein Interaction Motifs.

Guzina Jelena J Djordjevic Marko M

Journal of bacteriology 20160627 14

<h4>Unlabelled</h4>Extracytoplasmic function (ECF) σ factors are the largest and the most diverse group of alternative σ factors, but their mechanisms of transcription are poorly studied. This subfamily is considered to exhibit a rigid promoter structure and an absence of mixing and matching; both -35 and -10 elements are considered necessary for initiating transcription. This paradigm, however, is based on very limited data, which bias the analysis of diverse ECF σ subgroups. Here we investigat ...[more]

PMID: 27137497

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Project description:Bacteria must sense alterations in their environment and respond with changes in function and/or structure in order to cope. Extracytoplasmic function sigma factors (ECF ?s) modulate transcription in response to cellular and environmental signals. The symbiotic nitrogen-fixing alphaproteobacterium Sinorhizobium meliloti carries genes for 11 ECF-like ?s (RpoE1 to -E10 and FecI). We hypothesized that some of these play a role in mediating the interaction between the bacterium and its plant symbiotic partner. The bacterium senses changes in its immediate environment as it establishes contact with the plant root, initiates invasion of the plant as the root nodule is formed, traverses several root cell layers, and enters plant cortical cells via endocytosis. We used genetics, transcriptomics, and functionality to characterize the entire S. meliloti cohort of ECF ?s. We discovered new targets for individual ?s, confirmed others by overexpressing individual ECF ?s, and identified or confirmed putative promoter motifs for nine of them. We constructed precise deletions of each ECF ? gene and its demonstrated or putative anti-? gene and also a strain in which all 11 ECF ? and anti-? genes were deleted. This all-ECF ? deletion strain showed no major defects in free-living growth, in Biolog Phenotype MicroArray assays, or in response to multiple stresses. None of the ECF ?s were required for symbiosis on the host plants Medicago sativa and Medicago truncatula: the strain deleted for all ECF ? and anti-? genes was symbiotically normal.IMPORTANCE Fixed (reduced) soil nitrogen plays a critical role in soil fertility and successful food growth. Much soil fertility relies on symbiotic nitrogen fixation: the bacterial partner infects the host plant roots and reduces atmospheric dinitrogen in exchange for host metabolic fuel, a process that involves complex interactions between the partners mediated by changes in gene expression in each partner. Here we test the roles of a family of 11 extracytoplasmic function (ECF) gene regulatory proteins (sigma factors [?s]) that interact with RNA polymerase to determine if they play a significant role in establishing a nitrogen-fixing symbiosis or in responding to various stresses, including cell envelope stress. We discovered that symbiotic nitrogen fixation occurs even when all 11 of these regulatory genes are deleted, that most ECF sigma factors control accessory functions, and that none of the ECF sigma factors are required to survive envelope stress.

Project description:Protein sequences encompass tertiary structures and contain information about specific molecular interactions, which in turn determine biological functions of proteins. Knowledge about how protein sequences define interaction specificity is largely missing, in particular for paralogous protein families with high sequence similarity, such as the plant MADS domain transcription factor family. In comparison to the situation in mammalian species, this important family of transcription regulators has expanded enormously in plant species and contains over 100 members in the model plant species Arabidopsis thaliana. Here, we provide insight into the mechanisms that determine protein-protein interaction specificity for the Arabidopsis MADS domain transcription factor family, using an integrated computational and experimental approach. Plant MADS proteins have highly similar amino acid sequences, but their dimerization patterns vary substantially. Our computational analysis uncovered small sequence regions that explain observed differences in dimerization patterns with reasonable accuracy. Furthermore, we show the usefulness of the method for prediction of MADS domain transcription factor interaction networks in other plant species. Introduction of mutations in the predicted interaction motifs demonstrated that single amino acid mutations can have a large effect and lead to loss or gain of specific interactions. In addition, various performed bioinformatics analyses shed light on the way evolution has shaped MADS domain transcription factor interaction specificity. Identified protein-protein interaction motifs appeared to be strongly conserved among orthologs, indicating their evolutionary importance. We also provide evidence that mutations in these motifs can be a source for sub- or neo-functionalization. The analyses presented here take us a step forward in understanding protein-protein interactions and the interplay between protein sequences and network evolution.

Dataset Information

Promoter Recognition by Extracytoplasmic Function ? Factors: Analyzing DNA and Protein Interaction Motifs.

Publications

Promoter Recognition by Extracytoplasmic Function σ Factors: Analyzing DNA and Protein Interaction Motifs.

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