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Regulatory proteins and cis-acting elements involved in the transcriptional control of Rhizobium etli reiterated nifH genes.

ABSTRACT: In Rhizobium etli the nitrogenase reductase genes are reiterated. Strain CE3 has three copies; nifHa and nifHb form part of nifHDK operons with the nitrogenase structural genes, while nifHc is linked to a truncated nifD homolog. Their sequences are identical up to 6 residues upstream from a sigma54-dependent promoter. A remarkable difference among them is the absence of canonical NifA binding sites upstream of nifHc while a canonical binding site is located 200 bp upstream of nifHa and nifHb. To evaluate the transcriptional regulation of the reiterated nifH genes, we constructed fusions of nifHa and nifHc with the lacZ gene of Escherichia coli. Both genes were expressed at maximum levels under 1% oxygen in free-living cultures, and their expression declined as the oxygen concentration was increased. This expression was dependent on the integrity of nifA, and nifHc was expressed at higher levels than nifHa. The same pattern was observed with root nodule bacteroids. Expression of both genes in E. coli required sigma54 in addition to NifA bound to the upstream activator sequence. In vivo dimethyl sulfate footprinting analyses showed that NifA binds to the canonical site upstream of nifHa and to a TGT half-site 6 nucleotides further upstream. NifA protected an imperfect binding site upstream of nijHc at position 85 from the promoter. The integration host factor stimulated each gene differently, nifHa being more dependent on this protein. The above results correlate the asymmetric arrangement of cis-acting elements with a differential expression of the reiterated nifH genes, both in culture and during symbiosis with bean plants.

SUBMITTER: Valderrama B

PROVIDER: S-EPMC178061 | biostudies-other | 1996 Jun

REPOSITORIES: biostudies-other

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Regulatory proteins and cis-acting elements involved in the transcriptional control of Rhizobium etli reiterated nifH genes.

Valderrama B B Dávalos A A Girard L L Morett E E Mora J J

Journal of bacteriology 19960601 11

In Rhizobium etli the nitrogenase reductase genes are reiterated. Strain CE3 has three copies; nifHa and nifHb form part of nifHDK operons with the nitrogenase structural genes, while nifHc is linked to a truncated nifD homolog. Their sequences are identical up to 6 residues upstream from a sigma54-dependent promoter. A remarkable difference among them is the absence of canonical NifA binding sites upstream of nifHc while a canonical binding site is located 200 bp upstream of nifHa and nifHb. To ...[more]

PMID: 8655489

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Project description:Recently, Rhizobium etli, in addition to Agrobacterium spp., has emerged as a prokaryotic species whose genome encodes a functional machinery for DNA transfer to plant cells. To understand this R. etli-mediated genetic transformation, it would be useful to define how its vir genes respond to the host plants. Here, we explored the transcriptional activation of the vir genes contained on the R. etli p42a plasmid. Using a reporter construct harboring lacZ under the control of the R. etli virE promoter, we show that the signal phenolic molecule acetosyringone (AS) induces R. etli vir gene expression both in an R. etli background and in an Agrobacterium tumefaciens background. Furthermore, in both bacterial backgrounds, the p42a plasmid also promoted plant genetic transformation with a reporter transfer DNA (T-DNA). Importantly, the R. etli vir genes were transcriptionally activated by AS in a bacterial species-specific fashion in regard to the VirA/VirG signal sensor system, and this activation was induced by signals from the natural host species of this bacterium but not from nonhost plants. The early kinetics of transcriptional activation of the major vir genes of R. etli also revealed several features distinct from those known for A. tumefaciens: the expression of the virG gene reached saturation relatively quickly, and virB2, which in R. etli is located outside the virB operon, was expressed only at low levels and did not respond to AS. These differences in vir gene transcription may contribute to the lower efficiency of T-DNA transfer of R. etli p42a than of T-DNA transfer of pTiC58 of A. tumefaciensIMPORTANCE The region encoding homologs of Agrobacterium tumefaciens virulence genes in the Rhizobium etli CE3 p42a plasmid was the first endogenous virulence system encoded by the genome of a non-Agrobacterium species demonstrated to be functional in DNA transfer and stable integration into the plant cell genome. In this study, we explored the transcriptional regulation and induction of virulence genes in R. etli and show similarities to and differences from those of their A. tumefaciens counterparts, contributing to an understanding and a comparison of these two systems. Whereas most vir genes in R. etli follow an induction pattern similar to that of A. tumefaciens vir genes, a few significant differences may at least in part explain the variations in T-DNA transfer efficiency.

Project description:Post-transcriptional gene regulation controls the amount of protein produced from an individual mRNA by altering rates of decay and translation. Many sequence elements that direct post-transcriptional regulation have been found; in mammals, most such elements are located within the 3' untranslated regions (3'UTRs). Comparative genomic studies demonstrate that mammalian 3'UTRs contain extensive conserved sequence tracts, yet only a small fraction corresponds to recognized elements, implying that many additional novel elements exist. Despite a variety of computational, molecular, and biochemical approaches, identifying functional 3'UTRs elements remains difficult.We created a high-throughput cell-based screen that enables identification of functional post-transcriptional 3'UTR regulatory elements. Our system exploits integrated single-copy reporters, which are expressed and processed as endogenous genes. We screened many thousands of short random sequences for their regulatory potential. Control sequences with known effects were captured effectively using our approach, establishing that our methodology was robust. We found hundreds of functional sequences, which we validated in traditional reporter assays, including verifying their regulatory impact in native sequence contexts. Although 3'UTRs are typically considered repressive, most of the functional elements were activating, including ones that were preferentially conserved. Additionally, we adapted our screening approach to examine the effect of elements on RNA abundance, revealing that most elements act by altering mRNA stability.We developed and used a high-throughput approach to discover hundreds of post-transcriptional cis-regulatory elements. These results imply that most human 3'UTRs contain many previously unrecognized cis-regulatory elements, many of which are activating, and that the post-transcriptional fate of an mRNA is largely due to the actions of many individual cis-regulatory elements within its 3'UTR.

Project description:BACKGROUND: The MECP2 gene codes for methyl CpG binding protein 2 which regulates activities of other genes in the early development of the brain. Mutations in this gene have been associated with Rett syndrome, a form of autism. The purpose of this study was to investigate the role of evolutionarily conserved cis-elements in regulating the post-transcriptional expression of the MECP2 gene and to explore their possible correlations with a mutation that is known to cause mental retardation. RESULTS: A bioinformatics approach was used to map evolutionarily conserved cis-regulatory elements in the transcribed regions of the human MECP2 gene and its mammalian orthologs. Cis-regulatory motifs including G-quadruplexes, microRNA target sites, and AU-rich elements have gained significant importance because of their role in key biological processes and as therapeutic targets. We discovered in the 5'-UTR (untranslated region) of MECP2 mRNA a highly conserved G-quadruplex which overlapped a known deletion in Rett syndrome patients with decreased levels of MeCP2 protein. We believe that this 5'-UTR G-quadruplex could be involved in regulating MECP2 translation. We mapped additional evolutionarily conserved G-quadruplexes, microRNA target sites, and AU-rich elements in the key sections of both untranslated regions. Our studies suggest the regulation of translation, mRNA turnover, and development-related alternative MECP2 polyadenylation, putatively involving interactions of conserved cis-regulatory elements with their respective trans factors and complex interactions among the trans factors themselves. We discovered highly conserved G-quadruplex motifs that were more prevalent near alternative splice sites as compared to the constitutive sites of the MECP2 gene. We also identified a pair of overlapping G-quadruplexes at an alternative 5' splice site that could potentially regulate alternative splicing in a negative as well as a positive way in the MECP2 pre-mRNAs. CONCLUSIONS: A Rett syndrome mutation with decreased protein expression was found to be associated with a conserved G-quadruplex. Our studies suggest that MECP2 post-transcriptional gene expression could be regulated by several evolutionarily conserved cis-elements like G-quadruplex motifs, microRNA target sites, and AU-rich elements. This phylogenetic analysis has provided some interesting and valuable insights into the regulation of the MECP2 gene involved in autism.

Dataset Information

Regulatory proteins and cis-acting elements involved in the transcriptional control of Rhizobium etli reiterated nifH genes.

Publications

Regulatory proteins and cis-acting elements involved in the transcriptional control of Rhizobium etli reiterated nifH genes.

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