Recipient Organization
MISSISSIPPI STATE UNIV
(N/A)
MISSISSIPPI STATE,MS 39762
Performing Department
BIOCHEMISTRY & MOLECULAR BIOLOGY
Non Technical Summary
According to the central dogma of genetics, RNA is a mare messenger between DNA and protein and leaves the regulatory business to transcriptional and translational factors. However, in the last few years, as an increasing number of small regulatory RNA molecules discovered in bacteria, it has become obvious that RNA plays wide-ranging role in many genetic circuits. While characterizing an Azotobacter vinelandii mutant that does not produce Fur (ferric uptake regulator), we have found that the sodB gene encoding Fe-containing superoxide dismutase is positively regulated by Fur protein and this positive regulation involves the action of ArrF. This ArrF is itself shown to be negatively regulated by Fur, so net effect is positive regulation of sodB gene by Fur. Intriguingly, the feSII gene encoding FeSII, which has a function associated with biological nitrogen fixation, is also similarly regulated by Fur and ArrF. These results suggest that ArrF appears to function as a small RNA that regulates the expression of genes involving many biological processes and one of which appear to be biological nitrogen fixation. The goal of this work is to determine the function of putative small RNA ArrF in A. vinelandii, a potentially important organism to Agriculture because of its unique ability of converting nitrogen into useful ammonia. To meet this goal, we set the following specific objectives. 1. We will establish that the A. vinelandii ArrF is an iron-responsive sRNA that represses FeSOD and FeSII-encoding genes. 2. We will elucidate the mechanism by which ArrF regulates its target mRNAs 3. We will identify the target of the A. vinelandii ArrF To achieve these aims, a multidisciplinary approach will be used that employs mass spectrometry in conjunction with genetic, biochemical, and molecular biology methods. In vitro DNA assay will be used to study the interaction between small RNA ArrF and its target mRNAs in the presence or absence of Hfq protein, and A. vinelandii hfq mutant will be generated to establish in vivo role of Hfq protein in the action of ArrF. RNA footprint with nucleases will be used to monitor the change in secondary structure when they interact with each other, or they bind to Hfq protein. This study will allow drawing a model for ArrF-its target mRNA-Hfq interaction. Finally, differential gel electrophoresis techniques combined with mass spectroscopy will be used to identify the invisible proteins that are regulated by small RNA ArrF. The protein identified in this way will be further confirmed by real-time RT-PCR. Since ArrF and its regulatory network will bring an additional level of complexity to the cell physiology, the results from this study will help to better understand how cells respond and adapt to rapidly changing environments.
Animal Health Component
15%
Research Effort Categories
Basic
85%
Applied
15%
Developmental
(N/A)
Goals / Objectives
1. To establish that the A. vinelandii ArrF is an iron-responsive sRNA that represses FeSOD and FeSII-encoding genes. Since our results are obtained with the A. vinelandii fur mutant, this regulation of the two genes must be further confirmed in the arrF mutant. Even if two genes turn out to be negatively regulated by ArrF, this does not imply that this negative regulation is as the result of direct interaction between ArrF and its target mRNAs. To establish that ArrF functions a small RNA with an antisense RNA mechanism, the physical interaction between ArrF and its target mRNAs must also be directly demonstrated. 2. To elucidate the mechanism by which ArrF regulates its target mRNAs. Hfq is a small, ubiquitous protein that has shown to be a pleiotropic regulator of the expression of many genes by binding to RNAs. Some studies show that Hfq functions as RNA chaperone and is essential for the interaction of small RNA with its target mRNAs. In other cases, the Hfq protein did not mediate the interaction between small RNA and its target mRNA. A. vinelandii carries Hfq. However, it is not clear and needs to be established whether this protein is essential for the action of ArrF. If the protein does, also determined where the protein binds and how it mediates the interaction between two RNA partners. 3. To identify the target of the A. vinelandii. For some bacterial species, global transcriptome analyses of a mutant that did not produce the small RNA or Fur identified a number of potential target genes of the small RNA. Many of the genes encoded iron-containing proteins in the energy conversion, oxidative stress defense or [Fe-S] cluster formation and/or repair. Some of which, such as acnA coding for aconitase A, fumA for fumarase A, sdhCDAB operon for succinate dehydrogenase complex, or sodB for FeSOD, are highly conserved as small RNA-regulated genes among many species, while others are not. In A. vinelandii, almost all of those highly conserved genes are no longer ArrF-regulated, and the feSII gene, which is uniquely found in nitrogen-fixing bacterial species, is newly identified as a potential ArrF target, suggesting that A. vinelandii ArrF might have unique targets and some of them are associated with nitrogen fixation process. This study will answer some of exciting questions to be remained to address: What other physiological responses will sRNAs be found to play a role What new functions of sRNA regulators remain to be identified What constitutes productive basepairing and how does basepairing influence the regulatory outcome Furthermore, this study aimed at identifying ArrF regulatory network will help to better understanding how this RNA and its regulatory network was added to the complexity of the physiology of A. vinelandii under stress in order to fight for the survival.
Project Methods
1. To establish that the A. vinelandii ArrF is an iron-responsive sRNA that represses FeSOD and FeSII-encoding genes. The negative regulation of the two genes must be verified by generating a mutant that does not producing ArrF and investigating the effect of this mutation on the expression of sodB and feSII genes using real time RT-PCR. RNA gel mobility shift assay will be used to demonstrate the direct interaction between ArrF and its target mRNAs, sodB mRNA and feSII mRNA. For this study, the transcriptional start site of sodB and feSII genes will be determined by 5-RACE. RNA will be prepared at large scale from in vitro transcription assay using T7 promoter and RNA polymerase. In case Hfq protein is critical for the interaction, a recombinant E. coli that overproduces His-tagged A. vinelandii Hfq will be created using the pET expression system, and the protein will be isolated on Ni-NTA agarose and will be used in the binding assay. 2. To elucidate the mechanism by which ArrF regulates its target mRNAs. The essential role of Hfq protein from in vitro RNA binding study will be also confirmed in vivo by generating a mutant that has a deletion on the hfq gene and assessing this mutation effects on sodB and feSII expression using real-time RT-PCR or Northern blot. ArrF or its target mRNAs fragments with deletions of the 5 region will be constructed and used to compete against labeled undeleted RNA fragments in Hfq binding assays to determine the structural requirements of these RNAs for binding to Hfq protein. RNase footprinting will be used to monitor the conformational change of the RNA induced upon binding to Hfq and when two interacting RNA pairs. 3. To identify the targets of the A. vinelandii ArrF. To this end, the differential gel electrophoresis, which uses fluorescent dyes to differentially label protein samples prior to 2D gel electrophoresis, will be used to identify a potential proteins regulated by ArrF. The ArrF-regulated proteins must show underexpression in the fur mutants under iron-replete condition and in the wild type in response to iron depletion, but they must show overexpression in the ∆arrF mutant under iron-rich conditions. The proteins that meet these requirements will be picked by robotics and analyzed by using MALDI-TOF and TOF and TOF. They will be further confirmed by real-time RT-PCR.