Igor Y. Morozov

A novel sRNA component of the carbon storage regulatory system of Escherichia coli.

Small untranslated RNAs (sRNAs) perform a variety of important functions in bacteria. The 245 nucleotide sRNA of Escherichia coli, CsrC, was discovered using a genetic screen for factors that regulate glycogen biosynthesis. CsrC RNA binds multiple copies of CsrA, a protein that post‐transcriptionally regulates central carbon flux, biofilm formation and motility in E. coli. CsrC antagonizes the regulatory effects of CsrA, presumably by sequestering this protein. The discovery of CsrC is intriguing, in that a similar sRNA, CsrB, performs essentially the same function. Both sRNAs possess similar imperfect repeat sequences (18 in CsrB, nine in CsrC), primarily localized in the loops of predicted hairpins, which may serve as CsrA binding elements. Transcription of csrC increases as the culture approaches the stationary phase of growth and is indirectly activated by CsrA via the response regulator UvrY. Because CsrB and CsrC antagonize CsrA activity and depend on CsrA for their synthesis, a csrB null mutation causes a modest compensatory increase in CsrC levels and vice versa. Homologues of csrC are apparent in several Enterobacteriaceae. The regulatory and evolutionary implications of these findings are discussed.

CsrA regulates glycogen biosynthesis by preventing translation of glgC in Escherichia coli

The carbon storage regulatory system of Escherichia coli controls the expression of genes involved in carbohydrate metabolism and cell motility. CsrA binding to glgCAP transcripts inhibits glycogen metabolism by promoting glgCAP mRNA decay. CsrB RNA functions as an antagonist of CsrA by sequestering this protein and preventing its action. In this paper, we elucidate further the mechanism of CsrA‐mediated glgC regulation. Results from gel shift assays demonstrate that several molecules of CsrA can bind to each glgC transcript. RNA footprinting studies indicate that CsrA binds to the glgCAP leader transcript at two positions. One of these sites overlaps the glgC Shine–Dalgarno sequence, whereas the other CsrA target is located further upstream in an RNA hairpin. Results from toeprint and cell‐free translation experiments indicate that bound CsrA prevents ribosome binding to the glgC Shine–Dalgarno sequence and that this reduces GlgC synthesis. The effect of two deletions in the upstream binding site was examined. Both of these deletions reduced, but did not eliminate, CsrA binding in vitro and CsrA‐dependent regulation in vivo. Our findings establish that bound CsrA inhibits initiation of glgC translation, thereby reducing glycogen biosynthesis. This inhibition of translation probably contributes to destabilization of the glgC transcript that was observed previously.

Characterization of nitrogen metabolite signalling in Aspergillus via the regulated degradation of areA mRNA

AreA is the principal transcription factor involved in determining nitrogen utilization in Aspergillus nidulans. NH4+ and Gln are utilized preferentially but in their absence, AreA acts to facilitate the expression of genes involved in metabolizing alternative nitrogen sources. It is crucial to the function of AreA that its expression is tightly modulated by the quality and availability of nitrogen sources. One signalling mechanism involves regulated degradation of the areA transcript in response to NH4+ and Gln, which provides the first direct means of monitoring nitrogen signalling in this fungus. Here we assess the specificity of the transcript degradation response, determining that it responds qualitatively to a variety of additional nitrogen sources including Asn. Furthermore, the response to Gln and NH4+ requires the same discrete region of the areA 3′‐UTR but both NH4+ and Asn need to be metabolized to Gln before they are effective as a signal. However, NH4+ signalling is independent of AreA activity, unlike Gln and Asn signalling. A mutation in the structural gene for NADP‐linked glutamate dehydrogenase, gdhA, which disrupts metabolism of NH4+ to Glu, is additive with mutations in two distinct regions of areA that disrupt the previously identified signalling mechanisms. The triple mutant is both strongly derepressed and expresses very high levels of nitrate reductase activity. These data suggest nitrogen metabolism in A. nidulans is in part regulated in response to the intracellular levels of Gln via the regulated degradation of areA mRNA, but the intracellular Gln level is not the sole determinant of nitrogen metabolite repression.

Gene expression in a cell-free system on the preparative scale

A cell-free system for preparative gene expression is described. It is composed of DNA-free Escherichia coli extract and added plasmid DNA; coupled transcription-translation proceeds with a continuous flow of the feeding solution containing nucleoside triphosphates and amino acids. The system works at a high constant rate for tens of hours. The yield of synthesised proteins after 20-50 h is hundreds of micrograms from 1 ml of the reaction mixture. Electrophoretic analysis of translation products confirms synthesis of proteins of the expected molecular mass.

The GATA factor AreA regulates localization and in vivo binding site occupancy of the nitrate activator NirA

The GATA factor AreA is a wide‐domain regulator in Aspergillus nidulans with transcriptional activation and chromatin remodelling functions. AreA interacts with the nitrate‐specific Zn2‐C6 cluster protein NirA and both proteins cooperate to synergistically activate nitrate‐responsive genes. We have previously established that NirA in vivo DNA binding site occupancy is AreA dependent and in this report we provide a mechanistic explanation for our previous findings. We now show that AreA regulates NirA at two levels: (i) through the regulation of nitrate transporters, AreA affects indirectly the subcellular distribution of NirA which rapidly accumulates in the nucleus following induction; (ii) AreA directly stimulates NirA in vivo target DNA occupancy and does not act indirectly by chromatin remodelling. Simultaneous overexpression of NirA and the nitrate transporter CrnA bypasses the AreA requirement for NirA binding, permits utilization of nitrate and nitrite as sole N‐sources in an areA null strain and leads to an AreA‐independent nucleosome loss of positioning.

A defined sequence within the 3′ UTR of the areA transcript is sufficient to mediate nitrogen metabolite signalling via accelerated deadenylation

Nitrogen metabolism in Aspergillus nidulans is regulated by AREA, a member of the GATA family of transcription factors. One mechanism that modulates AREA activity involves the rapid degradation of the areA transcript when sufficient NH4+ or Gln are available. This signalling mechanism has been shown to require a region of 218 nucleotides within the 3′ untranslated region of areA mRNA. We demonstrate that this region functions independently in a heterologous transcript and acts to accelerate degradation of the poly(A) tail, which in turn leads to rapid transcript degradation in response to the addition of NH4+ or Gln to the growth medium. areA transcript degradation is inhibited by cycloheximide, but this is not a general consequence of translational inhibition. We believe that this is the first reported example in which specific physiological signals, acting through a defined sequence within a transcript, have been shown to promote accelerated poly(A) degradation, which in turn triggers transcript degradation.

Genetic analysis of the TOR pathway in Aspergillus nidulans.

ABSTRACT We identified five genes encoding components of the TOR signaling pathway within Aspergillus nidulans. Unlike the situation in Saccharomyces cerevisiae, there is only a single Tor kinase, as in plant and animal systems, and mutant phenotypes suggest that the TOR pathway plays only a minor role in regulating nitrogen metabolism.

CUCU Modification of mRNA Promotes Decapping and Transcript Degradation in Aspergillus nidulans

ABSTRACT In eukaryotes, mRNA decay is generally initiated by the shortening of the poly(A) tail mediated by the major deadenylase complex Ccr4-Caf1-Not. The deadenylated transcript is then rapidly degraded, primarily via the decapping-dependent pathway. Here we report that in Aspergillus nidulans both the Caf1 and Ccr4 orthologues are functionally distinct deadenylases in vivo: Caf1 is required for the regulated degradation of specific transcripts, and Ccr4 is responsible for basal degradation. Intriguingly disruption of the Ccr4-Caf1-Not complex leads to deadenylation-independent decapping. Additionally, decapping is correlated with a novel transcript modification, addition of a CUCU sequence. A member of the nucleotidyltransferase superfamily, CutA, is required for this modification, and its disruption leads to a reduced rate of decapping and subsequent transcript degradation. We propose that 3′ modification of adenylated mRNA, which is likely to represent a common eukaryotic process, primes the transcript for decapping and efficient degradation.

Opposing signals differentially regulate transcript stability in Aspergillus nidulans

A good model for gene regulation, requiring the organism to monitor a complex and changing environment and respond in a precise and rapid way, is nitrogen metabolism in Aspergillus nidulans. This involves co‐ordinated expression of hundreds of genes, many dependent on the transcription factor AreA, which monitors the nitrogen state of the cell. AreA activity is in part modulated by differential degradation of its transcript in response to intracellular glutamine. Here we report that glutamine triggers synchronized degradation of a large subset of transcripts involved in nitrogen metabolism. Among these are all four genes involved in the assimilation of nitrate. Significantly, we show that two of these transcripts, niaD and niiA, are stabilized by intracellular nitrate, directly reinforcing transcriptional regulation. Glutamine‐signalled degradation and the nitrate‐dependent stabilization of the niaD transcript are effected at the level of deadenylation and are dependent on its 3′ UTR. When glutamine and nitrate are both present, nitrate stabilization is predominant, ensuring that nitrate and the toxic intermediate nitrite are removed from the cell. Regulated transcript stability is therefore an integral part of the adaptive response. This represents the first example of distinct physiological signals competing to differentially regulate transcripts at the level of deadenylation.

The bZIP transcription factor MeaB mediates nitrogen metabolite repression at specific loci.

ABSTRACT In Fusarium fujikuroi, bikaverin (BIK) biosynthesis is subject to repression by nitrogen. Unlike most genes subject to nitrogen metabolite repression, it has been shown that transcription of bik biosynthetic genes is not AreA dependent. Searching for additional transcription factors that may be involved in nitrogen regulation, we cloned and characterized the orthologue of Aspergillus nidulansmeaB, which encodes a bZIP transcription factor. Two transcripts are derived from F. fujikuroimeaB: the large transcript (meaBL) predominates under nitrogen-sufficient conditions and the smaller transcript (meaBS) under nitrogen limitation, in an AreA-dependent manner. MeaB is specifically translocated to the nucleus under nitrogen-sufficient conditions in both F. fujikuroi and A. nidulans. Deletion of meaB resulted in partial upregulation of several nitrogen-regulated genes, but only in the ΔmeaB ΔareA double mutant were the bikaverin genes significantly upregulated in the presence of glutamine. These data demonstrate that MeaB and AreA coordinately mediate nitrogen metabolite repression and, importantly, that independently of AreA, MeaB can mediate nitrogen metabolite repression at specific loci in F. fujikuroi.