The Alpha Project: a model system for systems biology research.

One goal of systems biology is to understand how genome-encoded parts interact to produce quantitative phenotypes. The Alpha Project is a medium-scale, interdisciplinary systems biology effort that aims to achieve this goal by understanding fundamental quantitative behaviours of a prototypic signal transduction pathway, the yeast pheromone response system from Saccharomyces cerevisiae. The Alpha Project distinguishes itself from many other systems biology projects by studying a tightly bounded and well-characterised system that is easily modified by genetic means, and by focusing on deep understanding of a discrete number of important and accessible quantitative behaviours. During the project, the authors have developed tools to measure the appropriate data and develop models at appropriate levels of detail to study a number of these quantitative behaviours. The authors have also developed transportable experimental tools and conceptual frameworks for understanding other signalling systems. In particular, the authors have begun to interpret system behaviours and their underlying molecular mechanisms through the lens of information transmission, a principal function of signalling systems. The Alpha Project demonstrates that interdisciplinary studies that identify key quantitative behaviours and measure important quantities, in the context of well-articulated abstractions of system function and appropriate analytical frameworks, can lead to deeper biological understanding. The authors' experience may provide a productive template for systems biology investigations of other cellular systems.

Role of the sporulation protein BofA in regulating activation of the Bacillus subtilis developmental transcription factor sigmaK.

During sporulation, the Bacillus subtilis transcription factor sigmaK is activated by regulated proteolytic processing. I have used a system that facilitates the analysis of the contributions of a modified form of the processing enzyme, SpoIVFB-GFP, and the regulatory proteins BofA and SpoIVFA to the conversion of pro-sigmaK to sigmaK. The results show that in the presence of BofA, SpoIVFA levels increase by greater than 20-fold, SpoIVFA is substantially stabilized, and pro-sigmaK processing is inhibited. In addition, enhanced accumulation of the SpoIVFA protein in the absence of BofA (achieved through the use of an ftsH null mutation) substantially inhibits pro-sigmaK processing. These results suggest that during growth, increased accumulation of the SpoIVFA protein inhibits the activity of SpoIVFB-GFP and regulates the activation of sigmaK.

Negative regulation of the proteolytic activation of a developmental transcription factor in Bacillus subtilis.

The sporulation transcription factor sigmaK of Bacillus subtilis is controlled by a signal transduction pathway that operates at the level of the proteolytic processing of the inactive precursor protein pro-sigmaK. The conversion of pro-sigmaK to sigmaK requires the putative processing enzyme SpoIVFB and is governed by the regulatory proteins SpoIVFA and BofA. We engineered vegetative cells to carry out processing of pro-sigmaK by inducing the synthesis of the proprotein, a modified form of the putative processing enzyme, and its two regulators during growth. The results showed that (i) modified SpoIVFB was the only sporulation protein necessary to achieve processing of pro-sigmaK; (ii) SpoIVFA stimulated processing, apparently by protecting the processing enzyme from degradation; (iii) BofA inhibited processing in a manner that did not involve degradation of SpoIVFB; and (iv) the inhibition of SpoIVFB by BofA was dependent on SpoIVFA. We conclude that BofA and SpoIVFA act synergistically and are the only two sporulation proteins needed to inhibit the function of SpoIVFB. Our results are consistent with the idea that activation of pro-sigmaK occurs by a reversal of the BofA/SpoIVFA-mediated inhibition of the processing enzyme.

Subcellular localization of proteins governing the proteolytic activation of a developmental transcription factor in Bacillus subtilis.

BACKGROUND: Spore formation in Bacillus subtilis takes place in a sporangium consisting of two compartments called the forespore and the mother cell. Late in development, when the forespore is wholly contained within the mother cell, gene transcription is coordinated between the compartments by an intercellular signal transduction pathway. This pathway operates at the level of proteolytic processing of the proprotein precursor (pro-sigma K to the mother-cell transcription factor sigma K. The conversion of pro-sigma K to sigma K is governed by the putative processing enzyme SpoIVFB and its negative regulator SpoIVFA, which are produced in the mother cell. RESULTS: We used fluorescence microscopy in conjunction with antibodies against SpoIVFA and SpoIVFB and a fusion of SpoIVFB to the Green Fluorescent Protein from Aquorea victoria to visualize these proteins in the sporangium. Both proteins were found to co-localize with the forespore region of the sporangium, a finding consistent with the idea that SpoIVFA and SpoIVFB, which are inferred to be integral membrane proteins, are located in the mother cell membrane that surrounds the forespore. CONCLUSIONS: We conclude that SpoIVFA and SpoIVFB are situated at the boundary between the forespore and the mother cell, at which location SpoIVFB could be activated by a signalling protein produced in the forespore.

Repression of beta interferon gene expression in virus-infected cells is correlated with a poly(A) tail elongation.

Expression of beta interferon (IFN-beta) is transiently induced when Namalwa B cells (Burkitt lymphoma cell line) are infected by Sendai virus. In this study, we found that an elongation of the IFN-beta mRNA could be detected in virus-infected cells and that such a modification was not observed when the IFN-beta transcript was induced by a nonviral agent, poly(I-C). Treatment of the cells with a transcriptional inhibitor (actinomycin D or 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole) resulted in further elongation of the transcript. Characterization of the elongated IFN-beta transcript by primer extension and RNase H treatment showed that the modification was a result of an elongated poly(A) tail of up to 400 nucleotides. We conclude that the poly(A) tail elongation of the IFN-beta transcript is associated with the viral infection. Furthermore, the presence of the elongated IFN-beta transcript correlated with a decrease of IFN-beta protein in the medium and in cell extracts. Sucrose gradient analysis of cytoplasmic extracts showed that IFN-beta transcripts with elongated poly(A) tails were found in the nonpolysomal fractions, whereas the shorter transcripts could be detected in both polysomal and nonpolysomal fractions. A longer form of the IFN-beta mRNA was also found in the nonpolysomal fractions of cells not treated with transcriptional inhibitors. Thus, the observed regulation of IFN-beta mRNA is not entirely dependent on the inhibition of transcription. To our knowledge, this study provides the first example of a poly(A) tail elongation in somatic cells that negatively influences gene expression in vivo.

Identification and characterization of sporulation gene spoVS from Bacillus subtilis.

We report the identification and characterization of an additional sporulation gene from Bacillus subtilis called spoVS, which is induced early in sporulation under the control of sigma H. We show that spoVS is an 86-codon-long open reading frame and is capable of encoding a protein of 8,796 Da which exhibits little similarity to other proteins in the databases. Null mutations in spoVS have two contrasting phenotypes. In otherwise wild-type cells they block sporulation at stage V, impairing the development of heat resistance and coat assembly. However, the presence of a spoVS mutation in a spoIIB spoVG double mutant (which is blocked at the stage [II] of polar septation) acts as a partial suppressor, allowing sporulation to advance to a late stage. The implications of the contrasting phenotypes are discussed in the context of the formation and maturation of the polar septum.

Organization and regulation of the Bacillus subtilis odhAB operon, which encodes two of the subenzymes of the 2-oxoglutarate dehydrogenase complex.

The primary structure of Bacillus subtilis 105 kDa 2-oxoglutarate dehydrogenase (E10) was deduced from the nucleotide sequence of the odhA gene and confirmed by N-terminal sequence analysis. The protein is highly homologous to E1o of Azotobacter vinelandii and Escherichia coli and of bakers' yeast cells. The 5' end of the odhAB mRNA was determined and the promoter region for the odhAB operon was localized to a 375 bp DNA fragment. The cellular concentration of the 4.5 kb odhAB transcript was found to be growth stage dependent; its concentration during growth in nutrient sporulation medium decreased abruptly at the end of the exponential growth phase and it was not detectable in early stationary phase. This decrease in the cellular concentration of the transcript is not the result of an increased rate of decay of the full-length odhAB mRNA, suggesting that transcription is down-regulated at the end of the exponential growth phase. The cellular concentration of the odhA and odhB gene products, E1o and dihydrolipoamide transsuccinylase (E2o), remains essentially constant throughout the growth curve in nutrient sporulation medium, indicating that both are rather stable proteins. In exponentially growing cells, glucose in nutrient sporulation medium repressed the cellular concentration of the odhAB mRNA, as well as that of E1o and E2o, about four-fold. This effect is most likely the result of a decreased rate of transcription from the odhAB promoter, since neither the stability nor the 5'-end of the transcript were affected by glucose in the medium. It is concluded that the cellular concentration of the 2-oxoglutarate dehydrogenase multienzyme complex (E1o and E2o) is regulated mainly at the transcriptional level.

Elements involved in an in vitro block to transcription elongation at the end of the L1 mRNA family of adenovirus 2.

Using the 3' end of the L1 mRNA family of adenovirus 2 (Ad2) as a model system, we investigated transcription elongation following a poly(A) signal in a cell-free system. The results show that RNA polymerase II can halt transcription elongation at a T-rich stretch in the non-coding DNA strand 20 nucleotides downstream of the poly(A) signal. The block to transcription elongation is enhanced when Sarkosyl is included in the elongation reaction. Deletion studies narrowed the region which directs the elongation block at the T-rich stretch, to an upstream fragment of 53 nucleotides that is very dA-rich and also contains a functional poly(A) signal. The deletion studies and analysis by site-directed mutagenesis indicate that in the present system, RNA secondary structure, the stretch of T's and the poly(A) signal are not the dominant elements responsible for the elongation block. The block to transcription elongation at the T-rich stretch was also shown to be 5 times more effective in an uninfected extract than in an Ad2 infected extract, which is reminiscent of the in vivo situation and is consistent with the suggestion that a trans-acting factor is involved in modulating the elongation block at the T-rich stretch.

Changes in the stability of specific mRNA species in response to growth stage in Bacillus subtilis.

In this study we compared the cellular concentrations and stability of the mRNA transcribed from the aprE (subtilisin) gene (a gene preferentially expressed in stationary growth phase) with those of a vegetative mRNA, succinate dehydrogenase (SDH) mRNA. The subtilisin transcript was shown to be at least 3 times more stable in early stationary phase than it is 2 hr further into stationary phase. When cells were shifted from maximum expression of the subtilisin transcript in stationary phase to physiological conditions, which allowed for the resumption of vegetative growth, the cellular concentration of the subtilisin mRNA decreased rapidly. We conclude that mRNA degradation is one of the means by which the cellular concentrations of the SDH and subtilisin transcripts are adjusted in response to growth stage.

A cis downstream element participates in regulation of in vitro transcription initiation from the P38 promoter of minute virus of mice.

We report the use of a HeLa whole cell extract (WCE) runoff transcription system for the study of cis- and trans-acting elements, participating in the regulation of transcription initiation from the P38 promoter of the parvovirus minute virus of mice (MVM). Our initial studies with HeLa WCE indicated that transcription from the P38 promoter is very inefficient, compared with transcription from the P4 promoter. Supplementation of the HeLa WCE with WCE prepared from uninfected Ehrlich ascites cells enhanced transcription from the P38 promoter twofold, indicating a role for a cellular factor in transcription from the P38 promoter. Furthermore, supplementation with WCE prepared from MVM-infected Ehrlich ascites cells enhanced transcription from the P38 promoter about sixfold, indicating a role for a virally encoded or induced factor. Analyses of runoffs produced by transcription of DNA templates digested with various restriction enzymes defined a downstream promoter element (DPE) necessary for efficient transcription initiation from the P38 promoter. This element resides 282 to 647 base pairs 3' to the transcription initiation site, between the NarI site and the HindIII site (2287 to 2652, MVM numbering system). The virally encoded NS1 protein was shown by DNA precipitation to bind directly or indirectly through a cellular factor to the DPE. This interaction is suggested to be involved in the up regulation of the P38 promoter of MVM. Finally, with a DNase I protection assay performed on a fragment containing the DPE, we estimated the sequence involved in the binding of a factor present in uninfected and infected extracts. The correlation between the binding and transcription activation is discussed.

RNA secondary structure is an integral part of the in vitro mechanism of attenuation in simian virus 40.

At late times after infection with SV40, a prematurely terminated transcript that initiates at the major late promoter (MLP) and has a 3'-end about 95 nucleotides downstream has been identified and termed an attenuated RNA (Hay, N., Skolnik-David, H., and Aloni, Y. (1982) Cell 29, 183-193). The DNA template of the attenuated RNA has two regions of dyad symmetry, and the attenuated RNA can therefore fold into two hairpin elements. The hairpin element at the 3'-end of the attenuated RNA is followed by a stretch of Us and resembles a rho-independent terminator in prokaryotes. We have suggested that folding of the RNA into two hairpin elements will lead to a block of transcription elongation. Using site-directed mutagenesis, we created two templates that either strengthened or weakened the proposed hairpin structures. The mutated and wild-type templates were cloned downstream from the adenovirus 2 MLP, and transcription patterns were compared between the templates in a cell-free extract. We have shown that RNA polymerase II recognizes the SV40 sequence that leads to a block of transcription elongation, even when it is under the control of the MLP of adenovirus 2. The extent of the block of transcription elongation is directly dependent on the stability of the hairpin structure of the RNA as assessed by a comparison of transcription of the wild-type and mutated templates. The addition of Sarkosyl and transcription at an elevated temperature during the elongation reaction enhanced the production of the attenuated RNA from all templates.

RNA polymerase II is capable of pausing and prematurely terminating transcription at a precise location in vivo and in vitro.

By using the minute virus of mice, we have shown that in vivo and in vitro RNA polymerase II pauses or prematurely terminates transcription at a specific location 142-147 nucleotides downstream from the P4 promoter. The attenuated RNA was found and mapped in vivo in A9 cell late after infection in both the nuclear and cytoplasmic fractions, and the terminal nucleotide was shown to have a 3' OH group. The 3' end of the attenuated RNA is capable of forming a hairpin structure that is followed by a stretch of uridines. To distinguish whether the attenuated RNA is formed as a result of processing, pausing, or termination and to dissect structural elements, factors, or mechanisms that are involved in its formation, we used in vitro systems: isolated nuclei and cell-free extracts from HeLa cells. The results of the in vitro studies show that the attenuated RNA is a result of pausing or termination and not processing. Additionally, a salt-soluble factor and RNA secondary structure were implicated in the process of termination.

Transcription termination in animal viruses and cells.

Three experimental systems: isolated nuclei, cell-free reactions and whole cells were used for defining and characterizing cis and trans elements which regulate the block of transcription elongation in animal viruses and cells. In addition we have presented models for transcription termination within and at the end of a gene, which are consistent with the available information on the transcription bubble propagated during transcription elongation and can explain the modes of transcription termination described for various eukaryotic genes.