Proteome analysis of Aspergillus nidulans reveals proteins associated with the response to the antibiotic concanamycin A, produced by Streptomyces species.

Competition between microbes is common to all ecosystems, but the exact nature of the competition is in most cases unknown. We have previously studied the antagonism between Streptomyces halstedii and several fungi at both the organismal and gene expression levels. Here we analysed the effect of an antibiotic produced by Streptomyces, concanamycin A, on protein levels in the filamentous fungus Aspergillus nidulans. Two-dimensional gel electrophoresis revealed that 20 proteins either increased or decreased in abundance upon treatment of the fungus with the antibiotic. Five of the most prominent proteins which changed in abundance were identified based on peptide analysis by mass spectrometry. Two of these correspond to proteins previously described in A. nidulans, and three others are homologous to proteins found in other organisms. Of these, one down-regulated protein was identified as glyceraldehyde dehydrogenase, a protein involved in general metabolic pathways. A second down-regulated protein, CpcB, affects the initiation of sexual development. Among the proteins not previously described in A. nidulans, all of them up-regulated by concanamycin A, we found two proteins with described homologues in other fungal species. The first is homologous to a cadmium-induced protein in Candida sp. The second protein is homologous to LovC, an enoyl transferase involved in the biosynthesis of lovastatin, a secondary metabolite identified in A. terreus. A third protein has a homologue in A. niger, which is of unknown function. This study indicates that proteome analysis may be a useful method for studying effects on gene expression during competitive interactions between bacteria and filamentous fungi.

Novel small RNA-encoding genes in the intergenic regions of Escherichia coli.

BACKGROUND: Small, untranslated RNA molecules were identified initially in bacteria, but examples can be found in all kingdoms of life. These RNAs carry out diverse functions, and many of them are regulators of gene expression. Genes encoding small, untranslated RNAs are difficult to detect experimentally or to predict by traditional sequence analysis approaches. Thus, in spite of the rising recognition that such RNAs may play key roles in bacterial physiology, many of the small RNAs known to date were discovered fortuitously. RESULTS: To search the Escherichia coli genome sequence for genes encoding small RNAs, we developed a computational strategy employing transcription signals and genomic features of the known small RNA-encoding genes. The search, for which we used rather restrictive criteria, has led to the prediction of 24 putative sRNA-encoding genes, of which 23 were tested experimentally. Here we report on the discovery of 14 genes encoding novel small RNAs in E. coli and their expression patterns under a variety of physiological conditions. Most of the newly discovered RNAs are abundant. Interestingly, the expression level of a significant number of these RNAs increases upon entry into stationary phase. CONCLUSIONS: Based on our results, we conclude that small RNAs are much more widespread than previously imagined and that these versatile molecules may play important roles in the fine-tuning of cell responses to changing environments.

Bulged residues promote the progression of a loop-loop interaction to a stable and inhibitory antisense-target RNA complex.

In several groups of bacterial plasmids, antisense RNAs regulate copy number through inhibition of replication initiator protein synthesis. These RNAs are characterized by a long hairpin structure interrupted by several unpaired residues or bulged loops. In plasmid R1, the inhibitory complex between the antisense RNA (CopA) and its target mRNA (CopT) is characterized by a four-way junction structure and a side-by-side helical alignment. This topology facilitates the formation of a stabilizer intermolecular helix between distal regions of both RNAs, essential for in vivo control. The bulged residues in CopA/CopT were shown to be required for high in vitro binding rate and in vivo activity. This study addresses the question of why removal of bulged nucleotides blocks stable complex formation. Structure mapping, modification interference, and molecular modeling of bulged-less mutant CopA-CopT complexes suggests that, subsequent to loop-loop contact, helix propagation is prevented. Instead, a fully base paired loop-loop interaction is formed, inducing a continuous stacking of three helices. Consequently, the stabilizer helix cannot be formed, and stable complex formation is blocked. In contrast to the four-way junction topology, the loop-loop interaction alone failed to prevent ribosome binding at its loading site and, thus, inhibition of RepA translation was alleviated.

Four-way junctions in antisense RNA-mRNA complexes involved in plasmid replication control: a common theme?

In several groups of bacterial plasmids, antisense RNAs regulate copy number through inhibition of replication initiator protein synthesis. In plasmid R1, we have recently shown that the inhibitory complex between the antisense RNA (CopA) and its target mRNA (CopT) is characterized by the formation of two intermolecular helices, resulting in a four-way junction structure and a side-by-side helical alignment. Based on lead-induced cleavage and ribonuclease (RNase) V(1) probing combined with molecular modeling, a strikingly similar topology is supported for the complex formed between the antisense RNA (Inc) and mRNA (RepZ) of plasmid Col1b-P9. In particular, the position of the four-way junction and the location of divalent ion-binding site(s) indicate that the structural features of these two complexes are essentially the same in spite of sequence differences. Comparisons of several target and antisense RNAs in other plasmids further indicate that similar binding pathways are used to form the inhibitory antisense-target RNA complexes. Thus, in all these systems, the structural features of both antisense and target RNAs determine the topologically possible and kinetically favored pathway that is essential for efficient in vivo control.

Introduction of an RNA stability element at the 5'-end of an antisense RNA cassette increases the inhibition of target RNA translation.

This communication describes improvement strategies used on a previously described two-unit antisense RNA cassette system. This cassette system encodes RNA with noncontiguous regions of complementarity to a bacterial target RNA, lacI mRNA. One of the units of complementarity was contained within an RNA stem-loop resembling that of the very efficient, naturally occurring antisense RNA CopA. As relatively low inhibitory activity was obtained previously, we tested variants in which several stem-loops were combined within one RNA, each of them directed against a different stretch of target RNA. One to four stem-loop RNAs were tested and found to be relatively ineffective, likely because of low metabolic stability. To increase the intracellular stability of these and other antisense RNAs, a stabilizer element (stem-loop derived from gene 32 mRNA of phage T4) was inserted at their 5'-ends. The results indicate that addition of this element indeed increased antisense RNA efficiency in vivo. As expected, this effect was primarily due to a longer antisense RNA half-life, as shown by RNA abundance (Northern analysis) and decay rates (rifampicin runout experiments). In summary, the results reported indicate that rational design of antisense RNA is feasible, but that the degree of inhibition (approximately 75% maximum inhibition) accomplished here could still be improved.

Progression of a loop-loop complex to a four-way junction is crucial for the activity of a regulatory antisense RNA.

The antisense RNA, CopA, regulates the replication frequency of plasmid R1 through inhibition of RepA translation by rapid and specific binding to its target RNA (CopT). The stable CopA-CopT complex is characterized by a four-way junction structure and a side-by-side alignment of two long intramolecular helices. The significance of this structure for binding in vitro and control in vivo was tested by mutations in both CopA and CopT. High rates of stable complex formation in vitro and efficient inhibition in vivo required initial loop-loop complexes to be rapidly converted to extended interactions. These interactions involve asymmetric helix progression and melting of the upper stems of both RNAs to promote the formation of two intermolecular helices. Data presented here delineate the boundaries of these helices and emphasize the need for unimpeded helix propagation. This process is directional, i.e. one of the two intermolecular helices (B) must form first to allow formation of the other (B'). A binding pathway, characterized by a hierarchy of intermediates leading to an irreversible and inhibitory RNA-RNA complex, is proposed.

Antisense RNA-mediated transcriptional attenuation: an in vitro study of plasmid pT181.

Antisense RNAs regulate plasmid replication by several different mechanisms. One of these mechanisms, transcriptional attenuation, was first described for the staphylococcal plasmid pT181, and later for the streptococcal plasmids pIP501 and pAMbeta1. Previously, we performed detailed in vitro and in vivo analyses of the pIP501 system. Here, we present an in vitro analysis of the antisense system of plasmid pT181. The secondary structures of antisense and sense RNA species of different lengths were determined. Binding rate constants for sense/antisense RNA pairs were measured, and functional segments required for complex formation were determined. A single-round transcription assay was used for in vitro analysis of transcriptional attenuation. A comparison between pT181 and pIP501 revealed several differences; whereas a truncated derivative of pIP501 antisense RNA was sufficient for stable complex formation, both stem-loop structures of pT181-RNAI were required. In contrast to the sense RNA of pIP501, which showed an intrinsic propensity to terminate (30-50% in the absence of antisense RNA), the sense RNA of pT181 required antisense RNA for induced termination. Rate constants of formation of pT181 sense-antisense RNA complexes were similar to inhibition rate constants, in striking contrast to pIP501, in which inhibition occurred at least 10-fold faster than stable binding.

An unusual structure formed by antisense-target RNA binding involves an extended kissing complex with a four-way junction and a side-by-side helical alignment.

The antisense RNA CopA binds to the leader region of the repA mRNA (target: CopT). Previous studies on CopA-CopT pairing in vitro showed that the dominant product of antisense RNA-mRNA binding is not a full RNA duplex. We have studied here the structure of CopA-CopT complex, combining chemical and enzymatic probing and computer graphic modeling. CopI, a truncated derivative of CopA unable to bind CopT stably, was also analyzed. We show here that after initial loop-loop interaction (kissing), helix propagation resulted in an extended kissing complex that involves the formation of two intermolecular helices. By introducing mutations (base-pair inversions) into the upper stem regions of CopA and CopT, the boundaries of the two newly formed intermolecular helices were delimited. The resulting extended kissing complex represents a new type of four-way junction structure that adopts an asymmetrical X-shaped conformation formed by two helical domains, each one generated by coaxial stacking of two helices. This structure motif induces a side-by-side alignment of two long intramolecular helices that, in turn, facilitates the formation of an additional intermolecular helix that greatly stabilizes the inhibitory CopA-CopT RNA complex. This stabilizer helix cannot form in CopI-CopT complexes due to absence of the sequences involved. The functional significance of the three-dimensional models of the extended kissing complex (CopI-CopT) and the stable complex (CopA-CopT) are discussed.

The international space station: globalizing life sciences.

The ISS changes the scope of science activities for the future and links the US to its partners in technology, science, and the exploration of space in an unprecedented manner.

Antisense RNA regulation in prokaryotes: rapid RNA/RNA interaction facilitated by a general U-turn loop structure.

Efficient gene control by antisense RNA requires rapid bi-molecular interaction with a cognate target RNA. A comparative analysis revealed that a YUNR motif (Y=pyrimidine, R=purine) is ubiquitous in RNA recognition loops in antisense RNA-regulated gene systems. The (Y)UNR sequence motif specifies two intraloop hydrogen bonds forming U-turn structures in many anticodon-loops and all T-loops of tRNAs, the hammerhead ribozyme and in other conserved RNA loops. This structure creates a sharp bend in the RNA phosphate-backbone and presents the following three to four bases in a solvent-exposed, stacked configuration providing a scaffold for rapid interaction with complementary RNA. Sok antisense RNA from plasmid R1 inhibits translation of the hok mRNA by preventing ribosome entry at the mok Shine & Dalgarno element. The 5' single-stranded region of Sok-RNA recognizes a loop in the hok mRNA. We show here, that the initial pairing between Sok antisense RNA and its target in hok mRNA occurs with an observed second-order rate-constant of 2 x 10(6) M(-1) s(-1). Mutations that eliminate the YUNR motif in the target loop of hok mRNA resulted in reduced antisense RNA pairing kinetics, whereas mutations maintaining the YUNR motif were silent. In addition, RNA phosphate-backbone accessibility probing by ethylnitrosourea was consistent with a U-turn structure formation promoted by the YUNR motif. Since the YUNR U-turn motif is present in the recognition units of many antisense/target pairs, the motif is likely to be a generally employed enhancer of RNA pairing rates. This suggestion is consistent with the re-interpretation of the mutational analyses of several antisense control systems including RNAI/RNAII of ColE1, CopA/CopT of R1 and RNA-IN/RNA-OUT of IS10.

The plant-growth-promoting rhizobacterium Paenibacillus polymyxa induces changes in Arabidopsis thaliana gene expression: a possible connection between biotic and abiotic stress responses.

This paper addresses changes in plant gene expression induced by inoculation with plant-growth-promoting rhizobacteria (PGPR). A gnotobiotic system was established with Arabidopsis thaliana as model plant, and isolates of Paenibacillus polymyxa as PGPR. Subsequent challenge by either the pathogen Erwinia carotovora (biotic stress) or induction of drought (abiotic stress) indicated that inoculated plants were more resistant than control plants. With RNA differential display on parallel RNA preparations from P. polymyxa-treated or untreated plants, changes in gene expression were investigated. From a small number of candidate sequences obtained by this approach, one mRNA segment showed a strong inoculation-dependent increase in abundance. The corresponding gene was identified as ERD15, previously identified to be drought stress responsive. Quantification of mRNA levels of several stress-responsive genes indicated that P. polymyxa induced mild biotic stress. This suggests that genes and/or gene classes associated with plant defenses against abiotic and biotic stress may be co-regulated. Implications of the effects of PGPR on the induction of plant defense pathways are discussed.

Regulation of plasmid R1 replication: PcnB and RNase E expedite the decay of the antisense RNA, CopA.

The replication frequency of plasmid R1 is controlled by an unstable antisense RNA, CopA, which, by binding to its complementary target, blocks translation of the replication rate-limiting protein RepA. Since the degree of inhibition is directly correlated with the intracellular concentration of CopA, factors affecting CopA turnover can also alter plasmid copy number. We show here that PcnB (PAPl-a poly(A)polymerase of Escherichia coli) is such a factor. Previous studies have shown that the copy number of ColE1 is decreased in pcnB mutant strains because the stability of the RNase E processed form of RNAI, the antisense RNA regulator of ColE1 replication, is increased. We find that, analogously, the twofold reduction in R1 copy number caused by a pcnB lesion is associated with a corresponding increase in the stability of the RNase E-generated 3' cleavage product of CopA. These results suggest that CopA decay is initiated by RNase E cleavage and that PcnB is involved in the subsequent rapid decay of the 3' CopA stem-loop segment. We also find that, as predicted, under conditions in which CopA synthesis is unaffected, pcnB mutation reduces RepA translation and increases CopA stability to the same extent.

Dual function of the copR gene product of plasmid pIP501.

Replication of plasmid pIP501 is regulated at a step subsequent to transcription initiation by an antisense RNA (RNAIII) and transcriptionally by a repressor protein, CopR. Previously, it had been shown that CopR binds to a 44-bp DNA fragment upstream of and overlapping the repR promoter pII. Subsequently, we found that high-copy-number pIP501 derivatives lacking copR and low-copy-number derivatives containing copR produced the same intracellular amounts of RNAIII. This suggested a second, hitherto-unknown function of CopR. In this report, we show that CopR does not affect the half-life of RNAIII. Instead, we demonstrate in vivo that, in the presence of both pII and pIII, CopR provided in cis or in trans causes an increase in the intracellular concentration of RNAIII and that this effect is due to the function of the protein rather than its mRNA. We suggest that, in the absence of CopR, the increased (derepressed) RNAII transcription interferes, in cis, with initiation of transcription of RNAIII (convergent transcription), resulting in a lower RNAIII/plasmid ratio. When CopR is present, the pII promoter is repressed to >90%, so that convergent transcription is mostly abolished and RNAIII/plasmid ratios are high. The hypothesis that RNAII transcription influences promoter pIII through induced changes in DNA supercoiling is supported by the finding that the gyrase inhibitor novobiocin affects the accumulation of both sense and antisense RNA. The dual role of CopR in repression of RNAII transcription and in prevention of convergent transcription is discussed in the context of replication control of pIP501.

A two unit antisense RNA cassette test system for silencing of target genes.

This communication describes a two unit antisense RNA cassette system for use in gene silencing. Cassettes consist of a recognition unit and an inhibitory unit which are transcribed into a single RNA that carries sequences of non-contiguous complementarity to the chosen target RNA. The recognition unit is designed as a stem-loop for rapid formation of long- lived binding intermediates with target sequences and resembles the major stem-loop of a naturally occurring antisense RNA, CopA. The inhibitory unit consists of either a sequence complementary to a ribosome binding site or of a hairpin ribozyme targeted at a site within the chosen mRNA. The contributions of the individual units to inhibition was assessed using the lacI gene as a target. All possible combinations of recognition and inhibitory units were tested in either orientation. In general, inhibition of lacI expression was relatively low. Fifty per cent inhibition was obtained with the most effective of the constructs, carrying the recognition stem-loop in the antisense orientation and the inhibitory unit with an anti-RBS sequence. Several experiments were performed to assess activities of the RNAs in vitro and in vivo : antisense RNA binding assays, cleavage assays, secondary structure analysis as well as Northern blotting and primer extension analysis of antisense and target RNAs. The problems associated with this antisense RNA approach as well as its potential are discussed with respect to possible optimization strategies.

Antisense RNA control of plasmid R1 replication. The dominant product of the antisense rna-mrna binding is not a full RNA duplex.

The replication frequency of plasmid R1 is controlled by an antisense RNA (CopA) that binds to its target site (CopT) in the leader region of repA mRNA and inhibits the synthesis of the replication initiator protein RepA. Previous studies on CopA-CopT pairing in vitro revealed the existence of a primary loop-loop interaction (kissing complex) that is subsequently converted to an almost irreversible duplex. However, the structure of more stable binding intermediates that lead to the formation of a complete duplex was speculative. Here, we investigated the interaction between CopA and CopT by using Pb(II)-induced cleavages. The kissing complex was studied using a truncated antisense RNA (CopI) that is unable to form a full duplex with CopT. Furthermore, RNase III, which is known to process the CopA-CopT complex in vivo, was used to detect the existence of a full duplex. Our data indicate that the formation of a full CopA-CopT duplex appears to be a very slow process in vitro. Unexpectedly, we found that the loop-loop interaction persists in the predominant CopA-CopT complex and is stabilized by intermolecular base pairing involving the 5'-proximal 30 nucleotides of CopA and the complementary region of CopT. This almost irreversible complex suffices to inhibit ribosome binding at the tap ribosome binding site and may be the inhibitory complex in vivo.

An antisense/target RNA duplex or a strong intramolecular RNA structure 5' of a translation initiation signal blocks ribosome binding: the case of plasmid R1.

Antisense RNAs in prokaryotic systems often inhibit translation of mRNAs. In some cases, this involves sequestration of Shine-Dalgarno (SD) sequences and start codons. In other cases, antisense/target RNA duplexes do not overlap these signals, but form upstream. We have performed toeprinting analyses on repA mRNA of plasmid R1, both free and in duplex with the antisense RNA, CopA. An intermolecular RNA duplex 2 nt upstream of the tap SD prevents ribosome binding. An intrastrand stem-loop at this location yields the same inhibition. Thus, stable secondary structures immediately upstream of the tap SD sequence inhibit translation, as shown by toeprinting in vitro and repA-lacZ expression in vivo. Previous work showed that repA (initiator protein) expression requires tap (leader peptide) translation. Toeprinting data confirm that the tap ribosome binding site (RBS) is accessible, whereas the repA RBS, which is sequestered by a stable stem-loop, is weakly recognized by the ribosome. Truncated CopA RNA (CopI) is unable to pair completely with target RNA, but proceeds normally to a kissing intermediate. This mutant RNA species inhibits repA expression in vivo. By a kinetic toeprint inhibition protocol, we have shown that the structure of the kissing complex is sufficient to sterically prevent ribosome binding. These results are discussed in comparison with the effect of RNA structures elsewhere in the ribosome-binding region of an mRNA.

An unusually long-lived antisense RNA in plasmid copy number control: in vivo RNAs encoded by the streptococcal plasmid pIP501.

The main regulator of pIP501 replication is an antisense RNA (RNAIII) that induces transcriptional attenuation of the essential RNAII. Previous studies identified the termination point in vivo and demonstrated attenuation in vitro. This in vivo analysis confirms the appearance of attenuated RNAII dependent on RNAIII. Half-lives and intracellular levels of RNAII and RNAIII were determined: in a Bacillus subtilis cell harboring a wild-type pIP501 plasmid, approximately 50 molecules RNAII and 1000 to 2000 molecules of RNAIII were measured, respectively. The half-life of RNAII was in the range of that of other target RNAs, whereas that of RNAIII (approximately 30 minutes) was unusually long, representing a so far unprecedented case of a metabolically stable antisense RNA regulating plasmid copy number. Long antisense RNA half-life is predicted to yield sluggish control and instability of maintenance. We propose a model for how plasmid pIP501 may avoid this problem by using both the repressor CopR and the antisense RNAIII for control. Four stem-loop mutants of RNAII/RNAIII with elevated copy numbers were characterized for in vitro antisense/target RNA binding, RNAIII half-life, incompatibility, and attenuation in vivo. Two classes were found: interaction mutants and half-life mutants. The former suggest a key function for loop LIII of RNAIII as recognition loop in the primary steps of RNAII/RNAIII interaction.