Biomedical subjects

E G Wagner

Publications and source records attributed to E G Wagner.

At least 37 records · Page 2Linked to original sources

PubMed1996

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.

Bacterial Proteins↗

PubMed1995

Bulged-out nucleotides protect an antisense RNA from RNase III cleavage.

Bulged-out nucleotides or internal loops are present in the stem-loop structures of several antisense RNAs. We have used the antisense/target RNA system (CopA/CopT) that controls the copy number of plasmid R1 to examine the possible biological function of bulged-out nucleotides. Two regions within the major stem-loop of the antisense RNA, CopA, carry bulged-out nucleotides. Base pairing in either one or both of these regions of the stem was restored by site-specific mutagenesis and in one case a new internal loop was introduced. The set of mutant and wild-type CopA variants was characterized structurally in vitro. The results reported here indicate a possible function of the bulges: their presence protects CopA RNA from being a substrate for the double-strand-specific enzyme RNase III. In vitro cleavage rates were drastically increased when either the lower or both bulges were absent. This is paralleled by a similar, but not identical, effect of the bulges on metabolic stability of the CopA RNAs in vivo. The degradation pathways of wild-type and mutant CopA in various strain backgrounds are discussed. In the accompanying paper, we address the significance of bulges in CopA for binding to the target RNA in vitro and for its inhibitory efficiency in vivo.

Bacterial Proteins↗

PubMed1995

Bulged-out nucleotides in an antisense RNA are required for rapid target RNA binding in vitro and inhibition in vivo.

Naturally occurring antisense RNAs in prokaryotes are generally short, highly structured and untranslated. Stem-loops are always present, and loop regions serve as primary recognition structures in most cases. Single-stranded tails or internal unstructured regions are required for initiation of stable pairing between antisense and target RNA. Most antisense RNAs contain bulged-out nucleotides or small internal loops in upper stem regions. Here we investigated the role of the bulged-out nucleotides of CopA (the copy number regulator of plasmid R1) in determining the binding properties of this antisense RNA to its target in vitro and the efficiency of a translational inhibition in vivo. The introduction of perfect helicity in the region of the two bulges in CopA decreased pairing rate constants by up to 180-fold, increased equilibrium dissociation constants of the 'kissing intermediate' up to 14-fold, and severely impaired inhibition of repA expression. A previously described loop size mutant of CopA showed decreased pairing rates, but, in contrast to the bulge-less mutant CopAs, shows a decreased dissociation constant of the 'kissing complex'. We conclude that removal of the specific bulges/internal loops within the stem-loop II of CopA impairs the inhibitor, and that creation of an internal loop at a different position does not restore activity, emphasizing the optimal folding of wild-type CopA. The accompanying paper shows that an additional function of bulges can be protection from RNase III cleavage.

Bacterial Proteins↗

PubMed1994

Antisense RNA-mediated transcriptional attenuation occurs faster than stable antisense/target RNA pairing: an in vitro study of plasmid pIP501.

Antisense RNA-mediated transcriptional attenuation is the mode of replication control of several plasmids, among them pIP501. This mechanism implies that the repR mRNAs can fold into two mutually exclusive structures. The formation of one of these structures is induced by binding of the antisense RNA and results in premature termination. Since the fate of the nascent mRNA transcripts depends on the binding rate of the antisense RNA to its target, the control is kinetic. We have studied the antisense RNA, RNAIII, and target RNA, RNAII, whose interaction determines the replication frequency of plasmid pIP501. RNA secondary structures were analyzed using structure-specific RNases. RNA binding was studied in vitro with normal size and truncated RNAIII species. An in vitro single-round attenuation assay was developed that permits qualitative and quantitative assessment of inhibition by RNAIII. The effect of varying concentrations of RNAIII species on attenuation was tested and inhibition rate constants were calculated. The inhibition rate constants were at least 10 times higher than the pairing rate constants. Thus, steps preceding stable RNA duplex formation are sufficient to induce RNAIII-dependent termination of nascent RNAII transcripts.

Base Sequence↗

PubMed1994

Mechanism of post-segregational killing: Sok antisense RNA interacts with Hok mRNA via its 5'-end single-stranded leader and competes with the 3'-end of Hok mRNA for binding to the mok translational initiation region.

The hok/sok system of plasmid R1, which mediates plasmid stabilization by killing of plasmid-free segregants, codes for two RNA species, Hok mRNA and Sok antisense RNA. The lethal expression of hok is inhibited post-transcriptionally by the 67 nt Sok-RNA. In this paper, we analyse the secondary structure of Sok-RNA and the binding of Sok-RNA to Hok mRNA in vitro. The reaction between the two RNAs leads to the formation of a complete duplex in which Sok-RNA is hybridized over its entire length to Hok mRNA. The second-order rate constant of duplex formation was determined to be approximately 1 x 10(5) M-1s-1. Mutations in the 5'-end single-stranded leader of Sok-RNA severely reduced the binding rate to wt Hok mRNA, whereas loop mutations in Sok-RNA had no such effect. The reduced binding rates were paralleled by abolished in vivo regulatory properties. These results suggest that, unlike in other well-characterized antisense/target RNA systems, the initial recognition reaction between Sok-RNA and Hok mRNA takes place between the single-stranded 5'-end of Sok-RNA and the complementary region in Hok mRNA, without the involvement of an antisense loop in the initial binding step. Furthermore, the finding that Sok-RNA competes with the 3'-end of full-length Hok mRNA for binding to the mok translational initiation region adds to the complexity of killer gene regulation.

Bacterial Proteins↗

PubMed1994

Kinetic aspects of control of plasmid replication by antisense RNA.

Plasmids are replicating DNA molecules that are present in defined numbers of copies per cell. They encode systems that control their replication such that given steady-state values for their copy numbers are maintained. This is a special type of control, since it requires the genome to measure its concentration continuously and adjust its rate of replication to parallel the rate of growth of the cell mass. In this review we discuss the quantitative kinetic properties of copy-number control of the R1 plasmid, in which the control device is an antisense RNA that controls the synthesis of a protein that is rate-limiting for replication of the plasmid.

Bacterial Proteins↗

PubMed1994

Replication control of plasmid R1: disruption of an inhibitory RNA structure that sequesters the repA ribosome-binding site permits tap-independent RepA synthesis.

The replication frequency of plasmid R1 is controlled by an antisense RNA, CopA, that inhibits the synthesis of the replication initiator protein, RepA, at the post-transcriptional level. This inhibition is indirect and affects translation of a leader peptide reading frame (tap). Translation of tap is required for repA translation (Blomberg et al., 1992). Here we asked whether an RNA stem-loop sequestering the repA ribosome-binding site blocks tap translation-independent repA expression. Destabilization of this structure resulted in tap-independent RepA synthesis, concomitant with a loss of CopA-mediated inhibition; thus, CopA acts at the level of tap translation. Structure probing of RepA mRNAs confirmed that the introduced mutations induced a local destabilization in the repA ribosome-binding site stem-loop. An increased spacing between the repA Shine-Dalgarno region and the start codon permitted even higher repA expression. In Incl alpha/IncB plasmids, an RNA pseudoknot acts as an activator for rep translation. We suggest that the regulatory pathway in plasmid R1 does not involve an activator RNA pseudoknot.

Bacterial Proteins↗

PubMed1994

Antisense RNA control in bacteria, phages, and plasmids.

Antisense RNA control is now recognized as an efficient and specific means of regulating gene expression at the posttranscriptional level. Almost all naturally occurring cases have been found in prokaryotes, often in their accessory genetic elements. Several antisense RNA systems are now well-understood, and these display a spectrum of mechanisms of action, binding pathways, and kinetics. This review summarizes antisense RNA control in prokaryotes, emphasizing the biology of the systems involved.

Bacteria↗

PubMed1993

Structural and functional analyses of the FinP antisense RNA regulatory system of the F conjugative plasmid.

The efficiency of conjugation of F-like plasmids is regulated by the FinOP fertility inhibition system. The transfer (tra) operon is under the direct control of the TraJ transcriptional activator which, in turn, is negatively regulated by FinP, an antisense RNA, and FinO, a 22 kDa protein. Recently, FinO has been shown to extend the chemical stability of FinP in vivo in the absence of traJ mRNA. The in vitro secondary structures of both the FinP and TraJ RNAs were determined by the use of single- and double-strand-specific nucleases; both RNAs were found to have double stem-loop structures that are complementary to each other and, therefore, FinP RNA and TraJ RNA have the potential to form a duplex with each other. This was verified by in vitro binding experiments. The reaction was shown to be biomolecular with an apparent rate constant (kapp) of 5 x 10(5)M-1s-1, a value that is similar to those found for other natural antisense RNA systems. Preliminary evidence for the in vivo formation of the FinP-TraJ RNA duplex was obtained by primer extension of the traJ mRNA; the presence of both FinO and FinP was required to cause a dramatic reduction in the steady-state level of traJ mRNA, perhaps as a result of RNase III degradation of the resulting RNA duplex.

Bacterial Outer Membrane Proteins↗

PubMed1993

PcnB is required for the rapid degradation of RNAI, the antisense RNA that controls the copy number of ColE1-related plasmids.

The replication of ColE1-related plasmids is controlled by an unstable antisense RNA, RNAI, which can interfere with the successful processing of the RNAII primer of replication. We show here that a host protein, PcnB, supports replication by promoting the decay of RNAI. In bacterial strains deleted for PcnB a stable, active form of RNAI, RNAI*, which appears to be identical to the product of 5'-end processing by RNAase E, accumulates. This leads to a reduction in plasmid copy number. We show, using a GST-PcnB fusion protein, that PcnB does not interfere with RNAI/RNAII binding in vitro. The fusion protein, like PcnB, has polyadenylating activity and is able to polyadenylate RNAI (and also another antisense RNA, CopA) in vitro.

Ampicillin Resistance↗

PubMed1992

The effect of loop size in antisense and target RNAs on the efficiency of antisense RNA control.

Most natural antisense RNAs display a high degree of secondary structure with stem-loops as their most prominent feature. Mutations affecting the inhibitory activity of these RNAs most often map in or close to loop regions in both the antisense and target RNAs. The primary recognition loops often contain 5-7 unpaired nucleotides. Nucleotide changes in the loops affect the binding rate and, hence, the inhibitory effect on the activity of the target RNA. Here we address the question whether loop sizes affect binding rates between antisense and target RNAs, using the replication control system of plasmid R1 as a model system. By creating a series of loop size mutants we show that loop size alterations have strong effects on the binding rates between the two reactant RNAs in vitro, and that most of the mutations analyzed display corresponding effects on antisense RNA control in vivo. Our data suggest that the three-dimensional structures of antisense and target RNA stem-loops are crucial for determining binding rates. The implications of these results for the design of efficient artificial antisense RNA control systems are discussed.

Bacterial Proteins↗

PubMed1992

Mechanism of killer gene activation. Antisense RNA-dependent RNase III cleavage ensures rapid turn-over of the stable hok, srnB and pndA effector messenger RNAs.

The hok/sok, srnB and pnd systems of plasmids R1, F and R438 mediate plasmid maintenance by killing plasmid-free segregants. The systems encode exceptionally stable full-length mRNAs that code for potent cell toxins that kill the cells from within. The systems also produce truncated mRNAs whose appearance is correlated with killing activity. The truncated mRNAs are shortened by 35 to 70 nucleotides in the 3' ends, but have the same 5' ends as the full-length transcripts. Translation of the stable killer mRNAs is regulated by unstable antisense RNAs that are complementary to the leader regions of the full-length and truncated mRNAs. We show here, that both the presence of the antisense RNA and of the host enzyme RNase III is required for rapid cleavage of the truncated mRNAs, and we map the cleavage point in the Hok mRNA in vitro and in vivo to be located between nucleotides +245 and +246. The RNase III cleavage products of the Hok mRNA were found to be very unstable in vivo. Thus, RNase III cleavage seems to be the initial event leading to decay of the killer mRNAs. In an rnc- strain, the truncated mRNA species were found in steady-state cells. This observation indicates that the truncated mRNAs are formed constitutively and independently of the presence of the antisense RNAs. Thus, the antisense RNAs prevent the accumulation of the truncated mRNAs solely by mediating their rapid hydrolysis by RNase III. Furthermore, the generation of the truncated killer mRNAs in the rnc- host indicate that RNase III is dispensable for induction of the killer gene systems. Based on these and on observations obtained previously, we present a molecular model that explains the activation of the killer mRNAs in plasmid-free segregants and after addition of rifampicin.

Base Sequence↗

PubMed1992

Replication control in plasmid R1: duplex formation between the antisense RNA, CopA, and its target, CopT, is not required for inhibition of RepA synthesis.

The replication frequency of plasmid R1 is regulated by an antisense RNA, CopA, which inhibits the synthesis of the rate-limiting initiator protein RepA. The inhibition requires an interaction between the antisense RNA and its target, CopT, in the leader of the RepA mRNA. This binding reaction has previously been studied in vitro, and the formation of a complete RNA duplex between the two RNAs has been demonstrated in vitro and in vivo. Here we investigate whether complete duplex formation is required for CopA-mediated inhibition in vivo. A mutated copA gene was constructed, encoding a truncated CopA which is impaired in its ability to form a complete CopA/CopT duplex, but which forms a primary binding intermediate (the 'kissing complex'). The mutated CopA species (S-CopA) mediated incompatibility against wild-type R1 plasmids and inhibited RepA-LacZ fusion protein synthesis. Northern blot, primer extension and S1 analyses indicated that S-CopA did not form a complete duplex with CopT in vivo since bands corresponding to RNase III cleavage products were missing. An in vitro analysis supported the same conclusion. These data suggest that formation of the 'kissing complex' suffices to inhibit RepA synthesis, and that complete CopA/CopT duplex formation is not required. The implications of these findings are discussed.

Bacterial Proteins↗

PubMed1992

Replication control of plasmid R1: RepA synthesis is regulated by CopA RNA through inhibition of leader peptide translation.

The replication frequency of plasmid R1 is post-transcriptionally controlled by an antisense RNA, CopA, that binds to the leader region in the RepA mRNA, CopT, and ultimately inhibits the synthesis of the replication initiator protein RepA. We present results demonstrating that CopA controls RepA synthesis indirectly. A reading frame for a 24 amino acid leader peptide (Tap, translational activator peptide) is located in the region between the copA and repA genes. A translational fusion between the tap and lacZ genes was used to demonstrate that tap is translated and controlled by CopA. Stop codons (UAA, UAG and UGA) introduced at three different positions within the tap gene led to a severe decrease in repA expression. Specific suppression of the stop codons reversed the effect. This indicates that tap translation is required for RepA synthesis. Phylogenetic comparisons between IncFII-like plasmids, together with previous in vitro and in vivo results (Ohman and Wagner, 1989, 1991), suggest that a stable RNA stem-loop structure sequesters the repA ribosome binding site irrespective of CopA-CopT duplex formation. The results presented here show that ribosomes translating the tap reading frame have to terminate close to the start codon of repA to permit reinitiation (direct translational coupling), and that transient disruption of the inhibitory RNA stem-loop is insufficient for activation of repA translation. The possibility that direct translational coupling is required because of a suboptimal repA RBS cannot be excluded.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗

PubMed1991

Regulation of replication of plasmid R1: an analysis of the intergenic region between copA and repA.

The synthesis of the rate-limiting RepA replication initiator protein of plasmid R1 is negatively controlled by an antisense RNA, CopA. The regulation is posttranscriptional and involves an inhibitory effect on RepA translation mediated by the binding of CopA to its target (CopT) in the leader region of the RepA mRNA. The evolutionary conservation of the intergenic region between the copA gene and the repA reading frame among plasmids related to R1 may be indicative of an important function in this regulation. One possibility is that sequences/structures in this region might be required for the presumed distal effect of CopAQCopT binding. We have performed a mutational analysis of this region, starting with a mutant repA-lacZ fusion plasmid that shows decreased RepA-LacZ synthesis compared to a wild-type construct, and have identified five compensatory mutations that increase repA-lacZ expression. Two of these were single base-pair substitutions in the copA promoter leading to a decrease in CopA transcription. The other three mutations increased RepA synthesis in the presence as well as in the absence of functional CopA. Reconstructed plasmids carrying these mutations--in conjunction with the original down-mutation or in an otherwise wild-type background--show the expected increase in copy number. The effect of two of these mutations is consistent with the destabilization of a putative secondary structure which may be responsible for the normally low translation rate of the RepA reading frame. The implications of the types of mutations found in this study, as well as the absence of other classes of mutations, are discussed in terms of alternative possible models of CopA-mediated inhibition of RepA synthesis.

Bacterial Proteins↗

PubMed1991

The rifampicin-inducible genes srnB from F and pnd from R483 are regulated by antisense RNAs and mediate plasmid maintenance by killing of plasmid-free segregants.

The gene systems srnB of plasmid F and pnd of plasmid R483 were discovered because of their induction by rifampicin. Induction caused membrane damage, RNase I influx, degradation of stable RNA and, consequently, cell killing. We show here that the srnB and pnd systems mediate efficient stabilization of a mini-R1 test-plasmid. We also show that the killer genes srnB' and pndA are regulated by antisense RNAs, and that the srnC- and pndB-encoded antisense RNAs, denoted SrnC- and PndB-RNAs, are unstable molecules of approximately 60 nucleotides. The srnB and pndA mRNAs were found to be very stable. The differential decay rates of the inhibitory antisense RNAs and the killer-gene-encoding mRNAs explain the induction of these gene systems by rifampicin. Furthermore, the observed plasmid-stabilization phenotype associated with the srnB and pnd systems is a consequence of this differential RNA decay: the newborn plasmid-free cells inherit the stable mRNAs, which, after decay of the unstable antisense RNAs, are translated into killer proteins, thus leading to selective killing of the plasmid-free segregants. Thus our observations lead us to conclude that the F srnB and R483 pnd systems are phenotypically indistinguishable from the R1 hok/sok system, despite a 50% dissimilarity at the level of DNA sequence.

Base Sequence↗

PubMed1991

Control of replication of plasmid R1: the intergenic region between copA and repA modulates the level of expression of repA.

The RepA protein of plasmid R1 is rate-limiting for initiation of R1 replication. Its synthesis is mainly regulated by interactions of the antisense RNA, CopA, with the leader region of the RepA mRNA, CopT. This work describes the characterization of several mutants with sequence alterations in the intergenic region between the copA gene and the repA reading frame. The analysis showed that most of the mutations led both to a decrease in stability of maintenance of mini-R1 derivatives and to lowered repA expression assayed in translational repA-lacZ fusion constructs. Destruction of the copA gene and replacement of the upstream region by the tac promoter in the latter constructs indicated that these mutations per se alter the expression of repA. In addition, we show that particular mutations in this region can directly affect CopA-mediated control, either by changing the kinetics of interaction of CopA RNA with the RepA mRNA and/or by modifying the activity of the copA promoter. These data indicate the importance of the region analysed in the process that controls R1 replication.

Bacterial Proteins↗

PubMed1990

Control of replication of plasmid R1: the duplex between the antisense RNA, CopA, and its target, CopT, is processed specifically in vivo and in vitro by RNase III.

The replication frequency of IncFII plasmids is regulated through the availability of a rate-limiting protein, RepA. The synthesis of this protein is controlled post-transcriptionally by a small antisense RNA, CopA, which binds to the leader region of the RepA mRNA (CopT). In this communication we report studies of the IncFII plasmid R1. We show that the duplex between CopA and CopT is cleaved specifically in vivo. The in vivo cleavage maps to the same position as that resulting from in vitro cleavage of a CopA/CopT duplex by purified RNase III. By introducing plasmids carrying translational repA-lacZ fusions into cells deficient in RNase III we show that the expression of repA is elevated when RNase III activity is severely decreased. Hence, cleavage by RNase III seems to be a key event in the copy number control system of plasmid R1.

Bacterial Proteins↗