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T Franch

Publications and source records attributed to T Franch.

9 recordsLinked to original sources
PubMed2000

U-turns and regulatory RNAs.

Conventional antisense RNAs, such as those controlling plasmid replication and maintenance, inhibit the function of their target RNAs rapidly and efficiently. Novel findings show that a common U-turn loop structure mediates fast RNA pairing in the majority of these RNA controlled systems. Usually, an antisense RNA regulates a single, cognate target RNA only. Recent reports, however, show that antisense RNAs can act as promiscuous regulators that control multiple genes in concert to integrate complex physiological responses in Escherichia coli.

Base Pairing↗
PubMed1999

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.

Bacterial Proteins↗
PubMed1999

Ribonuclease III processing of coaxially stacked RNA helices.

The RNase III family of endoribonucleases participates in maturation and decay of cellular and viral transcripts by processing of double-stranded RNA. RNase III degradation is inherent to most antisense RNA-regulated gene systems in Escherichia coli. In the hok/sok system from plasmid R1, Sok antisense RNA targets the hok mRNA for RNase III-mediated degradation. An intermediate in the pairing reaction between Sok RNA and hok mRNA forms a three-way junction. A complex between a chimeric antisense RNA and hok mRNA that mimics the three-way junction was cleaved by RNase III both in vivo and in vitro. Footprinting using E117A RNase III binding to partially complementary RNAs showed protection of the 13 base pairs of interstrand duplex and of the bottom part of the transcriptional terminator hairpin of the antisense RNA. This suggests that the 13 base pairs of RNA duplex are coaxially stacked on the antisense RNA terminator stem-loop and that each stem forms a monomer half-site, allowing symmetrical binding of the RNase III dimer. This processing scheme shows an unanticipated diversity in RNase III substrates and may have a more general implication for RNA metabolism.

Bacterial Proteins↗
PubMed1998

Targeting of nucleic acid junctions: addressing to a branch point an oligodeoxynucleotide conjugated with an intercalator.

It is possible to enhance targeting of a DNA stem flank domain with a complementary DNA when it is conjugated with diphenyl ether at the branch point. The nucleoside 2'-deoxy-5-methyl- N 4-(4-phenoxyphenyl)cytidine (5) was synthesized from thymidineby tritylation, acetylation, amination via 2,4, 6-trimethylbenzenesulfonyl activation and subsequent de-protection. When a three-way junction is formed with a bulged nucleoside 5 at the branch point, the thermal melting temperature was increased by 9 degreesC when compared with wild-type DNA. When hybridizing to one of the flanks at a stem allowing coaxial stacking to the stem, modification at the branch point resulted in DeltaTm= 5.8 degreesC. For targeting to RNA the results were more ambiguous. RNase H activity was observed in some cases when an intercalating aromatic ring was addressed at the branch point. RNase H activity was observed even for a short 7mer ODN.

Base Sequence↗
PubMed1997

Programmed cell death by hok/sok of plasmid R1: processing at the hok mRNA 3'-end triggers structural rearrangements that allow translation and antisense RNA binding.

The hok/sok locus of plasmid R1 mediates plasmid stabilization by killing of plasmid-free cells. The locus specifies two RNAs, hok mRNA and Sok antisense RNA. The post-segregational killing mediated by hok/sok is governed by a complicated control mechanism that involves both post-transcriptional inhibition of translation by Sok-RNA and activation of hok translation by mRNA 3' processing. Sok-RNA inhibits translation of a reading frame (mok) that overlaps with hok, and translation of hok is coupled to translation of mok. In the inactive full-length hok mRNA, the translational activator element at the mRNA 5'-end (tac) is sequestered by the fold-back-inhibitory element located at the mRNA 3'-end (fbi). The 5' to 3' pairing locks the RNA in an inert configuration in which the SDmok and Sok-RNA target regions are sequestered. Here we show that the 3' processing leads to major structural rearrangements in the mRNA 5'-end. The structure of the refolded RNA explains activation of translation and antisense RNA binding. The refolded RNA contains an antisense RNA target stem-loop that presents the target nucleotides in a single-stranded conformation. The stem of the target hairpin contains SDmok and AUGmok in a paired configuration. Using toeprinting analysis, we show that this pairing keeps SDmok in an accessible configuration. Furthermore, a mutational analysis shows that an internal loop in the target stem is prerequisite for efficient translation and antisense RNA binding.

Apoptosis↗
PubMed1997

Programmed cell death by hok/sok of plasmid R1: coupled nucleotide covariations reveal a phylogenetically conserved folding pathway in the hok family of mRNAs.

The hok/sok system of plasmid R1 mediates plasmid maintenance by killing of plasmid-free cells. Translation of the stable toxin-encoding hok mRNA is repressed by the unstable Sok antisense RNA. Using genetic algorithm simulations and phylogenetic comparisons, we analyse five plasmid-encoded and two chromosome-encoded hok-homologous mRNAs. A similar folding pathway was found for all mRNAs. Metastable hairpins at the very 5'-ends of the mRNAs were predicted to prevent the formation of structures required for translation and antisense RNA binding. Thus the folding of the mRNA 5'-ends appears to explain the apparent inactivity of the nascent transcripts. In the full-length mRNAs, long-range 5' to 3' interactions were predicted in all cases. The 5' to 3' interactions lock the mRNAs in inactive configurations. Translation of the mRNAs is activated by 3' exonucleolytic processing. Simulation of the 3' processing predicted that it triggers rearrangements of the mRNA 5'-ends with the formation of translational activator and antisense RNA target hairpins. Alignment of the mRNA sequences revealed a large number of nucleotide covariations that support the existence of the proposed secondary structures. Furthermore, coupled covariations support the folding pathway and provide evidence that the mRNA 5'-ends pair with three different partners during the proposed series of dynamic RNA rearrangements.

Apoptosis↗
PubMed1997

Antisense RNA-regulated programmed cell death.

Eubacterial plasmids and chromosomes encode multiple killer genes belonging to the hok gene family. The plasmid-encoded killer genes mediate plasmid stabilization by killing plasmid-free cells. This review describes the genetics, molecular biology, and evolution of the hok gene family. The complicated antisense RNA-regulated control-loop that regulates posttranscriptional and postsegregational activation of killer mRNA translation in plasmid-free cells is described in detail. Nucleotide covariations in the mRNAs reveal metastable stem-loop structures that are formed at the mRNA 5' ends in the nascent transcripts. The metastable structures prevent translation and antisense RNA binding during transcription. Coupled nucleotide covariations provide evidence for a phylogenetically conserved mRNA folding pathway that involves sequential dynamic RNA rearrangements. Our analyses have elucidated an intricate mechanism by which translation of an antisense RNA-regulated mRNA can be conditionally activated. The complex phylogenetic relationships of the plasmid- and chromosome-encoded systems are also presented and discussed.

Apoptosis↗
PubMed1996

Programmed cell death in bacteria: translational repression by mRNA end-pairing.

The hok/sok and pnd systems of plasmids R1 and R483 mediate plasmid maintenance by killing plasmid-free cells. Translation of the exceptionally stable hok and pnd mRNAs is repressed by unstable antisense RNAs. The different stabilities of the killer mRNAs and their cognate repressors explain the onset of translation in plasmid-free cells. The full-length hok and pnd mRNAs are inert with respect to translation and antisense RNA binding. We have previously shown that the mRNAs contain two negative translational control elements. Thus, the mRNAs contain upstream anti-Shine-Dalgarno elements that repress translation by shielding the Shine-Dalgarno elements. The mRNAs also contain fold-back-inhibition elements (fbi) at their 3' ends that are required to maintain the inert mRNA configuration. Using genetic complementation, we show that the 3' fbi elements pair with the very 5' ends of the mRNAs. This pairing sets the low rate of 3' exonucleolytical processing, which is required for the accumulation of an activatable pool of mRNA. Unexpectedly, the hok and pnd mRNAs were found to contain translational activators at their 5' ends (termed tac). Thus, the fbi elements inhibit translation of the full-length mRNAs by sequestration of the tac elements. The fbi elements are removed by 3' exonucleolytical processing. Mutational analyses indicate that the 3' processing triggers refolding of the mRNA 5' ends into translatable configurations in which the 5' tac elements base pair with the anti-Shine-Dalgarno sequences.

Apoptosis↗