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Probing the sequence and structure of in vitro synthesized antisense and target RNAs from the replication control
Celeste López-Aguilar1, Gloria del Solar
1Molecular Microbiology and Infection Biology Department, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu, 9, 28040 Madrid, Spain.
Abstract:
Antisense RNAII is a replication control element encoded by promiscuous plasmid pMV158. RNAII binds to its complementary sequence in the copG-repB mRNA, thus inhibiting translation of the replication initiator repB gene. In order to initiate the biochemical characterization of the pMV158 antisense RNA-mediated control system, conditions for in vitro transcription by T7RNA polymerase were set up that yielded large amounts of antisense and target run-off products able to bind to each other. The run-off antisense transcript was expected, and confirmed, to span the entire RNAII as synthesized by the bacterial RNA polymerase, including the intrinsic transcription terminator at its 3'-terminus. On the other hand, two different target transcripts, mRNA₆₀ and mRNA₈₀, were produced, characterized and tested for efficient binding to the antisense product. The mRNA₆₀ and mRNA₈₀ run-off transcripts supposedly spanned 60 and 80 nucleotides, respectively, on the copG-repB mRNA and lacked terminator-like structures at their 3'-termini. Probing of the sequence and conformation of the main products, along with modeling of their secondary structures, showed that both target transcripts were actually longer-than-expected, and contained a 3'-terminal hairpin wherein the extra nucleotides base-paired to the expected 3'-terminus of the corresponding run-off transcript. These longer products were proposed to arise from the RNA-dependent polymerizing activity of T7RNA polymerase on correct run-off transcripts primed by extremely short 3'-selfcomplementarity. Seizing of the target mRNA sequence complementary to the 5'-terminus of RNAII in a stable 3'-terminal hairpin generated by this activity seemed to cause a 3-fold decrease in the efficiency of binding to the antisense RNA.
Insights
Antisense RNAII inhibits plasmid replication by binding to repB mRNA. Unexpected 3' terminal hairpins in target transcripts reduced binding efficiency by threefold, revealing a novel regulatory mechanism.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Antisense RNAII from plasmid pMV158 regulates replication by inhibiting repB gene translation.
- Understanding this antisense RNA-mediated control system requires biochemical characterization.
Purpose of the Study:
- To biochemically characterize the pMV158 antisense RNA-mediated control system.
- To investigate the in vitro transcription and binding of antisense RNAII and target mRNA.
Main Methods:
- In vitro transcription using T7 RNA polymerase to produce antisense RNAII and target transcripts (mRNA₆₀, mRNA₈₀).
- Characterization of transcript sequences and secondary structures using probing and modeling.
- Assessment of binding efficiency between antisense RNAII and target transcripts.
Main Results:
- Antisense and target run-off transcripts were successfully synthesized.
- Target transcripts (mRNA₆₀, mRNA₈₀) were longer than expected, forming 3'-terminal hairpins due to T7 RNA polymerase activity.
- The 3'-terminal hairpin formation in target transcripts decreased binding efficiency to antisense RNAII by threefold.
Conclusions:
- T7 RNA polymerase can produce extended transcripts with 3'-terminal hairpins.
- These hairpins sequester the target sequence, reducing antisense RNA binding and potentially modulating translational inhibition.
- This highlights a novel RNA-dependent mechanism influencing antisense RNA efficacy.
