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Characterization of the small antisense CI RNA that regulates bacteriophage P4 immunity
Francesca Forti1, Ilaria Dragoni, Federica Briani
1Dipartimento di Genetica e di Biologia dei microrganismi, Università degli Studi di Milano, Via Celoria 26, Milano, 20133, Italy.
Abstract:
In the immune state bacteriophage P4 prevents expression of the replication functions by premature termination of transcription. A small RNA, the CI RNA, is the trans acting factor that regulates P4 immunity, by pairing to complementary target sequences and causing premature transcription termination. The CI RNA is matured by RNAse P and PNPase from the leader region of the same operon it regulates. In this work we better characterize this molecule. CI RNA copy number was determined to be around 500 molecules per lysogenic cell. By S(1) mapping we defined the 3'-end at 8423(+/-1); thus CI RNA is 79(+/-1) nt long. The minimum region for correct processing requires two bases upstream of the CI RNA 5'-end and the CCA sequence at the 3'-end. Computer analysis by FOLD RNA of CI RNA sequence predicts a cloverleaf-like structure formed by a double-stranded stalk, a minor and a major stem loop, and a single-stranded bulge. We analysed several cI mutations, which fall either in the single or double-stranded CI RNA regions. Base substitutions in the main loop and in the single-stranded bulge apparently did not change CI RNA structure, but affected its activity by altering the complementarity with the target sequences, whereas a mutation in the secondary stem had a disruptive effect on CI RNA secondary structure. The effects of this latter mutation were suppressed by a base substitution that restored the complementarity with the corresponding base in the stem. Base substitutions in the main stem caused only local alterations in the secondary structure of CI. However, when the substitutions concerned either G8501 or its complementary base at the bottom of the stem, CI RNA was not correctly processed.
Insights
Bacteriophage P4 uses a small RNA molecule, CI RNA, to control its replication by stopping transcription early. This study details CI RNA
Area of Science:
- Molecular Biology
- Virology
- RNA Biology
Background:
- Bacteriophage P4 employs premature transcription termination to regulate replication functions during its immune state.
- A small trans-acting RNA, CI RNA, is the key regulator of P4 immunity, mediating transcription termination.
- CI RNA is processed from its own operon's leader region by RNase P and PNPase.
Purpose of the Study:
- To further characterize the structure and function of bacteriophage P4's CI RNA.
- To investigate the molecular mechanisms underlying CI RNA processing and activity.
- To determine the impact of specific mutations on CI RNA structure and function.
Main Methods:
- Quantification of CI RNA copy number per cell.
- S1 mapping to precisely define the 3'-end and length of CI RNA.
- Computational analysis (FOLD RNA) to predict secondary structure.
- Site-directed mutagenesis to analyze the effects of mutations on CI RNA.
Main Results:
- CI RNA copy number was found to be approximately 500 molecules per lysogenic cell.
- CI RNA was determined to be 79 nucleotides long, with specific requirements for 5' and 3' processing.
- Mutations in loop regions affected target complementarity, while stem mutations disrupted structure and processing, with some effects being suppressible.
Conclusions:
- CI RNA possesses a predicted cloverleaf-like secondary structure crucial for its function.
- Specific regions of CI RNA are essential for its processing and interaction with target sequences.
- Understanding CI RNA's structure-function relationship provides insights into bacteriophage regulatory mechanisms.