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Antimicrobial Peptides Produced by Selective Pressure Incorporation of Non-canonical Amino Acids
Published on: May 4, 2018
Proteobacterial ArfA peptides are synthesized from non-stop messenger RNAs
Ryan E Schaub1, Stephen J Poole, Fernando Garza-Sánchez
1Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, CA 93106-9625, USA.
Abstract:
The translation of non-stop mRNA (which lack in-frame stop codons) represents a significant quality control problem for all organisms. In eubacteria, the transfer-messenger RNA (tmRNA) system facilitates recycling of stalled ribosomes from non-stop mRNA in a process termed trans-translation or ribosome rescue. During rescue, the nascent chain is tagged with the tmRNA-encoded ssrA peptide, which promotes polypeptide degradation after release from the stalled ribosome. Escherichia coli possesses an additional ribosome rescue pathway mediated by the ArfA peptide. The E. coli arfA message contains a hairpin structure that is cleaved by RNase III to produce a non-stop transcript. Therefore, ArfA levels are controlled by tmRNA through ssrA-peptide tagging and proteolysis. Here, we examine whether ArfA homologues from other bacteria are also regulated by RNase III and tmRNA. We searched 431 arfA coding sequences for mRNA secondary structures and found that 82.8% of the transcripts contain predicted hairpins in their 3'-coding regions. The arfA hairpins from Haemophilus influenzae, Proteus mirabilis, Vibrio fischeri, and Pasteurella multocida are all cleaved by RNase III as predicted, whereas the hairpin from Neisseria gonorrhoeae functions as an intrinsic transcription terminator to generate non-stop mRNA. Each ArfA homologue is ssrA-tagged and degraded when expressed in wild-type E. coli cells, but accumulates in mutants lacking tmRNA. Together, these findings show that ArfA synthesis from non-stop mRNA is a conserved mechanism to regulate the alternative ribosome rescue pathway. This strategy ensures that ArfA homologues are only deployed when the tmRNA system is incapacitated or overwhelmed by stalled ribosomes.
Insights
Bacterial ArfA peptide synthesis is conserved across species, regulated by RNase III cleavage and tmRNA (transfer-messenger RNA) for ribosome rescue. This ensures ArfA is only active when tmRNA is overwhelmed, maintaining cellular quality control.
Area of Science:
- Molecular Biology
- Bacterial Genetics
- RNA Biology
Background:
- Non-stop messenger RNA (mRNA) translation poses a quality control challenge in bacteria.
- The transfer-messenger RNA (tmRNA) system rescues stalled ribosomes by tagging nascent peptides for degradation.
- Escherichia coli utilizes an alternative pathway involving the ArfA peptide for ribosome rescue.
Purpose of the Study:
- To investigate the conserved regulation of ArfA homologues across different bacterial species.
- To determine if ArfA mRNA processing and degradation are controlled by RNase III and tmRNA in various bacteria.
Main Methods:
- Bioinformatic analysis of 431 arfA coding sequences to predict mRNA secondary structures (hairpins).
- In vitro cleavage assays using RNase III on predicted arfA hairpins from selected bacterial species.
- Expression of bacterial ArfA homologues in wild-type and tmRNA-deficient Escherichia coli strains to assess stability and degradation.
Main Results:
- 82.8% of analyzed arfA transcripts contain predicted 3'-coding region hairpins.
- RNase III cleaved arfA hairpins from Haemophilus influenzae, Proteus mirabilis, Vibrio fischeri, and Pasteurella multocida.
- Neisseria gonorrhoeae arfA hairpin acted as a transcription terminator, generating non-stop mRNA.
- Bacterial ArfA homologues were ssrA-tagged and degraded in wild-type E. coli but accumulated in tmRNA-deficient mutants.
Conclusions:
- ArfA synthesis from non-stop mRNA is a conserved regulatory mechanism for the alternative ribosome rescue pathway.
- Regulation by RNase III cleavage and tmRNA-dependent degradation ensures ArfA homologues are deployed only when the tmRNA system is insufficient.
- This conserved strategy maintains cellular quality control by managing stalled ribosomes effectively across diverse bacterial species.
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