Proteobacterial ArfA peptides are synthesized from non-stop messenger RNAs

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.

FIGURE 1.

The arfA(Ngo) and arfA(Hin) genes support alternative ribosome rescue in E. coli. E. coli ΔarfA ssrA::cat cells carrying plasmid-borne arfA genes from E. coli K-12 (Eco), N. gonorrhoeae PID1 (Ngo), or H. influenzae Rd (Hin) under control of an arabinose-inducible promoter were spotted onto selective media containing l-arabinose to induce or d-glucose to repress arfA expression. Cells were adjusted to an optical density at 600 nm of 1.0 and then plated as a 10-fold dilution series.

FIGURE 2.

Predicted arfA hairpin structures. The predicted hairpins found in the 3′-coding region of arfA genes from E. coli K-12, P. mirabilis HI4320, H. influenzae Rd, P. multocida Pm70, V. fischeri ATCC 700601, and N. gonorrhoeae PID1 are depicted. All structures were determined using UNAfold. The stop codons for P. multocida, H. influenzae, and V. fischeri arfA transcripts are indicated and rendered in gray. The stop codons for the other messages are found downstream of the predicted hairpin structures. The underlined/italicized amino acid residues are ssrA-tagged by the tmRNA·SmpB system. Arrows indicate the RNase III cleavage sites identified within the arfA(Eco) hairpin (21).

FIGURE 3.

arfA transcripts are truncated in an rnc-dependent manner. A, schematic for flag-trxA-arfA′ expression constructs. Sequences encoding the predicted arfA hairpin structures from E. coli (Eco), V. fischeri (Vfi), and P. multocida (Pmu) were fused in-frame to a flag-trxA gene under control of the bacteriophage T7 promoter. The arfA′(recode) construct is synonymously recoded to disrupt the predicted hairpin structure. The binding site for the Northern blot hybridization probe and the position of the T7 transcription terminator are indicated. B, Northern blot analysis of flag-trxA-arfA′ transcripts. Each expression construct was induced in the indicated rnc and ssrA backgrounds and total RNA analyzed by Northern blot using an oligonucleotide probe that hybridizes to the 5′-noncoding region of each transcript. The gel migration positions of full-length and truncated transcripts are indicated. C, Western blot analysis of FLAG-TrxA-ArfA′ proteins. FLAG-TrxA-ArfA′ fusion proteins produced in the indicated rnc and ssrA genetic backgrounds were analyzed by immunoblot using monoclonal antibodies to the N-terminal FLAG peptide epitope.

FIGURE 4.

Full-length arfA(Ngo) transcript accumulates in rnc⁺ cells. A, Northern blot analysis of flag-trxA-arfA transcripts. Fusion constructs carrying the full-length arfA coding sequences from E. coli (Eco), N. gonorrhoeae (Ngo), P. mirabilis (Pmi), and H. influenzae (Hin) were induced from T7 promoters in the indicated rnc and ssrA genetic backgrounds, and total RNA was analyzed by Northern blot using an oligonucleotide probe that hybridizes to the 5′-noncoding region of each transcript. The gel migration positions of full-length and truncated transcripts are indicated. B, Western blot analysis of FLAG-TrxA-ArfA proteins. FLAG-TrxA-ArfA fusion proteins produced in the indicated rnc and ssrA genetic backgrounds were analyzed by Western blot using monoclonal antibodies to the N-terminal FLAG peptide epitope.

FIGURE 5.

RNase III activity on arfA transcripts in vivo. A, Northern blot analysis of his₆-arfA(Eco) transcripts. The his₆-arfA(Eco) construct was induced the T7 promoter in the indicated rnc and ssrA genetic backgrounds, and total RNA was analyzed by Northern blot using an oligonucleotide probe that hybridizes to the 5′-noncoding region. All cell lines also carried a plasmid-borne copy of the rnc(Ngo) gene under control of the arabinose-inducible P_BAD promoter. Induction of rnc(Ngo) expression with arabinose is indicated by plus (+) symbols. The gel migration positions of full-length and truncated his₆-arfA(Eco) transcripts are indicated. B, Northern blot analysis of his₆-arfA(Ngo) transcripts. The his₆-arfA(Ngo) construct was induced in the same cell lines described in panel A, and total RNA was analyzed by Northern blot using an oligonucleotide probe that hybridizes to the 5′-noncoding region. Control in vitro transcripts were also run on the gel to mark the migration positions of full-length message and transcript truncated after the codon for Ser⁶⁶ of ArfA(Ngo).

FIGURE 6.

The arfA(Ngo) transcript is resistant to RNase III cleavage in vitro. The arfA(Eco) and arfA(Ngo) transcripts were synthesized by in vitro transcription using phage T7 RNA polymerase, mixed with total RNA from E. coli, and digested with purified RNase III from E. coli or N. gonorrhoeae. arfA transcripts were detected by Northern hybridization using an oligonucleotide probe that hybridizes to a common sequence in the 5′-noncoding region of both transcripts.

FIGURE 7.

ArfA(Ngo) synthesis is regulated by tmRNA·SmpB activity. A, Northern blot analysis of flag-arfA transcripts produced by E. coli RNAP. The flag-arfA(Eco) and flag-arfA(Ngo) constructs were induced from the P_BAD promoter in the indicated rnc and ssrA genetic backgrounds, and total RNA was analyzed by Northern blot using an oligonucleotide probe that hybridizes to a common 5′-noncoding region. The migration positions of full-length and 3′-truncated flag-arfA(Eco) transcripts are indicated. B, Western blot analysis of FLAG-TrxA-ArfA proteins. FLAG-TrxA-ArfA production from the P_trc promoter and proteins were detected by immunoblot using monoclonal antibodies specific for the N-terminal FLAG peptide epitope.

FIGURE 8.

The arfA(Ngo) hairpin is an intrinsic transcription terminator. E. coli RNA polymerase holoenzyme in vitro transcription reactions were programmed with linear templates containing arfA genes under control of the P_trc promoter. Reactions were analyzed by Northern blot hybridization using an oligonucleotide probe that hybridizes to a common sequence in the 5′-noncoding region of all transcripts. Transcription termination efficiency was ∼77, 54, and 11% for arfA(Ngo), arfA(Eco), and arfA(recode) templates, respectively. Control arfA(Ngo) transcripts were also produced with phage T7 RNA polymerase to indicate the gel migration positions of full-length and truncated (at codon Ser⁶⁶) message.