tmRNA regulates synthesis of the ArfA ribosome rescue factor

Abstract

Translation of mRNA lacking an in-frame stop codon leads to ribosome arrest at the 3' end of the transcript. In bacteria, the tmRNA quality control system recycles these stalled ribosomes and tags the incomplete nascent chains for degradation. Although ubiquitous in eubacteria, the ssrA gene encoding tmRNA is not essential for the viability of Escherichia coli and other model bacterial species. ArfA (YhdL) is a mediator of tmRNA-independent ribosome rescue that is essential for the viability of E. coliΔssrA mutants. Here, we demonstrate that ArfA is synthesized from truncated mRNA and therefore regulated by tmRNA tagging activity. RNase III cleaves a hairpin structure within the arfA-coding sequence to produce transcripts that lack stop codons. In the absence of tmRNA tagging, truncated ArfA chains are released from the ribosome. The truncated ArfAΔ18 protein (which lacks 18 C-terminal residues) is functional in ribosome rescue and supports ΔssrA cell viability when expressed from the arfA locus. Other proteobacterial arfA genes also encode hairpins, and transcripts from Dickeya dadantii and Salmonella typhimurium are cleaved by RNase III when expressed in E. coli. Thus, synthesis of ArfA from truncated mRNA appears to be a general mechanism to regulate alternative ribosome rescue activity.

Fig. 1. ArfA protein is truncated and tagged by tmRNA

A) SDS-PAGE analysis of purified His₆-ArfA proteins. His₆-ArfA was produced from full-length arfA, arfAΔ12 and arfAΔ18 constructs in ΔssrA, ssrA⁺ and ssrA⁺ ΔclpP cells. ArfA variants were purified by Ni²⁺-affinity chromatography and equal amounts of purified protein were analyzed by SDS-PAGE and western blot using antibodies specific for the SsrA peptide tag. B) Mass spectrometry analysis of His₆-ArfA. His₆-ArfA was overproduced and purified by Ni²⁺-affinity and reverse phase HPLC for mass spectrometry. Truncated ArfA peptides were identified in clpP⁺ cells, and SsrA-tagged ArfA chains were detected in samples purified from ΔclpP cells. The observed and calculated masses are indicated for each ArfA species.

Fig. 2. The arfA message is truncated

A) Schematic of full-length arfA mRNA. The arfA transcript is depicted along with the 5´ northern blot and S1 nuclease protection probe binding sites. Control in vitro transcripts truncated after codons Ala53 and His60 were used as migration markers for northern analysis. B) Northern blot analysis of arfA mRNA. Transcripts from full-length arfA, arfAΔ12 and arfAΔ18 constructs were expressed in ΔssrA and ssrA⁺ cells and analyzed by northern blot using a radiolabeled probe that hybridizes to the 5´-UTR. Full-length and truncated arfA transcripts were synthesized in vitro and mixed for use as gel migration markers. C) S1 nuclease protection analysis. RNA samples were isolated from the indicated genetic backgrounds and the 3´-end of truncated arfA mRNA was mapped by S1 nuclease protection using a 3´-radiolabeled oligonucleotide probe. Purified RNase III cleaved the arfA transcript at two positions in vitro. The main in vitro cleavage corresponded to a minor product (indicated by the asterisk) detected in rnc⁺ cells. Full-length and Ala53 truncated in vitro transcripts were used as standards. Probe migration positions that correspond to mRNA cleavage after the Glu52, Ala53 and Gly55 codons are indicated.

Fig. 3. Primer extension analysis of arfA mRNA

A) The arfA hairpin structure as predicted by mfold. The RNase III cleavage sites are indicated, as are the positions of selected arfA codons. The binding site of the reverse transcriptase (RT) primer used for primer extension is shown schematically as an arrow. B) Primer extension analysis of arfA mRNA. RNA samples were isolated from cells overexpressing the arfA mRNA in the indicated genetic backgrounds. Samples were hybridized with 5´-radiolabeled RT primer and incubated with reverse transcriptase. Negative controls included the omission of reverse transcriptase (no RT) and analysis of RNA from uninduced cells (no IPTG). Full-length arfA transcript was digested with RNase III in vitro and analyzed. Molecular markers show the gel migration positions of primer extension products corresponding to cleavage after codons Phe62 and Thr63.

Fig. 4. RNase III cleaves arfA mRNA

A) Sequence of the synonymously recoded arfA hairpin. Mutated nucleotide residues are shown in red, and the codons corresponding to residues Glu52, Ala53, Ser54, Gly55 and His60 are indicated. B) Northern blot analysis of arfA and recoded arfA mRNA. Wild-type arfA and synonymously recoded arfA constructs were expressed in cells with the indicated genotype. Transcripts were analyzed by northern blot using radiolabeled probes that hybridize to either the 5´-UTR (5´-probe) or 3´-UTR (3´-probe). Full-length and truncated arfA transcripts were synthesized in vitro and mixed for use as migration markers on the 5´-UTR northern blot. The first lane of 3´-UTR blot contains full-length arfA in vitro transcript, and the second lane contains in vitro transcript digested with purified RNase III.

Fig. 5. Full-length ArfA is synthesized in Δrnc cells

A) SDS-PAGE and western blot analyses of His₆-ArfA. His₆-ArfA was overproduced from wild-type and recoded arfA constructs in cells of the indicated genetic background. His₆-ArfA was purified by Ni²⁺-affinity chromatography, and equal amounts of purified protein were analyzed by SDS-PAGE and western blot using antibodies specific for the SsrA peptide tag. B) In vitro synthesis of ArfA variants using defined transcription/translation reactions. PURExpress^® (New England Biolabs) translation reactions containing [³⁵S]-L-methionine were programmed with the indicated arfA plasmid constructs and incubated at 37 °C for 1 hr. Purified RNase III was added to translation reactions where indicated. All reactions were analyzed by SDS-PAGE and autoradiography.

Fig. 6. Endogenous arfA mRNA is cleaved by RNase III

A) Northern blot analyses of arfA mRNA transcribed by E. coli RNAP. The pCH450-arfA expression construct was induced with 0.2% L-arabinose in the indicated genetic backgrounds. RNA samples were run in duplicate on the same polyacrylamide gel and blotted for northern hybridization. After transfer, the membrane was cut in half and hybridized with radiolabeled oligonucleotide probes to the 5´-coding region (codons Arg3 – Lys12, 5´-probe) and 3´-UTR (3´-probe). The two membranes were then realigned for phosphorimaging. B) Northern blot analysis of endogenous transcripts produced from the chromosomal arfA locus. The arfA+(FRT), arfAΔ18 and arfA(recode) alleles were introduced into the indicated ssrA rnc genetic backgrounds and total RNA isolated for northern blot analysis. The blotted samples were hybridized to a radiolabeled oligonucleotide probe to the 5´ coding region of arfA.

Fig. 7. tmRNA regulates the synthesis of ArfA

A) Western blot analysis of FLAG-ArfA proteins. Arabinose-inducible flag-arfA, flag-arfAΔ18 and flag-arfA(recode) constructs were expressed in the indicated genetic backgrounds, and total protein was isolated for western blot analysis using anti-FLAG antibodies. B) Western blot analysis of FLAG-TrxA-ArfA(38). An arabinose-inducible flag-trxA-arfA(38) fusion construct was expressed in the indicated genetic backgrounds, and total protein was isolated for western blot analysis using anti-FLAG antibodies. C) Western blot analysis of FLAG-TrxA-(ArfA)-TrxA fusion proteins. Fusion constructs containing the wild-type arfA hairpin or the synonymously recoded sequence were expressed in the indicated genetic backgrounds, followed by western blot analysis using anti-FLAG antibodies. The migration positions of full-length and truncated proteins are indicated.

Fig. 8. ArfAΔ18 is functional in tmRNA-independent ribosome rescue

A) Release of nascent chains from stalled ribosomes. λN-trpAt was synthesized from non-stop mRNA in both arfA⁺ and ΔarfA cells that express tmRNA(DD). All λN-trpAt chains were purified by Ni²⁺-affinity and analyzed by SDS-PAGE. SsrA(DD)-tagged λN-trpAt protein is indicated, and the gel migration position of untagged λN-trpAt was ascertained in ΔssrA arfA⁺ cells. The activity of ArfA produced from arabinose-inducible arfA, arfAΔ18 and arfA(recode) constructs was compared to the empty plasmid vector. The Ala18Thr mutation (A18T) inactivates ArfA and was used as negative control. B) The arfAΔ18 and arfA(recode) alleles support the viability of ΔssrA cells. The growth of strains carrying the arfA⁺, ΔarfA, arfAΔ18 and arfA(recode) alleles were monitored by measuring the optical density of the culture at 600 nm (OD₆₀₀). The growth of cells containing the various arfA alleles in the ssrA⁺ rnc⁺ background was indistinguishable from one another. Cells carrying the arfA(recode) allele in the ΔssrA rnc⁺ background grew more slowly than isogenic arfA⁺ and arfAΔ18 strains. Cells carrying the wild-type arfA⁺ allele in the ΔssrA Δrnc background had a slight growth phenotype compared to the arfAΔ18 ΔssrA Δrnc strain.

Fig. 9. ArfA from other γ-proteobacteria

A) Predicted hairpin structures found within arfA mRNA from D. dadantii 3937 and S. typhimurium LT2. B) Northern blot analysis of arfA messages. Full-length constructs encoding his₆-arfA from D. dadantii and S. typhimurium were expressed in rnc⁺ and Δrnc strains, followed by northern blot analysis using a radiolabeled probe to the 5´-UTR. C) SDS-PAGE analysis of His₆-ArfA. His₆-ArfA proteins from D. dadantii and S. typhimurium were overproduced and purified from E. coli rnc⁺ and Δrnc cells. Equal amounts of purified protein were analyzed by SDS-PAGE.