Transfer-messenger RNA controls the translation of cell-cycle and stress proteins in Streptomyces

Abstract

The transfer-messenger RNA (tmRNA)-mediated trans-translation mechanism is highly conserved in bacteria and functions primarily as a system for the rescue of stalled ribosomes and the removal of aberrantly produced proteins. Here, we show that in the antibiotic-producing soil bacterium Streptomyces coelicolor, trans-translation has a specialized role in stress management. Analysis of proteins that were carboxy-terminally His(8)-tagged by a recombinant tmRNA identified only 10 targets, including the stress proteins: DnaK heat-shock protein 70, thiostrepton-induced protein A, universal stress protein A, elongation factor Tu3, and the cell-cycle control proteins DasR, SsgA, SsgF and SsgR. Although tmRNA-tagged proteins are degraded swiftly, the translation of dnaK and dasR messenger RNAs (mRNAs) depends fully on tmRNA, whereas transcription is unaffected. The data unveil a surprisingly dedicated functionality for tmRNA, promoting the translation of the same mRNA it targets, at the expense of sacrificing the first nascent protein. In streptomycetes, tmRNA has evolved into a dedicated task force that ensures the instantaneous response to the exposure to stress.

Figure 1

Specific tagging by tmRNA in Streptomyces coelicolor. Two-dimensional gel electrophoresis. The numbers correspond to the identified proteins and indicate the SCO reference number in the Streptomyces database. The amount of protein loaded for this experiment was equivalent to 100 ml of culture. DasR, development of aerial mycelium and spores regulator; tmRNA, transfer–messenger RNA.

Figure 2

Translation, but not transcription, of dasR and dnaK depends on tmRNA tagging. (A) Proteins were analysed by western blotting using DasR, DnaK or EF-Tu1 antibodies. Escherichia coli overexpressing DasR (pET-dasR) was used as a control (E. coli DnaK is smaller than that of Streptomyces coelicolor). (B) RT–PCR of total RNA from the different strains, demonstrating that transcription of the dasR and dnaK genes is not affected in ssrA mutants. (C) Engineered carboxy-terminally tagged glucose kinase is degraded in a canonical manner. Protein extracts from transformants expressing wild-type Glk, Glk^AA or Glk^DD were separated by 12% SDS–PAGE and immunodetected with Glk antibodies. As a control, EF-Tu1 antibodies were used. The experiment demonstrates that tmRNA-tagged proteins are efficiently degraded in Streptomyces. A schematic representation of the translational fusion of wild-type tmRNA tag (AA; giving Glk^AA) or a mutated tmRNA tag (DD; giving Glk^DD) to glucose kinase is shown. DasR, development of aerial mycelium and spores regulator; EF, elongation factor; Glk, glucose kinase; RT–PCR, reverse transcriptase PCR; SDS–PAGE, sodium dodecyl sulphate–polyacrylamide gel electrophoresis; tmRNA, transfer–messenger RNA.

Figure 3

The dasR 3′ UTR dictates tmRNA dependence of egfp translation. Differential expression of a reporter (for DNA sequence see supplementary Fig S5 online), consisting of egfp fused to the 3′ UTR of dasR expressed from the ftsZ promoter in M600 (top) and its ssrA null mutant (bottom). In all hyphae analysed, the expression was much higher in the parent S. coelicolor M600 than in its ssrA mutant derivative (fivefold to sixfold; P<0.005), whereas in the absence of the dasR tag the EGFP expression was only 1.3-fold higher. This strongly suggests that the region around the dasR stop codon contains an intrinsic element that makes gene expression dependent on tmRNA (see Fig 5). EGFP, enhanced green fluorescent protein; tmRNA, transfer–messenger RNA; UTR, untranslated region.

Figure 4

Efficiency of tagging of tmRNA targets. ΔssrA expressing tmRNA-His was used to estimate the fraction of DasR protein, which becomes tagged by the tmRNA-mediated trans-translation mechanism. (A) Western analysis using His antibodies of tmRNA-tagged proteins and of purified EF-Tu1(His)₆ (excess of 10 pmol; control for column capacity). (B) Analysis of tmRNA-tagged fractions using DasR and EF-Tu1 antibodies and compared with Streptomyces coelicolor M600 and its ssrA mutant. The amount of protein loaded for this experiment was equivalent to 100 μl of culture. DasR, development of aerial mycelium and spores regulator; EF, elongation factor; pellet, bound fraction; sup., unbound fraction; tmRNA, transfer–messenger RNA; total, extract before separation.

Figure 5

Model for tmRNA-mediated translational control in Streptomyces coelicolor. Ribosomes translating mRNAs, such as that of dasR or dnaK, are stalled at the 3′ end, and the first nascent peptide chain becomes tagged for proteolytic degradation. During this tagging process the mRNA is cleaved to remove a yet unknown structural element in the mRNA. As a result, the ribosomes are able to finish translation without further tmRNA tagging. This mechanism allows the instantaneous production of crucial stress proteins after stress. See Discussion section for details. mRNA, messenger RNA; tmRNA, transfer–messenger RNA.