The tmRNA-tagging mechanism and the control of gene expression: a review.

The tmRNA-mediated trans-translation system is a unique quality control system in eubacteria that combines translational surveillance with the rescue of stalled ribosomes. During trans-translation, the chimeric tmRNA molecule--which acts as both tRNA and mRNA--is delivered to the ribosomal A site by a ribonucleoprotein complex of SmpB and EF-Tu-GTP, allowing the stalled ribosome to switch template and resume translation on a small coding sequence inside the tmRNA molecule. As a result, the aberrant protein becomes tagged by a sequence that is a target for proteolytic degradation. Thus, the system elegantly combines ribosome recycling with a clean-up function when triggered by truncated transcripts or rare codons. In addition, recent observations point to a specific regulation of the translation of a small number of genes by tmRNA-mediated inhibition or stimulation. In this review, we discuss the most prominent biochemical and structural aspects of trans-translation and then focus on the specific role of tmRNA in stress management and cell-cycle control of morphologically complex bacteria.

Unlocking Streptomyces spp. for use as sustainable industrial production platforms by morphological engineering.

Filamentous actinomycetes are commercially widely used as producers of natural products (in particular antibiotics) and of industrial enzymes. However, the mycelial lifestyle of actinomycetes, resulting in highly viscous broths and unfavorable pellet formation, has been a major bottleneck in their commercialization. Here we describe the successful morphological engineering of industrially important streptomycetes through controlled expression of the morphogene ssgA. This led to improved growth of many industrial and reference streptomycetes, with fragmentation of the mycelial clumps resulting in significantly enhanced growth rates in batch fermentations of Streptomyces coelicolor and Streptomyces lividans. Product formation was also stimulated, with a twofold increase in yield of enzyme production by S. lividans. We anticipate that the use of the presented methodology will make actinomycetes significantly more attractive as industrial and sustainable production hosts.

tRNA-like structure regulates translation of Brome mosaic virus RNA.

For various groups of plant viruses, the genomic RNAs end with a tRNA-like structure (TLS) instead of the 3' poly(A) tail of common mRNAs. The actual function of these TLSs has long been enigmatic. Recently, however, it became clear that for turnip yellow mosaic virus, a tymovirus, the valylated TLS(TYMV) of the single genomic RNA functions as a bait for host ribosomes and directs them to the internal initiation site of translation (with N-terminal valine) of the second open reading frame for the polyprotein. This discovery prompted us to investigate whether the much larger TLSs of a different genus of viruses have a comparable function in translation. Brome mosaic virus (BMV), a bromovirus, has a tripartite RNA genome with a subgenomic RNA4 for coat protein expression. All four RNAs carry a highly conserved and bulky 3' TLS(BMV) (about 200 nucleotides) with determinants for tyrosylation. We discovered TLS(BMV)-catalyzed self-tyrosylation of the tyrosyl-tRNA synthetase but could not clearly detect tyrosine incorporation into any virus-encoded protein. We established that BMV proteins do not need TLS(BMV) tyrosylation for their initiation. However, disruption of the TLSs strongly reduced the translation of genomic RNA1, RNA2, and less strongly, RNA3, whereas coat protein expression from RNA4 remained unaffected. This aberrant translation could be partially restored by providing the TLS(BMV) in trans. Intriguingly, a subdomain of the TLS(BMV) could even almost fully restore translation to the original pattern. We discuss here a model with a central and dominant role for the TLS(BMV) during the BMV infection cycle.

Qbeta-phage resistance by deletion of the coiled-coil motif in elongation factor Ts.

Elongation factor Ts (EF-Ts) is the guanine-nucleotide exchange factor of elongation factor Tu (EF-Tu), which promotes the binding of aminoacyl-tRNA to the mRNA-programmed ribosome in prokaryotes. The EF-Tu.EF-Ts complex, one of the EF-Tu complexes during protein synthesis, is also a component of RNA-dependent RNA polymerases like the polymerase from coliphage Qbeta. The present study shows that the Escherichia coli mutant GRd.tsf lacking the coiled-coil motif of EF-Ts is completely resistant to phage Qbeta and that Qbeta-polymerase complex formation is not observed. GRd.tsf is the first E. coli mutant ever described that is unable to form a Qbeta-polymerase complex while still maintaining an almost normal growth behavior. The phage resistance correlates with an observed instability of the mutant EF-Tu.EF-Ts complex in the presence of guanine nucleotides. Thus, the mutant EF-Tu.EF-Ts is the first EF-Tu.EF-Ts complex ever described that is completely inactive in the Qbeta-polymerase complex despite its almost full activity in protein synthesis. We propose that the role of EF-Ts in the Qbeta-polymerase complex is to control and trap EF-Tu in a stable conformation with affinity for RNA templates while unable to bind aminoacyl-tRNA.

The Streptomyces coelicolor ssgB gene is required for early stages of sporulation.

ssgB was identified as a novel early sporulation gene in Streptomyces coelicolor. An ssgB deletion mutant failed to sporulate, over-produced actinorhodin, and its colonies were significantly larger than those of the parental strain, suggesting an important role for the ssgB gene product in the process of growth cessation prior to sporulation-specific cell division. This places ssgB temporally before the paralogous sporulation gene ssgA. Analysis of ssgB mutant hyphae by electron microscopy and by confocal fluorescence microscopy showed that it was defective in the initiation of sporulation, as no sporulation septa could be identified, and DNA segregation had not yet been initiated in the mutant.

Isolation of Qbeta polymerase complexes containing mutant species of elongation factor Tu.

The RNA genome of coliphage Qbeta is replicated by a complex of four proteins, one of them being the translation elongation factor Tu. The role of EF-Tu in this RNA polymerase complex is still unclear, but the obligate presence of translationally functional EF-Tu in the cell hampers the use of conventional mutational analysis. Therefore, we designed a system based on affinity chromatography and could separate two types of complexes by placing an affinity tag on mutated EF-Tu species. Thus, we were able to show a direct link between the vital tRNA binding property of EF-Tu and polymerase activity.

Entrapping ribosomes for viral translation: tRNA mimicry as a molecular Trojan horse.

Turnip yellow mosaic virus (TYMV) has a genomic plus-strand RNA with a 5' cap followed by overlapping and different reading frames for the movement protein and polyprotein, while the distal coat protein cistron is translated from a subgenomic RNA. The 3'-untranslated region harbors a tRNA-like structure (TLS) to which a valine moiety can be added and it is indispensable for virus viability. Here, we report about a surprising interaction between TYMV-RNA-programmed ribosomes and 3'-valylated TLS that yields polyprotein with the valine N terminally incorporated by a translation mechanism resistant to regular initiation inhibitors. Disruption of the TLS exclusively abolishes polyprotein synthesis, which can be restored by adding excess TLS in trans. Our observations imply a novel eukaryotic mechanism for internal initiation of mRNA translation.

Structural variation and functional importance of a D-loop-T-loop interaction in valine-accepting tRNA-like structures of plant viral RNAs.

Valine-accepting tRNA-like structures (TLSs) are found at the 3' ends of the genomic RNAs of most plant viruses belonging to the genera Tymovirus, Furovirus, Pomovirus and Pecluvirus, and of one Tobamovirus species. Sequence alignment of these TLSs suggests the existence of a tertiary D-loop-T-loop interaction consisting of 2 bp, analogous to those in the elbow region of canonical tRNAs. The conserved G(18).Psi(55) pair of regular tRNAs is found to covary in these TLSs between G.U (possibly also modified to G.Psi) and A.G. We have mutated the relevant bases in turnip yellow mosaic virus (TYMV) and examined the mutants for symptom development on Chinese cabbage plants and for accumulation of genetic reversions. Development of symptoms is shown to rely on the presence of either A.G or G.U in the original mutants or in revertants. This finding supports the existence and functional importance of this tertiary interaction. The fact that only G.U and A.G are accepted at this position appears to result from steric and energetic limitations related to the highly compact nature of the elbow region. We discuss the implications of these findings for the various possible functions of the valine-accepting TLS.

The unique tuf2 gene from the kirromycin producer Streptomyces ramocissimus encodes a minor and kirromycin-sensitive elongation factor Tu.

Streptomyces ramocissimus, the producer of elongation factor Tu (EF-Tu)-targeted antibiotic kirromycin, contains three divergent tuf-like genes, with tuf1 encoding regular kirromycin-sensitive EF-Tu1; the functions of tuf2 and tuf3 are unknown. Analysis of the tuf gene organization in nine producers of kirromycin-type antibiotics revealed that they all contain homologues of tuf1 and sometimes of tuf3 but that tuf2 was found in S. ramocissimus only. The tuf2-flanking regions were sequenced, and the two tuf2-surrounding open reading frames were shown to be oriented in opposite directions. In vivo transcription analysis of the tuf2 gene displayed an upstream region with bidirectional promoter activity. The transcription start site of tuf2 was located approximately 290 nucleotides upstream of the coding sequence. Very small amounts of tuf2 transcripts were detected in both liquid- and surface-grown cultures of S. ramocissimus, consistent with the apparent absence of EF-Tu2 in total protein extracts. The tuf2 transcript level was not influenced by the addition of kirromycin to exponentially growing cultures. To assess the function of S. ramocissimus EF-Tu2, the protein was overexpressed in Streptomyces coelicolor LT2. This strain is a J1501 derivative containing His(6)-tagged EF-Tu1 as the sole EF-Tu species, which facilitated the separation of EF-Tu2 from the interfering EF-Tu1. S. ramocissimus EF-Tu1 and EF-Tu2 were indistinguishable in their ability to stimulate protein synthesis in vitro and exhibited the same kirromycin sensitivity, which excludes the possibility that EF-Tu2 is directly involved in the kirromycin resistance mechanism of S. ramocissimus.

Functional evidence for D- and T-loop interactions in tmRNA.

During bacterial protein synthesis, stalled ribosomes can be rescued by tmRNA, a molecule with both tRNA and mRNA features. The tRNA region of tmRNA has sequence similarity with tRNA(Ala) and also has a clover-leaf structure folded similarly as in canonical tRNAs. Here we propose the L-shape of tmRNA to be stabilized by two tertiary interactions between its D- and T-loop on the basis of phylogenetic and experimental evidence. Mutational analysis clearly demonstrates a tertiary interaction between G(13) and U(342). Strikingly, this in evolution conserved interaction is not primarily important for tmRNA alanylation and for binding to elongation factor Tu, but especially for a proper functioning of SmpB.

Evidence that a single EF-Ts suffices for the recycling of multiple and divergent EF-Tu species in Streptomyces coelicolor A3(2) and Streptomyces ramocissimus.

The tsf genes from Streptomyces coelicolor A3(2) and Streptomyces ramocissimus, encoding the guanine-nucleotide exchange factor EF-Ts, were cloned and sequenced. Streptomycetes have multiple and highly divergent EF-Tu species, with EF-Tu1 and EF-Tu3 showing only about 65% amino acid sequence identity, and yet these can apparently interact with a single EF-Ts species. tsf lies in an operon with rpsB, which encodes ribosomal protein S2. The amino acid sequence of S2 from S. coelicolor differs from most other bacterial S2 homologues in having a C-terminal extension of 70 aa residues with a highly repetitive organization, the function of which is unknown. Transcription analysis of the rpsB-tsf operon of S. coelicolor by promoter probing, nuclease S1 mapping and Northern blotting revealed that the genes give rise to a bicistronic transcript from a single promoter upstream of rpsB. An attenuator was identified in the rpsB-tsf intergenic region; it results in an approximately 2:1 ratio of rpsB vs tsf transcripts. Although tuf1, encoding the major EF-Tu, is located in the rpsL ribosomal protein operon, an additional promoter in the fus-tuf1 intergenic region leads to a significant excess of EF-Tu over ribosomes. Most amino acid residues known from the Escherichia coli crystal structure of the EF-Tu-EF-Ts complex to be directly involved in interaction between the two elongation factors are conserved between E. coli and Streptomyces. However, whenever interaction residues in the EF-Tu moiety show divergence among Streptomyces EF-Tu1, EF-Tu2 and EF-Tu3, the single Streptomyces EF-Ts exhibits compensatory substitutions of the corresponding residues. These apparently enable productive interaction to occur with all three EF-Tus.

A kirromycin-resistant EF-Tu species reverses streptomycin dependence of Escherichia coli strains mutated in ribosomal protein S12.

Streptomycin dependence can be caused by mutations in ribosomal protein S12. Mutations suppressing such streptomycin dependence have been found in ribosomal proteins S4 and S5, and in 16S rRNA. Here a new suppressor mutation localized in elongation factor Tu (EF-Tu) is described, consistent with recent models of ribosome-EF-Tu-tRNA interaction at the decoding centre. The EF-Tu mutation was obtained by genetic selection for streptomycin independence; it was identified as Ala375 --> Thr, previously described as EF-TuA(R) and known to confer a kirromycin-resistant, error-prone phenotype. Also, other streptomycin-dependent (SmD) S12 mutations could be complemented by this mutation. The streptomycin-independent (Sm1) strain grows more slowly than the wild-type (wt), suggesting that not all the defects of the S12 mutation can be complemented by EF-Tu[A375T]. Moreover, this strain is more susceptible than wt to reduction in the cellular EF-Tu concentration, and disruption of tufB led to considerable growth-rate impairment. Expression of EF-Tu from tufB, not only of wt EF-Tu and EF-Tu[A375T] but, remarkably, also of EF-Tu[G222D], known as EF-TuB0 and defective in protein synthesis, equally contributed to cell growth. In vitro analysis revealed a decreased translational activity of wt EF-Tu with SmD ribosomes as compared to EF-Tu[A375T], while EF-Tu[G222D] showed no activity at all, just as with wt ribosomes. Possible mechanisms are discussed for the improved growth rate observed in such Sm1 strains when they include wt EF-Tu or EF-Tu[G222D].