The wrong translation of the genetic code , called mistranslation, is traditionally considered to be detrimental to cell growth and survival. However, it has been proved that organisms deliberately mistranslate the genetic code in response to stress, and more and more evidences show that many microbial pathogens can deliberately mistranslate their genetic code to help invade the host or escape the host’s immune response. Mistranslations have been found in many microbial pathogens. This kind of deliberate mistranslation is not random, and is usually limited to a specific type of translation error. This kind of error is due to the existence of an unusual and dedicated protein synthesis factor that gives organisms the ability to deliberately change the rules of the genetic code . Due to the lack of a general strategy for identifying abnormal translation factors in nature, the study of mistranslation in pathogens has been hindered.
In a recently reported study, the author solved the above problem by studying the code aminoacyl-tRNA synthetase ( aaRS) by deliberately translating a copy of the span 8span gene. , The results were published in " PNAS " .
The plant pathogenic Streptomyces encodes an unusual ProRS subtype p_6span8 p_span8 p_span8Prolyl-tRNA synthetase ( ProRS ) has two subtypes, which perform the same basic functions, but have different structures and domain arrangements. But recently, people discovered a third type of ProRS, named ProRSx . Through BLAST comparison, it was found that ProRSx mainly appeared in 4 Streptomyces genus, but they all pose a threat to several crops, mainly potatoes. In terms of protein structure, ProRSx has the same domain organization as bacterial ProRS, which indicates that ProRSx may have evolved through the replication of bacterial ProRS genes in Streptomyces. However, there are significant differences between the ProRSx sequence and the bacterial ProRS, especially the C-terminal domain that recognizes the antisense codon of tRNAPro, which lacks the characteristic motif of bacterial ProRS (Figure 1B) .
ProRSx is flanked by unusual proline tRNA genes with multiple characteristic elements _strongp10span
6span _span. The genomic background of the ProRSx gene in Streptomyces species is described. Interestingly, the authors found that ProRSx is encoded by a conserved operon and includes a tRNA gene adjacent to ProRSx. The author performed a predictive analysis of the secondary structure of the tRNA gene (Figure 3A) ,It was found that the tRNA has a highly atypical structure. It has the alanine anti-codon AGC , but it lacks the unique G3:U70 base pair of tRNAAla, but has the proline tRNA characteristic element C1:G72 base pair. Because of the mixing of these characteristic elements, the author named the tRNA tRNAProA . Because tRNAProA mixes multiple characteristics, the authors next tested whether tRNAProA can cross-react with typical ProRS or AlaRS. The use of in vitro aminoacylation experiments showed that typical ProRS or AlaRS cannot aminoacylate tRNAProA. These data and the co-localization of the tRNAProA and ProRSx genes indicate that tRNAProA is only a ProRSx substrate.
Co-expression of S.prospan_span_span_span_span_span_span_span
_span_ Co-expression of s_span_span_span_span_span_span_span_span_span_span_span_span_ To further study the functional activity of S.turgidiscabies ProRSs in bacteria, the author next established a temperature-sensitive experimental protocol. The author uses the temperature-sensitive E. coli strain UQ27, whose ProRS will be inactivated at 42°C and the bacteria will die.But it can be remedied by a plasmid carrying a fully functional ProRS gene. Experimental results show that ProRSx cannot remedy the UQ27 strain at 42°C, that is, ProRSx cannot aminoacylate tRNAPro in E. coli. The author next tried to test whether tRNAProA can be aminoacylated by ProRSx directly in vitro. But in the process of prokaryotic expression, ProRSx forms inclusion bodies, so it is impossible to obtain active ProRSx. To solve this problem, the authors developed a highly sensitive alternative to determine the activity of ProRSx and tRNAProA in E. coli cells. This assay method is based on the β-lactamase reporter gene. If the conservative Pro residue at position 65 is replaced with Ala, the activity of β-lactamase will be greatly weakened, so it can be used to monitor the GCU alanine codon Wrong translation with proline (Ala→Pro wrong translation) (Figure 4A) . Experimental results show that ProRSx can use proline to acylate tRNAProA, resulting in the use of proline to decode GCU alanine codons.
ProRS-Ec-5ABD's in vivo aminoacylation of tRNAProA leads to the wrong translation of alanine codon 6 _span ProA Avoid cross-reaction with typical ProRS, but still maintain specificity for ProRSx.
in T.thermophilus ,The recognition of tRNAPro by ProRS mainly depends on the five conserved residues of K353, D354, E340, R347 and K369, but all of them are mutated in ProRSx. Therefore, according to the sequence of ProRSx, the author made a point mutation of the ProRS of Escherichia coli, and obtained a mutant of Escherichia coli ProRS ( ProRS-Ec-5ABD ). The experimental results found that ProRS-Ec-5ABD retains the ability of E. coli to aminoacylate typical tRNAPro, and at the same time obtains the ability to aminoacylate tRNAProA with proline (Figure 5C and D ) .
To facilitate quantification, the author established a detection protocol based on the chimeric protein fusion between the green fluorescent protein GFP and the red fluorescent protein mCherry (Figure 6A) _6span8span. Previous studies have shown that substituting proline for serine at position 65 will weaken GFP fluorescence, but substituting alanine for a fluorescent GFP. Therefore, by comparing the fluorescence intensity of GFP and mCherry, it can be judged whether mistranslation occurs. The mass spectrometry analysis of showed that the cells expressing ProRS-Ec-5ABD and tRNAProA did contain the Ala→Pro mutation in the GFP/mCherry reporter protein (at GFP position 65). In contrast, cells expressing ProRS-Ec5ABD without expressing tRNAProA only contained wild-type GFP (Figure 6D) .
ProRS-Ec-5ABD and tRNAspan ProRS-Ec-5ABD and tRNAProA were used to evaluate the protein growth of Ec-5ABD and tRNAspan strong. How does the mistranslation of the alanine codon affect the fitness of bacteria in the group? The authors observed that when tRNAProA or ProRS-Ec-5ADB is expressed alone, neither of them is toxic to the growth of E. coli. However, when tRNAProA and ProRS-Ec-5ADB were expressed at the same time, the growth of E. coli was significantly inhibited, which further indicated that the two may be the product of co-evolution.
Summary
The author took the bacterial prolyl-tRNA synthetase (ProRS) gene as an example to identify an abnormal ProRS subtype ProRSx and the corresponding tRNA--tRNAProA, which mainly exist in Among plant pathogens from the genus Streptomyces. Then the authors discovered that tRNAProA has an unusual mixed structure, allowing this tRNA to mistranslate the alanine codon into proline. Finally, the authors provided biochemical, genetic and mass spectrometric evidence that cells expressing ProRSx and tRNAProA can translate GCU alanine codons into alanine and proline. Due to random Ala→Pro mutations in the protein sequence, this double use of alanine codons creates hidden proteome diversity. This discovery reveals the first example of the use of natural tRNA synthetase/tRNA for the special mistranslation of sense codons.
original link:
https://www.pnas.org/content/118/35/e2110797118
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