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A frame-shifted gene, which rescued its function by non-natural start codons and its application in constructing synthetic gene circuits.

ABSTRACT: Background:Frame-shifted genes results in non-functional peptides. Because of this complete loss of function, frame-shifted genes have never been used in constructing synthetic gene circuits. Results:Here we report that the function of gene circuits is rescued by a frame-shifted gene, which functions by translating from a non-natural start codon. We report a single nucleotide deletion mutation that developed in the ?-repressor cI within a synthetic genetic NOT gate in Escherichia coli during growth and through this mutation, a non-functional synthetic gene circuit became functional. This mutation resulted in a frame-shifted cI, which showed effective functionality among genetic NOT-gates in Escherichia coli with high regulatory ranges (>?300) and Hill coefficient (>?6.5). The cI worked over a large range of relative copy numbers between the frame-shifted gene and its target promoter. These properties make this frame-shifted gene an excellent candidate for building synthetic gene circuits. We hypothesized a new operating mechanism and showed evidence that frame-shifted cI was translated from non-natural start codon. We have engineered and tested a series of NOT gates made from a library of cI genes, each of which starts from a different codon within the first several amino acids of the frame-shifted cI. It is found that one form with start codon ACA, starting from the 3rd codon had similar repression behavior as the whole frame-shifted gene. We demonstrated synthetic genetic NAND and NOR logic-gates with frame-shifted cI. This is the first report of synthetic-gene-circuits made from a frame-shifted gene. Conclusions:This study inspires a new view on frame-shifted gene and may serve as a novel way of building and optimizing synthetic-gene-circuits. This work may also have significance in the understanding of non-directed evolution of synthetic genetic circuits.

SUBMITTER: Sarkar K

PROVIDER: S-EPMC6397469 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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A frame-shifted gene, which rescued its function by non-natural start codons and its application in constructing synthetic gene circuits.

Sarkar Kathakali K Mukhopadhyay Sayak S Bonnerjee Deepro D Srivastava Rajkamal R Bagh Sangram S

Journal of biological engineering 20190301

<h4>Background</h4>Frame-shifted genes results in non-functional peptides. Because of this complete loss of function, frame-shifted genes have never been used in constructing synthetic gene circuits.<h4>Results</h4>Here we report that the function of gene circuits is rescued by a frame-shifted gene, which functions by translating from a non-natural start codon. We report a single nucleotide deletion mutation that developed in the λ-repressor cI within a synthetic genetic NOT gate in <i>Escherich ...[more]

PMID: 30867677

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Project description:Recent studies have shown that haloarchaea employ leaderless and Shine-Dalgarno (SD)-less mechanisms for translation initiation of leaderless transcripts with a 5' untranslated region (5' UTR) of <10 nucleotides (nt) and leadered transcripts with a 5' UTR of ?10 nt, respectively. However, whether the two mechanisms can operate on the same naturally occurring haloarchaeal transcript carrying multiple potential start codons is unknown. In this study, the transcript of the sptA gene (encoding an extracellular serine protease of Natrinema sp. strain J7-2) was experimentally determined and found to contain two potential in-frame AUG codons (AUG(1) and AUG(2)) located 5 and 29 nt, respectively, downstream of the transcription start site. Mutational analysis revealed that both AUGs can function as the translation start codon for production of active SptA, although AUG(1) is more efficient than AUG(2) for translation initiation. Insertion of a stable stem-loop structure between the two AUGs completely abolished initiation at AUG(1) but did not affect initiation at AUG(2), indicating that AUG(2)-initiated translation does not involve ribosome scanning from the 5' end of the transcript. Furthermore, the efficiency of AUG(2)-initiated translation was not influenced by an upstream SD-like sequence. In addition, both AUG(1) and AUG(2) contribute to transcript stability, probably by recruiting ribosomes to protect the transcript against degradation. These data suggest that depending on which of two in-frame start codons is used, the sptA transcript can act as either a leaderless or a leadered transcript for SptA production in haloarchaea.In eukaryotes and bacteria, alternative translation start sites contribute to proteome complexity and can be used as a functional mechanism to increase translation efficiency. However, little is known about alternative translation initiation in archaea. Our results demonstrate that leaderless and SD-less mechanisms can be used for translation initiation of the sptA transcript from two in-frame start codons, raising the possibility that in haloarchaea, alternative translation initiation on one transcript functions to increase translation efficiency and/or contribute to proteome complexity.

Project description:BackgroundOut-of-frame stop codons (OSCs) occur naturally in coding sequences of all organisms, providing a mechanism of early termination of translation in incorrect reading frame so that the metabolic cost associated with frameshift events can be reduced. Given such a functional significance, we expect statistically overrepresented OSCs in coding sequences as a result of a widespread selection. Accordingly, we examined available prokaryotic genomes to look for evidence of this selection.ResultsThe complete genome sequences of 990 prokaryotes were obtained from NCBI GenBank. We found that low G+C content coding sequences contain significantly more OSCs and G+C content at specific codon positions were the principal determinants of OSC usage bias in the different reading frames. To investigate if there is overrepresentation of OSCs, we modeled the trinucleotide and hexanucleotide biases of the coding sequences using Markov models, and calculated the expected OSC frequencies for each organism using a Monte Carlo approach. More than 93% of 342 phylogenetically representative prokaryotic genomes contain excess OSCs. Interestingly the degree of OSC overrepresentation correlates positively with G+C content, which may represent a compensatory mechanism for the negative correlation of OSC frequency with G+C content. We extended the analysis using additional compositional bias models and showed that lower-order bias like codon usage and dipeptide bias could not explain the OSC overrepresentation. The degree of OSC overrepresentation was found to correlate negatively with the optimal growth temperature of the organism after correcting for the G+C% and AT skew of the coding sequence.ConclusionsThe present study uses approaches with statistical rigor to show that OSC overrepresentation is a widespread phenomenon among prokaryotes. Our results support the hypothesis that OSCs carry functional significance and have been selected in the course of genome evolution to act against unintended frameshift occurrences. Some results also hint that OSC overrepresentation being a compensatory mechanism to make up for the decrease in OSCs in high G+C organisms, thus revealing the interplay between two different determinants of OSC frequency.

Dataset Information

A frame-shifted gene, which rescued its function by non-natural start codons and its application in constructing synthetic gene circuits.

Publications

A frame-shifted gene, which rescued its function by non-natural start codons and its application in constructing synthetic gene circuits.

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