Comparison of mRNA features affecting translation initiation and reinitiation

Osterman, Ilya А.; Evfratov, Sergey; Сергиев, Петр В.; Dontsova, Olga А.

doi:10.1093/nar/gks989

Cited by 123 publications

(151 citation statements)

References 41 publications

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“…Bandmann et al developed a mutation library of TrpL-fused eGFP proteins mutated at the first eight AAs and then studied the influence of different kinds of start codons on translation initiation rate [20]. They found that ATG was the best start codon, consistent with another observation that also surprisingly found a high translation initiation rate for the less frequent GTG codon [47]. The integrated nature of these features makes it difficult to perform truly isolated studies of any one of these single variables, but not impossible.…”

Section: Mrna Propertiesmentioning

confidence: 72%

Prediction of soluble heterologous protein expression levels inEscherichia colifrom sequence-based features and its potential in biopharmaceutical process development

Dai¹,

Guo²,

Long

et al. 2014

Pharmaceutical Bioprocessing

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Prediction of soluble protein expression levels in Escherichia coli based on the nature of protein itself remains a challenge for bioprocess development (BD). This review will critically discuss the current efforts and achievements that employ computational approaches to develop prediction models for soluble protein expression in E. coli. The contrast between the remarkable progresses made on the predictive models achieved by bioinformatics and their relatively infrequent application in BD will be explained. The effects of process-relevant variables at four different levels on the expression of heterologous proteins, for example, gene, vector, host cell and cultivation process, and also a critical comparison of several established bioinformatics tools for predicting expression levels will be presented. The potential utility of this emergent technology to increase the efficiency of BD strategies and thereby to reduce the cost of establishing a process for soluble protein expression are critically examined.Keywords: amino acid sequence-derived features • Escherichia coli culture • mathematic prediction model • process development • soluble protein expression

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Section: Mrna Propertiesmentioning

confidence: 72%

Prediction of soluble heterologous protein expression levels inEscherichia colifrom sequence-based features and its potential in biopharmaceutical process development

Dai¹,

Guo²,

Long

et al. 2014

Pharmaceutical Bioprocessing

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“…Adenine-rich regions have been shown to enhance expression by increasing the rate and amount of ternary complex formation during initiation (34), likely due to ribosomal protein S1's high affinity for polypyrimidines (43,44). S1-mediated initiation (45,46) occurs in the absence of an SD sequence; therefore, it is possible that S1 also plays a role in ptrB regulation.…”

Section: Discussionmentioning

confidence: 99%

Novel Translation Initiation Regulation Mechanism in Escherichia coli ptrB Mediated by a 5′-Terminal AUG

Beck

Janssen

2017

J Bacteriol

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Alternative translation initiation mechanisms, distinct from the ShineDalgarno (SD) sequence-dependent mechanism, are more prevalent in bacteria than once anticipated. Translation of Escherichia coli ptrB instead requires an AUG triplet at the 5= terminus of its mRNA. The 5=-terminal AUG (5=-uAUG) acts as a ribosomal recognition signal to attract ribosomes to the ptrB mRNA rather than functioning as an initiation codon to support translation of an upstream open reading frame. ptrB expression exhibits a stronger dependence on the 5=-uAUG than the predicted SD sequence; however, strengthening the predicted ptrB SD sequence relieves the necessity for the 5=-uAUG. Additional sequences within the ptrB 5= untranslated region (5=-UTR) work cumulatively with the 5=-uAUG to control expression of the downstream ptrB coding sequence (CDS), thereby compensating for the weak SD sequence. Replacement of 5=-UTRs from other mRNAs with the ptrB 5=-UTR sequence showed a similar dependence on the 5=-uAUG for CDS expression, suggesting that the regulatory features contained within the ptrB 5=-UTR are sufficient to control the expression of other E. coli CDSs. Demonstration that the 5=-uAUG present on the ptrB leader mRNA is involved in ribosome binding and expression of the downstream ptrB CDS revealed a novel form of translational regulation. Due to the abundance of AUG triplets at the 5= termini of E. coli mRNAs and the ability of ptrB 5=-UTR regulation to function independently of gene context, the regulatory effects of 5=-uAUGs on downstream CDSs may be widespread throughout the E. coli genome.IMPORTANCE As the field of synthetic biology continues to grow, a complete understanding of basic biological principles will be necessary. The increasing complexity of the synthetic systems highlights the gaps in our current knowledge of RNA regulation. This study demonstrates that there are novel ways to regulate canonical Shine-Dalgarno-led mRNAs in Escherichia coli, illustrating that our understanding of the fundamental processes of translation and RNA regulation is still incomplete. Even for E. coli, one of the most-studied model organisms, genes with translation initiation mechanisms that do not fit the canonical Shine-Dalgarno sequence paradigm are being revealed. Uncovering diverse mechanisms that control translational expression will allow synthetic biologists to finely tune protein production of desired gene products.KEYWORDS 5= upstream AUG, Shine-Dalgarno, noncanonical initiation, translation initiation, translational regulation, upstream open reading frame P rotein synthesis, in which the ribosome translates an mRNA sequence to form polypeptides, is a four-step process that includes initiation, elongation, termination, and ribosome recycling. Translation initiation is energy dependent and the most highly regulated phase of translation (1). The translational machinery interacts with the mRNA at its ribosome binding site (RBS) along with initiator tRNA to form a ternary complex that is equipped for polypeptide productio...

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“…4a). Based on a recent report that described the effect of upstream secondary structure within the 5′UTR on translation efficiency [27], we proposed that the aptamer domain of the theophylline riboswitch naturally forming a stem-loop upstream of the SD sequence after transcription augments the accessibility of the ribosome to the SD, eventually producing higher levels of GFP. These results are consistent with previously reported data in which the induced expression levels altered after combining different promoters with the same riboswitch [22, 23].…”

Section: Discussionmentioning

confidence: 99%

Engineering an inducible gene expression system for Bacillus subtilis from a strong constitutive promoter and a theophylline-activated synthetic riboswitch

et al. 2016

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BackgroundSynthetic riboswitches have been increasingly used to control and tune gene expression in diverse organisms. Although a set of theophylline-responsive riboswitches have been developed for bacteria, fully functional expression elements mediated by synthetic riboswitches in Bacillus subtilis are rarely used because of the host-dependent compatibility between the promoters and riboswitches.ResultsA novel genetic element composed of the promoter P43 and a theophylline-riboswitch was developed and characterized in B. subtilis. When combined with a P43 promoter (P43′-riboE1), the theophylline-riboswitch successfully switched the constitutive expression pattern of P43 to an induced pattern. The expression mediated by the novel element could be activated at the translational level by theophylline with a relatively high induction ratio. The induction ratios for P43′-riboE1 by 4-mM theophylline were elevated during the induction period. The level of induced expression was dependent on the theophylline dose. Correspondingly, the induction ratios gradually increased in parallel with the elevated dose of theophylline. Importantly, the induced expression level was higher than three other strong constitutive promoters including PsrfA, PaprE, and the native P43. It was found that the distance between the SD sequence within the expression element and the start codon significantly influenced both the level of induced expression and the induction ratio. A 9-bp spacer was suitable for producing desirable expression level and induction ratio. Longer spacer reduced the activation efficiency. Importantly, the system successfully overexpressed β-glucuronidase at equal levels, and induction ratio was similar to that of GFP.ConclusionThe constructed theophylline-inducible gene expression system has broad compatibility and robustness, which has great potential in over-production of pharmaceutical and industrial proteins and utilization in building more complex gene circuits.

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Comparison of mRNA features affecting translation initiation and reinitiation

Cited by 123 publications

References 41 publications

Prediction of soluble heterologous protein expression levels inEscherichia colifrom sequence-based features and its potential in biopharmaceutical process development

Prediction of soluble heterologous protein expression levels inEscherichia colifrom sequence-based features and its potential in biopharmaceutical process development

Novel Translation Initiation Regulation Mechanism in Escherichia coli ptrB Mediated by a 5′-Terminal AUG

Engineering an inducible gene expression system for Bacillus subtilis from a strong constitutive promoter and a theophylline-activated synthetic riboswitch

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