A possible universal role for mRNA secondary structure in bacterial translation revealed using a synthetic operon

Chemla, Yonatan; Peeri, Michael; Heltberg, Mathias L.; Eichler, Jerry; Jensen, Mogens H.; Tuller, Tamir; Alfonta, Lital

doi:10.1038/s41467-020-18577-4

Cited by 14 publications

(12 citation statements)

References 36 publications

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“…Moreover, translation efficiency depends on translation initiation and bias in codon usage (Chemla et al, 2020; Maier et al, 2009). The third level is post‐translational control, which affects the activity and half‐life of proteins.…”

Section: Discussionmentioning

confidence: 99%

Global proteomic response of unicellular cyanobacterium Synechocystis sp. PCC 6803 to fluctuating light upon CO ₂ step‐down

Mustila

Muth-Pawlak

Aro

et al. 2021

Physiologia Plantarum

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Photosynthetic cyanobacteria are exposed to rapid changes in light intensity in their natural habitats, as well as in photobioreactors. To understand the effects of such fluctuations on Synechocystis sp. PCC 6803, the global proteome of cells grown under a fluctuating light condition (low background light interrupted with high light pulses) was compared to the proteome of cells grown under constant light with concomitant acclimation of cells to low CO2 level. The untargeted global proteome of Synechocystis sp. PCC 6803 was analyzed by data‐dependent acquisition (DDA), which relies on the high mass accuracy and sensitivity of orbitrap‐based tandem mass spectrometry. In addition, a targeted selected reaction monitoring (SRM) approach was applied to monitor the proteomic changes in a strain lacking flavodiiron proteins Flv1 and Flv3. This strain is characterized by impaired growth and photosynthetic activity under fluctuating light. An obvious reprogramming of cell metabolism was observed in this study and was compared to a previous transcriptional analysis performed under the same fluctuating light regime. Cyanobacterial responses to fluctuating light correlated at mRNA and protein levels to some extent, but discrepancies indicate that several proteins are post‐transcriptionally regulated (affecting observed protein abundances). The data suggest that Synechocystis sp. PCC 6803 maintain higher nitrogen assimilation, serving as an electron valve, for long‐term acclimation to fluctuating light upon CO2 step‐down. Although Flv1 and Flv3 are known to be crucial for the cells at the onset of illumination, the flavodiiron proteins, as well as components of carbon assimilation pathways, were less abundant under fluctuating light.

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Section: Discussionmentioning

confidence: 99%

Global proteomic response of unicellular cyanobacterium Synechocystis sp. PCC 6803 to fluctuating light upon CO ₂ step‐down

Mustila

Muth-Pawlak

Aro

et al. 2021

Physiologia Plantarum

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“…For more detailed insights into codon usage, refer to [44]. The more usual approach now is the use of synthetic genes that can be codon optimized for the expression host, while simultaneously avoiding internal rbs, internal restriction sites, and factors that influence mRNA structure and stability [45,46]. As prices have rapidly dropped a synthetic gene can cost less than the labour and material costs associated with cloning a gene from a cDNA library.…”

Section: The Gene and Its Propertiesmentioning

confidence: 99%

So you want to express your protein in Escherichia coli?

Tungekar

Castillo-Corujo

Ruddock

2021

Essays in Biochemistry

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Recombinant proteins have been extensively employed as therapeutics for the treatment of various critical and life-threatening diseases and as industrial enzymes in high-value industrial processes. Advances in genetic engineering and synthetic biology have broadened the horizon of heterologous protein production using multiple expression platforms. Selection of a suitable expression system depends on a variety of factors ranging from the physicochemical properties of the target protein to economic considerations. For more than 40 years, Escherichia coli has been an established organism of choice for protein production. This review aims to provide a stepwise approach for any researcher embarking on the journey of recombinant protein production in E. coli. We present an overview of the challenges associated with heterologous protein expression, fundamental considerations connected to the protein of interest (POI) and designing expression constructs, as well as insights into recently developed technologies that have contributed to this ever-growing field.

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“…The different gene products are then translated from the same mRNA molecule and these genes are usually separated by less than 20 base pairs [6]. The translation of genes clustered in operons is often dependent on the space between genes [7]. Consequently, DNA and mRNA sequence features are expected to play an important role in gene expression and regulation in operons.…”

Section: Introductionmentioning

confidence: 99%

The high mutational sensitivity of ccdA antitoxin is linked to codon optimality

Chandra

Gupta

Khare

et al. 2022

Preprint

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Deep mutational scanning studies suggest that single synonymous mutations are typically silent and that most exposed, non active-site residues are tolerant to mutations. Here we show that the ccdA antitoxin component of the E.coli ccdAB toxin-antitoxin operonic system is unusually sensitive to mutations when studied in the operonic context. A large fraction (~80%) of single codon mutations, including many synonymous mutations in the ccdA gene show inactive phenotypes that are correlated with the E.coli codon usage frequency but retain native-like binding affinity towards cognate toxin, CcdB. Therefore, the observed phenotypic effects are largely not due to alterations in protein structure or stability, consistent with the fact that a large region of CcdA is intrinsically disordered. In select cases, proteomics studies reveal altered ratios of CcdA:CcdB protein levels in vivo, suggesting that these mutations likely alter relative translation efficiencies of the two genes in the operon. We extend these results by predicting and validating single synonymous mutations that lead to loss of function phenotypes in the relBE operon upon introduction of rarer codons. Thus, in their native context, genes are likely to be more sensitive to both synonymous and non-synonymous point mutations than inferred from previous saturation mutagenesis studies.

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A possible universal role for mRNA secondary structure in bacterial translation revealed using a synthetic operon

Cited by 14 publications

References 36 publications

Global proteomic response of unicellular cyanobacterium Synechocystis sp. PCC 6803 to fluctuating light upon CO ₂ step‐down

Global proteomic response of unicellular cyanobacterium Synechocystis sp. PCC 6803 to fluctuating light upon CO ₂ step‐down

So you want to express your protein in Escherichia coli?

The high mutational sensitivity of ccdA antitoxin is linked to codon optimality

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A possible universal role for mRNA secondary structure in bacterial translation revealed using a synthetic operon

Cited by 14 publications

References 36 publications

Global proteomic response of unicellular cyanobacterium Synechocystis sp. PCC 6803 to fluctuating light upon CO 2 step‐down

Global proteomic response of unicellular cyanobacterium Synechocystis sp. PCC 6803 to fluctuating light upon CO 2 step‐down

So you want to express your protein in Escherichia coli?

The high mutational sensitivity of ccdA antitoxin is linked to codon optimality

Contact Info

Product

Resources

About

Global proteomic response of unicellular cyanobacterium Synechocystis sp. PCC 6803 to fluctuating light upon CO ₂ step‐down

Global proteomic response of unicellular cyanobacterium Synechocystis sp. PCC 6803 to fluctuating light upon CO ₂ step‐down