Context effects of genetic code expansion by stop codon suppression

Chemla, Yonatan; Ozer, Eden; Algov, Itay; Alfonta, Lital

doi:10.1016/j.cbpa.2018.07.012

Cited by 50 publications

(59 citation statements)

References 59 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The efficiency of NAA incorporation in response to a stop codon is known to depend on the position of the mutation site and the nature of the recombinant protein and the context dependence. The thorough analysis demonstrated [118] that mRNA, operon, and cellular context effects define the permissivity of the site for the NAAs incorporation.…”

Section: Discussionmentioning

confidence: 99%

Site-Specific Incorporation of Unnatural Amino Acids into Escherichia coli Recombinant Protein: Methodology Development and Recent Achievement

Smolskaya

Andreev

2019

Biomolecules

View full text Add to dashboard Cite

More than two decades ago a general method to genetically encode noncanonical or unnatural amino acids (NAAs) with diverse physical, chemical, or biological properties in bacteria, yeast, animals and mammalian cells was developed. More than 200 NAAs have been incorporated into recombinant proteins by means of non-endogenous aminoacyl-tRNA synthetase (aa-RS)/tRNA pair, an orthogonal pair, that directs site-specific incorporation of NAA encoded by a unique codon. The most established method to genetically encode NAAs in Escherichia coli is based on the usage of the desired mutant of Methanocaldococcus janaschii tyrosyl-tRNA synthetase (MjTyrRS) and cognate suppressor tRNA. The amber codon, the least-used stop codon in E. coli, assigns NAA. Until very recently the genetic code expansion technology suffered from a low yield of targeted proteins due to both incompatibilities of orthogonal pair with host cell translational machinery and the competition of suppressor tRNA with release factor (RF) for binding to nonsense codons. Here we describe the latest progress made to enhance nonsense suppression in E. coli with the emphasis on the improved expression vectors encoding for an orthogonal aa-RA/tRNA pair, enhancement of aa-RS and suppressor tRNA efficiency, the evolution of orthogonal EF-Tu and attempts to reduce the effect of RF1.

show abstract

Section: Discussionmentioning

confidence: 99%

Site-Specific Incorporation of Unnatural Amino Acids into Escherichia coli Recombinant Protein: Methodology Development and Recent Achievement

Smolskaya

Andreev

2019

Biomolecules

View full text Add to dashboard Cite

show abstract

“…Expression of the synthetic operon was controlled by the lac operator so as not to affect bacterial fitness, given the variability of the random sequence, which is only expressed when IPTG (1 mM) is added to the growth media. To control for known stop codon context effects 24 , the first six nucleotides in this variable region (ACUAGU) were fixed. After assembly, the library was transformed into E. coli DH5α, where library complexity was measured as~10 4 by counting colony-forming units.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2020

Self Cite

View full text Add to dashboard Cite

In bacteria, translation re-initiation is crucial for synthesizing proteins encoded by genes that are organized into operons. The mechanisms regulating translation re-initiation remain, however, poorly understood. We now describe the ribosome termination structure (RTS), a conserved and stable mRNA secondary structure localized immediately downstream of stop codons, and provide experimental evidence for its role in governing re-initiation efficiency in a synthetic Escherichia coli operon. We further report that RTSs are abundant, being associated with 18%–65% of genes in 128 analyzed bacterial genomes representing all phyla, and are selectively depleted when translation re-initiation is advantageous yet selectively enriched so as to insulate translation when re-initiation is deleterious. Our results support a potentially universal role for the RTS in controlling translation termination-insulation and re-initiation across bacteria.

show abstract

“…However, it is important to discuss two sources of this variability, which could be a common impediment in the employment of this system. First, the context and location of the site chosen and mutated to facilitate Uaa incorporation have a significant effect on the efficiency of the system [35,36], but notably, from our experience, the context, and locations effects could be different between in-vivo and cell-free systems [data not shown]. This subject warrants a systematic investigation which is outside the scope of this study.…”

Section: Simplified Extract Preparation and Cell-free Reaction For Gementioning

confidence: 95%

Simplified Methodology for a Modular and Genetically Expanded Protein Synthesis in Cell-Free Systems

Chemla

Ozer

Alfonta

et al. 2019

Preprint

Self Cite

View full text Add to dashboard Cite

Genetic code expansion, which enables the site-specific incorporation of unnatural amino acids into proteins, has emerged as a new and powerful tool for protein engineering. Currently, it is mainly utilized inside living cells for a myriad of applications. However, utilization of this technology in a cell-free, reconstituted platform has several advantages over living systems. The common limitations to the employment of these systems are the laborious and complex nature of its preparation and utilization. Herein, we describe a simplified method for the preparation of this system from Escherichia coli cells, which is specifically adapted for the expression of the components needed for cell-free genetic code expansion. In addition, we propose and demonstrate a modular approach to its utilization. By this approach, it is possible to prepare and store different extracts, harboring various translational components, and mix and match them as needed for more than four years retaining its high efficiency. We demonstrate this with the simultaneous incorporation of two different unnatural amino acids into a reporter protein. Finally, we demonstrate the advantage of cell-free systems over living cells for the incorporation of δ-thioboc-lysine into ubiquitin by using the methanosarcina mazei wild-type pyrrolysyl tRNACUA and tRNA-synthetase pair, which can not be achieved in a living cell.

show abstract

Context effects of genetic code expansion by stop codon suppression

Cited by 50 publications

References 59 publications

Site-Specific Incorporation of Unnatural Amino Acids into Escherichia coli Recombinant Protein: Methodology Development and Recent Achievement

Site-Specific Incorporation of Unnatural Amino Acids into Escherichia coli Recombinant Protein: Methodology Development and Recent Achievement

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

Simplified Methodology for a Modular and Genetically Expanded Protein Synthesis in Cell-Free Systems

Contact Info

Product

Resources

About