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Rare codon recoding for selective and efficient noncanonical amino acid incorporation [RNA-seq]

ABSTRACT: Rare codon recoding for selective and efficient noncanonical amino acid incorporation [RNA-seq]

PROVIDER: PRJNA1075072 | ENA |

REPOSITORIES: ENA

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Rare codon recoding for selective and efficient noncanonical amino acid incorporation [Ribo-seq]

Project description:The ability to genetically encode noncanonical amino acids (ncAAs) has empowered proteins with improved or novel properties. However, existing strategies in mammalian cells rely on the introduction of blank codon for incorporating ncAAs, which is very inefficient and limits their widespread applications. Here, we develop a rare codon recoding strategy that takes advantage of the relative rarity of the TCG codon to achieve highly selective and efficient ncAA incorporation through systematic engineering and big data model predictions. We highlight the broad utility of this strategy for the incorporation of dozens of ncAAs into various functional proteins at the wild-type protein expression levels, as well as the synthesis of proteins with up to 6-site ncAAs or 4 distinct ncAAs in mammalian cells for downstream applications.

2025-01-01 | GSE255465 | GEO

Rare codon recoding for selective and efficient noncanonical amino acid incorporation [RNA-seq]

2025-01-01 | GSE255464 | GEO

Rare codon recoding for selective and efficient noncanonical amino acid incorporation [Ribo-seq]

Project description:Rare codon recoding for selective and efficient noncanonical amino acid incorporation [Ribo-seq]

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Rare codon recoding for selective and efficient unnatural amino acid incorporation in mammalian cells

Project description:Mass spectromery for DiZSeK residue specific incorporation on TCG codon.

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ENVIRONMENT MODULATES PROTEIN HETEROGENEITY THROUGH TRANSCRIPTIONAL AND TRANSLATIONAL STOP CODON RECODING

Project description:In order to systematically assess the frequency and origin of stop codon recoding events, we designed a library of reporters. We introduced premature stop codons into mScarlet that enabled high-throughput quantification of protein synthesis termination errors in E. coli using fluorescent microscopy. We found that under stress conditions, stop codon recoding may occur with a rate as high as 80%, depending on the nucleotide context, suggesting that evolution frequently samples stop codon recoding events. The analysis of selected reporters by mass spectrometry and RNA-seq showed that not only translation but also transcription errors contribute to stop codon recoding. The RNA polymerase is more likely to misincorporate a nucleotide at premature stop codons. Proteome-wide detection of stop codon recoding by mass spectrometry revealed that temperature regulates the expression of cryptic sequences generated by stop codon recoding in E. coli. Overall, our findings suggest that the environment influences the accuracy of protein production which increases protein heterogeneity when the organisms need to adapt to new conditions.

2024-03-28 | GSE226936 | GEO

Environment Modulates Protein Heterogeneity Through Transcriptional and Translational Stop Codon Recoding

Project description:Stop codon recoding events give rise to longer proteins, which may alter the proteins function and thereby generate short-lasting phenotypic variability from a single gene. In order to systematically assess the frequency and origin of recoding events, we designed a library of reporters. We introduced premature stop codons into mScarlet that enabled high-throughput quantification of protein synthesis termination errors in E.coli using fluorescent microscopy. We found that under stress conditions, stop codon recoding may occur as high as 80 percent of the time, depending on the genetic context, suggesting that evolution frequently samples stop codon recoding events. Targeted mass spectrometry and RNA-seq analyses showed that not only translational but also transcriptional errors contribute to stop codon recoding. The RNA polymerase is more likely to misincorporate a nucleotide at premature stop codons. Proteome-wide mass -spectrometry revealed that temperature regulates the expression of cryptic peptides generated by stop codon recoding in E.coli. Overall, our findings suggest that the environment influences the accuracy of protein production which increases protein heterogeneity when the organisms need to adapt to new conditions.

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Site-specific incorporation of two noncanonical amino acids in GFP

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Stop codon recoding is widespread in diverse phage lineages and has the potential to regulate translation of late stage and lytic genes

Project description:Stop codon recoding in bacteriophages

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ENVIRONMENT MODULATES PROTEIN HETEROGENEITY THROUGH TRANSCRIPTIONAL AND TRANSLATIONAL STOP CODON RECODING

Project description:ENVIRONMENT MODULATES PROTEIN HETEROGENEITY THROUGH TRANSCRIPTIONAL AND TRANSLATIONAL STOP CODON RECODING

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Sense codon emancipation for proteome-wide incorporation of noncanonical amino acids: rare isoleucine codon AUA as a target for genetic code expansion.

Project description:One of the major challenges in contemporary synthetic biology is to find a route to engineer synthetic organisms with altered chemical constitution. In terms of core reaction types, nature uses an astonishingly limited repertoire of chemistries when compared with the exceptionally rich and diverse methods of organic chemistry. In this context, the most promising route to change and expand the fundamental chemistry of life is the inclusion of amino acid building blocks beyond the canonical 20 (i.e. expanding the genetic code). This strategy would allow the transfer of numerous chemical functionalities and reactions from the synthetic laboratory into the cellular environment. Due to limitations in terms of both efficiency and practical applicability, state-of-the-art nonsense suppression- or frameshift suppression-based methods are less suitable for such engineering. Consequently, we set out to achieve this goal by sense codon emancipation, that is, liberation from its natural decoding function - a prerequisite for the reassignment of degenerate sense codons to a new 21st amino acid. We have achieved this by redesigning of several features of the post-transcriptional modification machinery which are directly involved in the decoding process. In particular, we report first steps towards the reassignment of 5797 AUA isoleucine codons in Escherichia coli using efficient tools for tRNA nucleotide modification pathway engineering.

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