Dissecting the Contribution of Release Factor Interactions to Amber Stop Codon Reassignment Efficiencies of the <i>Methanocaldococcus jannaschii</i> Orthogonal Pair
Non-canonical amino acids (ncAAs) are finding increasing use in basic biochemical studies and biomedical applications. The efficiency of ncAA incorporation is highly variable, as a result of competing system composition and codon context effects. The relative quantitative contribution of the multipl...
Main Authors: | , , |
---|---|
Format: | Article |
Language: | English |
Published: |
MDPI AG
2018-11-01
|
Series: | Genes |
Subjects: | |
Online Access: | https://www.mdpi.com/2073-4425/9/11/546 |
id |
doaj-b303d1b30ead42eb8360f1b0ac538d59 |
---|---|
record_format |
Article |
spelling |
doaj-b303d1b30ead42eb8360f1b0ac538d592020-11-25T00:49:12ZengMDPI AGGenes2073-44252018-11-0191154610.3390/genes9110546genes9110546Dissecting the Contribution of Release Factor Interactions to Amber Stop Codon Reassignment Efficiencies of the <i>Methanocaldococcus jannaschii</i> Orthogonal PairDavid G. Schwark0Margaret A. Schmitt1John D. Fisk2Department of Chemistry, University of Colorado Denver, Campus Box 194, P.O. Box 173364, Denver, CO 80217-3364, USADepartment of Chemistry, University of Colorado Denver, Campus Box 194, P.O. Box 173364, Denver, CO 80217-3364, USADepartment of Chemistry, University of Colorado Denver, Campus Box 194, P.O. Box 173364, Denver, CO 80217-3364, USANon-canonical amino acids (ncAAs) are finding increasing use in basic biochemical studies and biomedical applications. The efficiency of ncAA incorporation is highly variable, as a result of competing system composition and codon context effects. The relative quantitative contribution of the multiple factors affecting incorporation efficiency are largely unknown. This manuscript describes the use of green fluorescent protein (GFP) reporters to quantify the efficiency of amber codon reassignment using the <i>Methanocaldococcus jannaschii</i> orthogonal pair system, commonly employed for ncAA incorporation, and quantify the contribution of release factor 1 (RF1) to the overall efficiency of amino acid incorporation. The efficiencies of amber codon reassignments were quantified at eight positions in GFP and evaluated in multiple combinations. The quantitative contribution of RF1 competition to reassignment efficiency was evaluated through comparisons of amber codon suppression efficiencies in normal and genomically recoded <i>Escherichia coli</i> strains. Measured amber stop codon reassignment efficiencies for eight single stop codon GFP variants ranged from 51 to 117% in <i>E. coli</i> DH10B and 76 to 104% in the RF1 deleted <i>E. coli</i> C321.ΔA.exp. Evaluation of efficiency changes in specific sequence contexts in the presence and absence of RF1 suggested that RF1 specifically interacts with +4 Cs and that the RF1 interactions contributed approximately half of the observed sequence context-dependent variation in measured reassignment efficiency. Evaluation of multisite suppression efficiencies suggests that increasing demand for translation system components limits multisite incorporation in cells with competing RF1.https://www.mdpi.com/2073-4425/9/11/546genetic code expansionrelease factor 1amber stop codon suppression<i>M. jannaschii</i> orthogonal pairfluorescence-based screen |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
David G. Schwark Margaret A. Schmitt John D. Fisk |
spellingShingle |
David G. Schwark Margaret A. Schmitt John D. Fisk Dissecting the Contribution of Release Factor Interactions to Amber Stop Codon Reassignment Efficiencies of the <i>Methanocaldococcus jannaschii</i> Orthogonal Pair Genes genetic code expansion release factor 1 amber stop codon suppression <i>M. jannaschii</i> orthogonal pair fluorescence-based screen |
author_facet |
David G. Schwark Margaret A. Schmitt John D. Fisk |
author_sort |
David G. Schwark |
title |
Dissecting the Contribution of Release Factor Interactions to Amber Stop Codon Reassignment Efficiencies of the <i>Methanocaldococcus jannaschii</i> Orthogonal Pair |
title_short |
Dissecting the Contribution of Release Factor Interactions to Amber Stop Codon Reassignment Efficiencies of the <i>Methanocaldococcus jannaschii</i> Orthogonal Pair |
title_full |
Dissecting the Contribution of Release Factor Interactions to Amber Stop Codon Reassignment Efficiencies of the <i>Methanocaldococcus jannaschii</i> Orthogonal Pair |
title_fullStr |
Dissecting the Contribution of Release Factor Interactions to Amber Stop Codon Reassignment Efficiencies of the <i>Methanocaldococcus jannaschii</i> Orthogonal Pair |
title_full_unstemmed |
Dissecting the Contribution of Release Factor Interactions to Amber Stop Codon Reassignment Efficiencies of the <i>Methanocaldococcus jannaschii</i> Orthogonal Pair |
title_sort |
dissecting the contribution of release factor interactions to amber stop codon reassignment efficiencies of the <i>methanocaldococcus jannaschii</i> orthogonal pair |
publisher |
MDPI AG |
series |
Genes |
issn |
2073-4425 |
publishDate |
2018-11-01 |
description |
Non-canonical amino acids (ncAAs) are finding increasing use in basic biochemical studies and biomedical applications. The efficiency of ncAA incorporation is highly variable, as a result of competing system composition and codon context effects. The relative quantitative contribution of the multiple factors affecting incorporation efficiency are largely unknown. This manuscript describes the use of green fluorescent protein (GFP) reporters to quantify the efficiency of amber codon reassignment using the <i>Methanocaldococcus jannaschii</i> orthogonal pair system, commonly employed for ncAA incorporation, and quantify the contribution of release factor 1 (RF1) to the overall efficiency of amino acid incorporation. The efficiencies of amber codon reassignments were quantified at eight positions in GFP and evaluated in multiple combinations. The quantitative contribution of RF1 competition to reassignment efficiency was evaluated through comparisons of amber codon suppression efficiencies in normal and genomically recoded <i>Escherichia coli</i> strains. Measured amber stop codon reassignment efficiencies for eight single stop codon GFP variants ranged from 51 to 117% in <i>E. coli</i> DH10B and 76 to 104% in the RF1 deleted <i>E. coli</i> C321.ΔA.exp. Evaluation of efficiency changes in specific sequence contexts in the presence and absence of RF1 suggested that RF1 specifically interacts with +4 Cs and that the RF1 interactions contributed approximately half of the observed sequence context-dependent variation in measured reassignment efficiency. Evaluation of multisite suppression efficiencies suggests that increasing demand for translation system components limits multisite incorporation in cells with competing RF1. |
topic |
genetic code expansion release factor 1 amber stop codon suppression <i>M. jannaschii</i> orthogonal pair fluorescence-based screen |
url |
https://www.mdpi.com/2073-4425/9/11/546 |
work_keys_str_mv |
AT davidgschwark dissectingthecontributionofreleasefactorinteractionstoamberstopcodonreassignmentefficienciesoftheimethanocaldococcusjannaschiiiorthogonalpair AT margaretaschmitt dissectingthecontributionofreleasefactorinteractionstoamberstopcodonreassignmentefficienciesoftheimethanocaldococcusjannaschiiiorthogonalpair AT johndfisk dissectingthecontributionofreleasefactorinteractionstoamberstopcodonreassignmentefficienciesoftheimethanocaldococcusjannaschiiiorthogonalpair |
_version_ |
1725252437252505600 |