Document Detail


Synthetic approach to stop-codon scanning mutagenesis.
MedLine Citation:
PMID:  21452871     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
A general combinatorial mutagenesis strategy using common dimethoxytrityl-protected mononucleotide phosphoramidites and a single orthogonally protected trinucleotide phosphoramidite (Fmoc-TAG; Fmoc = 9-fluorenylmethoxycarbonyl) was developed to scan a gene with the TAG amber stop codon with complete synthetic control. In combination with stop-codon suppressors that insert natural (e.g., alanine) or unnatural (e.g., p-benzoylphenylalanine, Bpa) amino acids, a single DNA library can be used to incorporate different amino acids for diverse purposes. Here, we scanned TAG codons through part of the gene for a model four-helix bundle protein, Rop, which regulates the copy number of ColE1 plasmids. Alanine was incorporated into Rop for mapping its binding site using an in vivo activity screen, and subtle but important differences from in vitro gel-shift studies of Rop function are evident. As a test, Bpa was incorporated using a Phe14 amber mutant isolated from the scanning library. Surprisingly, Phe14Bpa-Rop is weakly active, despite the critical role of Phe14 in Rop activity. Bpa is a photoaffinity label unnatural amino acid that can form covalent bonds with adjacent molecules upon UV irradiation. Irradiation of Phe14Bpa-Rop, which is a dimer in solution like wild-type Rop, results in covalent dimers, trimers, and tetramers. This suggests that Phe14Bpa-Rop weakly associates as a tetramer in solution and highlights the use of Bpa cross-linking as a means of trapping weak and transient interactions.
Authors:
Lihua Nie; Jason J Lavinder; Mohosin Sarkar; Kimberly Stephany; Thomas J Magliery
Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't     Date:  2011-03-31
Journal Detail:
Title:  Journal of the American Chemical Society     Volume:  133     ISSN:  1520-5126     ISO Abbreviation:  J. Am. Chem. Soc.     Publication Date:  2011 Apr 
Date Detail:
Created Date:  2011-04-20     Completed Date:  2011-08-01     Revised Date:  2014-09-17    
Medline Journal Info:
Nlm Unique ID:  7503056     Medline TA:  J Am Chem Soc     Country:  United States    
Other Details:
Languages:  eng     Pagination:  6177-86     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
Base Sequence
Codon, Terminator*
DNA Primers
Magnetic Resonance Spectroscopy
Mass Spectrometry
Models, Molecular
Mutagenesis
Plasmids
Grant Support
ID/Acronym/Agency:
R01 GM083114/GM/NIGMS NIH HHS; R01 GM083114/GM/NIGMS NIH HHS; R01 GM083114-03/GM/NIGMS NIH HHS
Chemical
Reg. No./Substance:
0/Codon, Terminator; 0/DNA Primers
Comments/Corrections

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