A simplified reconstitution of mRNA-directed peptide synthesis: activity of the epsilon enhancer and an unnatural amino acid

Analytical Biochemistry
A C ForsterStephen C Blacklow

Abstract

The study of the early events in translation would be greatly facilitated by reconstitution with easily purified components. Here, Escherichia coli oligopeptide synthesis has been reconstituted using five purified recombinant His-tagged E. coli initiation and elongation factors. Highly purified ribosomes are required to yield products with strong dependencies on the translation factors. Based on HPLC separation of radiolabeled translation products from an mRNA encoding a tetrapeptide, approximately 80% of peptide products are full length, and the remaining 20% are the dipeptide and tripeptide products resulting from pausing or premature termination. Oligopeptide synthesis is enhanced when a commonly used epsilon (enhancer of protein synthesis initiation) sequence is included in the mRNA. The system incorporates a selectable, large, unnatural amino acid and may ultimately form the basis of a pure translation display technology for the directed evolution of peptidomimetic ligands and drug candidates. The recombinant clones can be exploited to prepare initiation factors and initiation complexes for structural studies, to study initiation and elongation in ribosomal peptide synthesis, and to screen for eubacterial-specific drugs.

References

Jul 1, 1979·Proceedings of the National Academy of Sciences of the United States of America·P C Jelenc, C G Kurland
Aug 1, 1977·Archives of Biochemistry and Biophysics·J W HersheyJ L Fakunding
Jul 1, 1991·Biochimie·Kim Kusk MortensenHans Uffe Sperling-Petersen
Jul 20, 1991·Journal of Molecular Biology·S LaalamiM Grunberg-Manago
Oct 29, 1990·FEBS Letters·C ClaessonT Borén
Jan 31, 1985·Biochemical and Biophysical Research Communications·R H GreenM C Ganoza
Mar 1, 1985·Proceedings of the National Academy of Sciences of the United States of America·M C GanozaR M Green
Jan 15, 1973·European Journal of Biochemistry·R BenneH O Voorma
Jun 1, 1974·Archives of Biochemistry and Biophysics·H F KungH Weissbach
Oct 1, 1984·Proceedings of the National Academy of Sciences of the United States of America·S PeacockH A Nash
Jul 1, 1981·Proceedings of the National Academy of Sciences of the United States of America·N RobakisH Weissbach
Sep 13, 1994·Proceedings of the National Academy of Sciences of the United States of America·L C MattheakisW J Dower
Mar 14, 1995·Proceedings of the National Academy of Sciences of the United States of America·M V Rodnina, W Wintermeyer
Oct 29, 1996·Proceedings of the National Academy of Sciences of the United States of America·Y P SemenkovW Wintermeyer
May 13, 1997·Proceedings of the National Academy of Sciences of the United States of America·J Hanes, A Plückthun
Nov 14, 1997·Proceedings of the National Academy of Sciences of the United States of America·R W Roberts, J W Szostak
Feb 26, 1998·Cell·K S Wilson, H F Noller
Feb 27, 1999·Current Opinion in Structural Biology·Boyd HardestyG Kramer
Apr 27, 1999·The International Journal of Biochemistry & Cell Biology·J LuM C Ganoza
Aug 4, 1999·Proceedings of the National Academy of Sciences of the United States of America·M O'ConnorA E Dahlberg
Sep 29, 1999·Trends in Biochemical Sciences·T E Dever
May 3, 2000·The EMBO Journal·J TomsicC O Gualerzi
Jun 1, 2000·Cell·Y NakamuraM Ehrenberg

Citations

Aug 28, 2007·Journal of the American Chemical Society·Baolin ZhangAnthony C Forster
Jun 28, 2012·ACS Synthetic Biology·Harris H WangGeorge M Church
Apr 21, 2009·Nucleic Acids Research·Anthony C Forster
Jun 18, 2003·RNA·Adam Frankel, Richard W Roberts
May 20, 2003·Proceedings of the National Academy of Sciences of the United States of America·Anthony C ForsterStephen C Blacklow
Mar 16, 2006·Proceedings of the National Academy of Sciences of the United States of America·Matthew C T HartmanJ W Szostak
Apr 23, 2004·Biological Procedures Online·Ayman AntounM åNs Ehrenberg M
Mar 25, 2015·International Journal of Molecular Sciences·Naohiro TerasakaHiroaki Suga
Jan 26, 2016·Journal of the American Chemical Society·Tomoshige FujinoHiroshi Murakami
Oct 26, 2013·Drug Discovery Today·Kristopher JosephsonJ W Szostak
Nov 19, 2011·Biotechnology and Bioengineering·Liping DuAnthony C Forster
Aug 4, 2009·Biotechnology and Bioengineering·Liping DuAnthony C Forster
Sep 29, 2004·Analytical Biochemistry·Anthony C ForsterStephen C Blacklow
Nov 16, 2004·Biochemical and Biophysical Research Communications·Christoph H RöhrigRichard R Schmidt
Dec 4, 2003·Chemistry & Biology·Adam FrankelRichard W Roberts
Aug 18, 2005·Journal of the American Chemical Society·Kristopher JosephsonJ W Szostak
Aug 4, 2005·Methods : a Companion to Methods in Enzymology·Zhongping TanAnthony C Forster
Dec 4, 2012·Biopolymers·Matthew T WeinstockMichael S Kay
Aug 23, 2006·Molecular Systems Biology·Anthony C Forster, George M Church
Nov 22, 2007·Molecular Microbiology·Stella H NorthHiroshi Nakai
Nov 6, 2008·Chembiochem : a European Journal of Chemical Biology·Atsushi OhtaHiroaki Suga
Apr 17, 2009·Chembiochem : a European Journal of Chemical Biology·Eiji NakajimaHiroaki Suga
Nov 15, 2012·Scientific Reports·Vladimir A MitkevichVasili Hauryliuk

Related Concepts

High Pressure Liquid Chromatography Procedure
Alkalescens-Dispar Group
Histidine
Lysine Hydrochloride
Peptide Chain Initiation, Translational
Elongation Factor
Peptide Initiation Factors
Peptide Biosynthesis
Polypeptides
Chimeric Proteins, Recombinant

Related Feeds

CREs: Gene & Cell Therapy

Gene and cell therapy advances have shown promising outcomes for several diseases. The role of cis-regulatory elements (CREs) is crucial in the design of gene therapy vectors. Here is the latest research on CREs in gene and cell therapy.

Related Papers

Analytical Biochemistry
Anthony C ForsterStephen C Blacklow
Methods : a Companion to Methods in Enzymology
Zhongping TanAnthony C Forster
Proceedings of the National Academy of Sciences of the United States of America
Anthony C ForsterStephen C Blacklow
© 2021 Meta ULC. All rights reserved