Fast in vitro translation system immobilized on a surface via specific biotinylation of the ribosome

Biological Chemistry
Romualdas StapulionisCharlotte R Knudsen

Abstract

The ribosome is the macromolecular machine responsible for translating the genetic code into polypeptide chains. Despite impressive structural and kinetic studies of the translation process, a number of challenges remain with respect to understanding the dynamic properties of the translation apparatus. Single-molecule techniques hold the potential of characterizing the structural and mechanical properties of macromolecules during their functional cycles in real time. These techniques often necessitate the specific coupling of biologically active molecules to a surface. Here, we describe a procedure for such coupling of functionally active ribosomes that permits single-molecule studies of protein synthesis. Oxidation with NaIO4 at the 3' end of 23S rRNA and subsequent reaction with a biotin hydrazide produces biotinylated 70S ribosomes, which can be immobilized on a streptavidin-coated surface. The surface-attached ribosomes are fully active in poly(U) translation in vitro, synthesizing poly(Phe) at a rate of 3-6 peptide bonds/s per active ribosome at 37 degrees C. Specificity of binding of biotinylated ribosomes to a streptavidin-coated quartz surface was confirmed by observation of individual fluorescently labeled, biotinylate...Continue Reading

References

Jan 1, 1974·Methods in Enzymology·S Rappoport, Y Lapidot
Jul 1, 1980·Analytical Biochemistry·P C Jelenc
Sep 1, 1981·Proceedings of the National Academy of Sciences of the United States of America·M Stöffler-MeilickeB Hardesty
Feb 1, 1982·European Journal of Biochemistry·E G WagnerC G Kurland
Aug 1, 1995·Proceedings of the National Academy of Sciences of the United States of America·R Stapulionis, M P Deutscher
Mar 14, 1995·Proceedings of the National Academy of Sciences of the United States of America·M V Rodnina, W Wintermeyer
Jan 8, 1999·Journal of Molecular Biology·A SytnikR M Hochstrasser
Feb 19, 2000·Current Opinion in Biotechnology·R L Rich, D G Myszka
Aug 11, 2000·Biological Chemistry·M V RodninaW Wintermeyer
Dec 7, 2000·Progress in Biophysics and Molecular Biology·J N ForkeyY E Goldman
Jun 11, 2002·Methods : a Companion to Methods in Enzymology·Chatchawan Srisawat, David R Engelke
Jan 7, 2003·The EMBO Journal·Lev KisselevLudmila Frolova
Apr 18, 2003·Nature·Manfred Schliwa, Günther Woehlke
May 3, 2003·Current Opinion in Structural Biology·John W WeiselRustem I Litvinov
May 6, 2003·The Journal of Biological Chemistry·Andrei A LeonovOlga A Dontsova
Jul 11, 2003·Proceedings of the National Academy of Sciences of the United States of America·Antón Vila-SanjurjoJamie H Doudna Cate
Aug 23, 2003·Trends in Biochemical Sciences·Gregers R AndersenJens Nyborg
Sep 18, 2003·RNA·Francesco VanziBarry S Cooperman
Nov 19, 2003·European Journal of Biochemistry·Henrik KarringCharlotte R Knudsen
Aug 20, 2004·Proceedings of the National Academy of Sciences of the United States of America·Scott C BlanchardSteven Chu
Sep 28, 2004·Nature Structural & Molecular Biology·Scott C BlanchardJoseph D Puglisi
Oct 21, 2004·Biochemical Society Transactions·W WintermeyerM V Rodnina
Jan 5, 2005·Microscopy Research and Technique·M CapitanioF S Pavone
May 17, 2005·Molecular Cell·Frank PeskeWolfgang Wintermeyer
Jun 14, 2005·Biophysical Journal·Francesco VanziYale E Goldman
Aug 4, 2005·Methods : a Companion to Methods in Enzymology·Elaine M Youngman, Rachel Green
Jan 7, 2006·Nature·Jeff GoreCarlos Bustamante
Mar 15, 2006·Nature Structural & Molecular Biology·Dongli PanBarry S Cooperman
Feb 24, 2007·Molecular Cell·Dongli PanBarry S Cooperman
Jul 27, 2007·RNA·Thu BetteridgeYa-Ming Hou
Sep 18, 2007·Journal of Molecular Biology·Christina GrigoriadouBarry S Cooperman

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Citations

Dec 18, 2009·Journal of Molecular Recognition : JMR·Rebecca L Rich, David G Myszka
Apr 25, 2017·Scientific Reports·Noémie KempfJörg Fitter

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