Name: isolation of ribosome bound nascent polypeptides in vitro to identify translational pause sites along mrna
Uploaded: 2012-07-06T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA at JoVE.com

Dette arbejde blev finansieret af Human Frontier Science Program tilskud RGP0024.

1. DNA-template Fremstilling og in vitro-transskription <ol><li> Genet af interesse klones under T7 og / eller f.eks SP6 transkriptionel promotor. </li><li> Til in vitro-transkription af template-DNA lineariseret med et passende restriktionsenzym skære nedstrøms for ORF stopkodon og / eller mRNA 3&#39;-enden. Et behov for at kontrollere fuldstændig linearisering af plasmid-DNA ved at køre restriktionsfordøjelse produktet agarosegelelektroforese. </li><li> Det lineariserede plasmid anven...

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For reproducerbare resultater, og koncentration af de komponenter, der anvendes til in vitro-transkriptions-og translationsreaktioner er kritiske. I den aktuelle undersøgelse har vi anvendt kommercielt tilgængelige kits og ekstrakter, der tilvejebringer meget reproducerbare data, hvis den håndteres forsigtigt. Imidlertid kan translation-kompetente ekstrakter kan fremstilles ud fra cellen af ​​et valg, hvis det er nødvendigt. Kvaliteten af mRNA kan påvirke translation, så er det af yderste vigtighed fo...

<table border="1"><tbody><tr><td> Navnet reagens / Kit </td><td> Firma </td><td> Katalog nummer </td></tr><tr><td> MEGAscript T7 Højt udbytte Transcription Kit </td><td> Ambion </td><td> AM1333 </td></tr><tr><td> Ribonucleaseinhibitor </td><td> Invitrogen </td><td> 15518012 </td></tr><tr><td> Trans [35S]-Label </td><td> MP Biomedicals </td><td> 0151006 </td></tr><tr><td> Ribonuklease-A </td><td> Invitrogen </td><td> 12091 </td></tr><tr><td> Kanin-reticulocytlysat System nuklease Behandlet </td><td> Promega </td><td> L4960 </td></tr><tr><td> E. coli S30 Extract System for lineære skabeloner </td><td> Promega </td><td> L1030 </td></tr><tr><td> Centrifugering </td><td> Beckman Coulter </td><td> Optima TLX Ultracentrifuge </td></tr><tr><td> Opbevaring fosfor Autoradiografieny </td><td> GE Healthcare </td><td> Typhoon 9410 variabel tilstand imager </td></tr><tr><td> Software til spirende polypeptid analyse </td><td> GE Healthcare </td><td> Billede Quant TL, v2005 </td></tr></tbody></table>

isolation of ribosome bound nascent polypeptides in vitro to identify translational pause sites along mrna

Hastigheden af ​​translationelle forlængelse er ikke ensartet. mRNA sekundær struktur, codonanvendelsen og mRNA associerede proteiner kan ændre ribosom bevægelse på meddelelsen for review se 1. Men er det nu bredt accepteret, at synonyme codonanvendelsen er den primære årsag til ikke-ensartede translationelle brudforlængelse vækst1. Synonyme kodoner ikke anvendes med samme frekvens. En forspænding findes i anvendelsen af synonyme kodoner med nogle codons, der anvendes oftere end andre 2. Kodon-bias er organisme, samt vævsspecifikke 2,3. Desuden frekvens kodonanvendelse er direkte proportional med koncentrationen af beslægtet tRNA&#39;er 4. Således vil en hyppigt anvendt kodon har højere mængde af tilsvarende tRNA&#39;er, som yderligere antyder, at en hyppig kodon vil blive omsat hurtigere end en sjælden én. Således vil regionerne på mRNA beriget i sjældne codons (potentielle pause-steder), som hovedregel bremse ribosom bevægelse på Message og forårsage akkumulering af nascente peptider fra de respektive størrelser 5-8. Disse pause-steder kan have funktionel effekt på proteinekspression, mRNA-stabilitet og proteinfoldning for en oversigt se 9. Faktisk blev det vist, at lettelse af sådanne pause-steder kan ændre ribosom bevægelse mRNA og efterfølgende kan påvirke effektiviteten af co-translationel (in vivo) proteinfoldning 1,7,10,11. At forstå processen af proteinfoldning in vivo, i cellen, der i sidste ende koblet til processen af proteinsyntese er det vigtigt at opnå omfattende indsigt i virkningen af kodonanvendelse / tRNA indhold på bevægelsen af ribosomer langs mRNA under translationel brudforlængelse . Her beskriver vi en enkel teknik, der kan anvendes til at lokalisere større oversættelse pause-steder for et givet mRNA translateres i forskellige cellefrie systemer 6-8. Denne procedure er baseret på isolering af spirende polypeptider accumulating på ribosomer under in vitro-translation af et mål-mRNA. Rationalet er, at lavfrekvente kodoner, stigningen i opholdstiden af ribosomerne resulterer i forøgede mængder af nascente peptider til de tilsvarende størrelser. In vitro-transkriberet mRNA anvendes til in vitro translationelle reaktioner i nærvær af radioaktivt mærkede aminosyrer at tillade påvisning af spirende kæder. For at isolere ribosom bundne nascente polypeptidkomplekser oversættelsen reaktionen lag oven på 30% glycerol-opløsning efterfulgt af centrifugering. Spirende polypeptider i polysomalt pellet behandles yderligere med ribonuclease A og adskilt ved SDS PAGE. Denne teknik kan potentielt anvendes til hvilket som helst protein og tillader analyse af ribosomet bevægelse langs mRNA og påvisning af de store pause-steder. Derudover kan denne protokol tilpasses til at studere faktorer og forhold, der kan ændre ribosom bevægelse og dermed potentielt kan også ændre the funktion / konformation af proteinet.

Watch this Scientific Journal Video about Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA at JoVE.com

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

En teknik til at identificere translationelle pause-steder på mRNA, er beskrevet. Denne procedure er baseret på isolering af nascente polypeptider akkumuleres på ribosomer under in vitro-translation af et mål-mRNA, efterfulgt af størrelses-analyse af nascente kæder under anvendelse af en denaturerende gelelektroforese.

Isolering af ribo Bundet Gryende polypeptider<em&gt; In vitro</em&gt; Til at identificere Translationelle Pause steder langs mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

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Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

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