RNA affinity column

Nick Theodorakis nicholas_theodorakis at urmc.rochester.edu
Fri Sep 17 10:09:50 EST 1999


In article <7rrtlk$5n0$1 at panix3.panix.com>,
  iayork at panix.com (Ian A. York) wrote:
>
> This is something I'm thinking about as a concept rather than an
> experiment for tomorrow, if you see the difference.  Let's say I have an
> RNA that I suspect of interacting with some unknown proteins.  One way of
> purifying the proteins would be to make an affinity column:  bind the RNA
> to the column, pour cell lysates over it, wash, and elute.
>

Harford, Klausner, and colleagues did this for the purification of the
IRE binding protein. They biotinylated the RNA, mixed it with cell
extract, and bound it to avidin.

---------------------------------------------------------

Proc Natl Acad Sci U S A 1989 Aug;86(15):5768-72

The iron-responsive element binding protein: a method for the affinity
purification of a regulatory RNA-binding protein.

Rouault TA, Hentze MW, Haile DJ, Harford JB, Klausner RD

Cell Biology and Metabolism Branch, National Institute of Child Health
and Human Development, Bethesda, MD 20892.

A method of affinity purification of a regulatory protein that binds
specific RNA sequences is described. RNAs containing the regulatory
sequences are transcribed in vitro from oligonucleotide templates,
biotinylated, and incubated with unfractionated cytosol. Specific RNA-
protein complexes are bound in solution to avidin, and the resulting
complex is bound to biotin-agarose beads. The cytosolic binding protein
is released from the RNA in high salt, and a second round of
purification yields an essentially homogeneous protein. Using this
method, we have identified the protein in human liver that binds iron
responsive RNA regulatory sequences. Iron-responsive elements (IREs) are
RNA stem-loops present in the mRNAs encoding ferritin and the
transferrin receptor. IREs form the basis for the translational
regulation of ferritin gene expression and the regulation of transferrin
receptor mRNA degradation rates. The IRE binding protein purified by
this technique migrates as a 90-kDa polypeptide on SDS/PAGE. The
interaction of the purified protein with IRE-containing RNAs can be
detected by gel-mobility shift assays or by covalent crosslinking
induced by UV irradiation.

PMID: 2474819, UI: 89345548

----------

There is also a yeast 3-hybrid system around, but so far I think only W.
Marzluff has gotten it to work (with a histone mRNA stem-loop binding
protein), but I could be wrong.

--
 _______________________________________________
| Nick Theodorakis                              |
| nicholas_theodorakis at urmc.rochester.edu       |
| (previously theodorn at gusun.georgetown.edu)    |


Sent via Deja.com http://www.deja.com/
Share what you know. Learn what you don't.



More information about the Methods mailing list