Methods for Studying the Interactions of Translation Factors with the Ribosome - Biophysical Approaches to Translational Control of Gene Expression - page 83

bridge an amino and a carboxyl groups would be appropriate. Cross-linking reagents
have variable spacer lengths. UV cross-linking involves no spacer and is thus zero-
length cross-linking. A cross-linking agent with a long spacer (long distance between
its two reactive sites) can bridge the distance between reactive groups located on
two different molecules, yielding higher efficiency of cross-linking. Increased cross-
linker length can also allow cross-linking of molecules that are not in direct contact
with each other, leading to lower specificity. It should be noted however that
specificity may not be a major issue with long-length cross-linkers because com-
parisons of cross-linking data with the crystal structure of the ribosome did not find
significant correlation between the length of the cross-linker and the distance
between the cross-linked species within the ribosome (Whirl-Carrillo et al. 2002 ) .

5.3 Limitations and Challenges

Any two noninteracting proteins can be nonspecifically cross-linked to each other if
mixed at high enough concentration and under appropriate conditions, just from
random collisions. Therefore, proper controls are required to avoid false positive
results. Conversely, absence of cross-linking is not in itself proof that two molecules
do not interact with each other.
With the ribosomal complexes containing large ribosomal RNAs (rRNAs) and
tens of proteins, identifying the cross-linked molecules and the specific positions
can be challenging. Cross-links in proteins can be identified using 2D gels and Mass
Spectrometry. The exact position of cross-links in RNA are easier to determine
using reverse transcriptase (RT) since the cross-links block the enzyme, causing
termination in front of the cross-link, in the same way as footprinting (see below).
Several approaches are used to simplify the task of identifying cross-links in com-
plex mixtures:

- Adding an affinity tag on a specific molecule in order to be able to isolate cross-

linked species that contain it and/or labeling the molecule in order to be able to
detect the complexes.
Using reversible cross-linkers or cleavable cross-linkers. The latter type can be

-

used to transfer a label (e.g. biotin) to the molecules that were cross-linked, in
order to aid in their further identification.
Using an experimental setup where a specific molecule is activated for cross- -

linking. This can be achieved by pretreatment of a protein with a bifunctional
cross-linker before assembling the ribosomal complexes, ensuring that the only
cross-linkers available in the reaction mix are already covalently attached to the
molecule of interest. For RNAs, specific cross-linking can be achieved by incor-
porating modified nucleotides, such as 4-thiouracil (4-thioU) or 6-thioguanine
(6-thioG) at selected positions. Cross-linking with halogenated nucleotide ana-
logs can be performed using longer-wavelength UV light (360 nm) that does not
affect the protein or RNA backbone, both ensuring specific cross-linking and
minimizing damage to the interacting molecules (Favre et al. 1998 ) .