Methods for Studying the Interactions of Translation Factors with the Ribosome - Biophysical Approaches to Translational Control of Gene Expression - page 84

5.3.1 Applications

Cross-linking with halogenated nucleotide analogs was used successfully to study
the interactions of mRNA with the mammalian ribosome and components of the
translation initiation complex (Pisarev et al. 2006, 2008 ). In eukaryotes, to be
efficiently recognized by the translation initiation machinery, the start codon needs
to be in an optimum sequence context: GCC(A/G)CCAUGG (Kozak 1986 ) . The
nucleotides at positions −3 and +4 (where the A in AUG is +1) are the most impor-
tant determinants. Cross-linking using mRNA containing 4-thioU or 6-thioG at
position −3 or +4 allowed identifying nucleotides in 18S rRNA and ribosomal pro-
teins interacting with the mRNA at these positions, with some of the interactions
being dependent on, or modulated by the nature of the nucleotide at these mRNA
positions. In addition to ribosomal components, eukaryotic translation initiation
factor (eIF) 2a was also efficiently cross-linked to mRNA at position −3. The
authors showed that eIF2a has a role in recognition of the proper sequence context,
thus obtaining information both about the ribosomal position of eIF2a and its func-
tion (Pisarev et al. 2006 ). The same approach was also used to determine the path of
mRNA in the eukaryotic 48S complex (Pisarev et al. 2008 ) . Cross-linking in 48S
ribosomal complexes with a series of model mRNAs with 4-thioU incorporated at
specific positions showed that the mRNA path in eukaryotic 48S initiation com-
plexes is very similar to that in elongating bacterial 70S ribosomes (Selmer et al.
2006 ; Yusupova et al. 2001 ). Two eIF3 subunits, eIF3a and eIF3d, were found to
cross-link to mRNA upstream from the start codon and extend the mRNA Exit
channel on the 40S ribosomal subunit (Pisarev et al. 2008 ) . Footprinting and
hydroxyl radical probing (see below) showed that eIF3 also interacts with 18S
rRNA helix 16 (h16) near the mRNA Entry channel of the small ribosomal subunit
(Pisarev et al. 2008 ), which together with yeast two-hybrid assays, and in vitro bind-
ing data (Chiu et al. 2010 ; Elantak et al. 2010 ; Valasek et al. 2003 ) identi fi ed several
points of contact between eIF3 subunits and ribosomal components spanning the
solvent surface of the 40S subunit between the mRNA Entry and Exit channels.
Analysis of these results in the context of the Cryo-EM reconstruction of eIF3
(Siridechadilok et al. 2005 ) provided valuable information about the overall orien-
tation of eIF3 on the 40S ribosomal subunit.

5.3.2 Outlook

In summary, cross-linking can be used to identify pairs of molecules that are in close
proximity on the ribosome or directly interacting with each other. Observing cross-
links between a translation factor and a ribosomal component or another translation
factor allows mapping that protein's approximate binding site in ribosomal com-
plexes. Where the exact positions of the cross-links can be determined, the approach
also provides some information about the orientation of the bound translation factor.