Methods for Studying the Interactions of Translation Factors with the Ribosome - Biophysical Approaches to Translational Control of Gene Expression - page 82

5.1 Introduction

Translation is a multistep process involving a number of RNA and protein molecules.
At every stage, individual proteins bind to the ribosome, change their position, or
are released from the complex. Therefore, to understand the mechanisms of transla-
tion, one would need to know the structure of the translation complexes at every
step of the process. Recent breakthroughs have yielded a number of high-resolution
crystal structures of ribosomes and ribosomal subunits (reviewed in Schmeing and
Ramakrishnan 2009 ), including the first crystal structures of eukaryotic ribosomes
(Ben-Shem et al. 2010 ; Rabl et al. 2011 ). However, only a handful of structures are
available for translation factor—ribosome complexes. To a great extent, this is due
to the fact that most of these complexes are dynamic and/or unstable, making it
difficult to obtain crystals or even cryo-electron microscopy (Cryo-EM) reconstruc-
tions; although the increasing number of crystal structures of ribosomal complexes
reported in recent years is a source of optimism (reviewed in Schmeing and
Ramakrishnan 2009 ) .
The limited information about the structure of the ribosomal complexes has
always been an obstacle in our understanding of the mechanisms of translation.
Therefore, a number of alternative biochemical and biophysical approaches have
been used successfully to study the interactions of translation factors with the ribo-
some and determine their approximate binding sites. These include cross-linking,
footprinting, directed hydroxyl radical probing, and more recently fluorescence
resonance energy transfer (FRET). The goal of this chapter is to provide a descrip-
tion of these methods, the information obtained with them, their limitations, and
how they complement the results from Cryo-EM and crystallography. Other binding
assays, such as ultracentrifugation or fluorescence anisotropy, which do not provide
information about the ribosomal location of the proteins, are not discussed.

5.2 Cross-Linking

5.2.1 Method Description

Chemical and UV cross-linking has been used for several decades to determine the
positions of ribosomal proteins, translation factors, and RNAs on the ribosome
(reviewed in Fraser and Doudna 2007 ; Green and Noller 1997 ) . Individual compo-
nents of molecular complexes directly interact with one another, or at least close in
space, are linked with covalent bonds using a compound that reacts with both mol-
ecules simultaneously (chemical cross-linking) or through direct covalent bond for-
mation induced by UV irradiation (UV cross-linking). Depending on the specific
reagent used, protein-protein, protein-RNA, or RNA-RNA cross-links can be
observed. Multiple factors are taken into consideration in selection of an appropriate
cross-linking reagent. For example, if an interaction involves a surface rich in Lys
and Arg and a surface rich in Asp and Glu, then a bifunctional cross-linker that can