3.7 Protein
Production
Pure RNA and RNase-free protein (greater than 95% purity based
on SDS-PAGE analysis) are required for downstream RNA–protein
complex formation and crystallization experiments. Care must be
taken when producing recombinant RBPs and RBDs for crystallization experiments to ensure that protein samples are homogeneous and that host RNA contaminants are not carried over from
the purification. RNA contamination can be removed using a gel
filtration and desalting step to separate the larger protein from RNA
contaminants and/or anion (MonoQ, Q-Sepharose HP) or cation
(MonoS, SP sepharose) exchange chromatography. Anion
exchange will bind the RNA contaminants to the resin whereas
cation exchange will typically bind the protein with RNA contaminants eluting in the flow through. The 260/280 nm absorbance
ratio determined using NanoDrop™ (see Subheading 3.5) provides
a useful indication whether there is RNA contamination in the
protein solution during purification. The A 260 /A 280 ratio should
be 0.57 for pure proteins, whereas the ratio rises quickly if contamination exists (see Fig. 3). Furthermore, it is critical to ensure that
proteins are free from RNases, which can degrade RNA when the
RNA–protein complex is formed. The Ambion RNaseAlert
® lab
test kit provides a simple and sensitive RNase contamination assay
to evaluate chromatographic fractions (see Subheading 3.3). Subsequently, fractions identified as having RNase contamination can be
excluded from downstream purification steps.
3.8 RNA Binding
Assays
Proteins interact with RNA through electrostatic interactions,
hydrophobic interactions, hydrogen bonding, and base stacking
in a similar fashion to protein–DNA interactions. Protein–RNA
interactions are also influenced by the tertiary structure of the
RNA. Therefore, both the RNA and protein(s) must be correctly
folded in order to facilitate the correct RNA–protein interactions.
RNA is prone to degradation and care must be taken not to
introduce RNases into the reaction (see Note 1 and Subheading
3.3). The most common methods for confirming and studying
RNA–protein interactions are described below.
3.8.1 EMSA
EMSA (also known as the gel retardation assay or band shift assay)
is an in vitro technique that can be used to detect RNA–protein
interactions by monitoring the electrophoretic mobility of RNA
and to estimate kinetic parameters, e.g., dissociation constants, for
RNA–protein complexes [17]. RNA–protein complexes usually
remain intact when fractionated by gel electrophoresis, provided
that the affinity of the complex is high enough, and migrate with
reduced mobility on non-denaturing gels compared with free
(unbound) RNA (see Fig. 4; for a comprehensive method for running electrophoretic mobility shift assays, see ref. 14 and
Chapter 14). RNAs can be labeled with radioisotopes, covalent or
non-covalent fluorophores, or biotin and detected using
430
Andrew P. Turnbull and Xiaoqiu Wu
Production
Pure RNA and RNase-free protein (greater than 95% purity based
on SDS-PAGE analysis) are required for downstream RNA–protein
complex formation and crystallization experiments. Care must be
taken when producing recombinant RBPs and RBDs for crystallization experiments to ensure that protein samples are homogeneous and that host RNA contaminants are not carried over from
the purification. RNA contamination can be removed using a gel
filtration and desalting step to separate the larger protein from RNA
contaminants and/or anion (MonoQ, Q-Sepharose HP) or cation
(MonoS, SP sepharose) exchange chromatography. Anion
exchange will bind the RNA contaminants to the resin whereas
cation exchange will typically bind the protein with RNA contaminants eluting in the flow through. The 260/280 nm absorbance
ratio determined using NanoDrop™ (see Subheading 3.5) provides
a useful indication whether there is RNA contamination in the
protein solution during purification. The A 260 /A 280 ratio should
be 0.57 for pure proteins, whereas the ratio rises quickly if contamination exists (see Fig. 3). Furthermore, it is critical to ensure that
proteins are free from RNases, which can degrade RNA when the
RNA–protein complex is formed. The Ambion RNaseAlert
® lab
test kit provides a simple and sensitive RNase contamination assay
to evaluate chromatographic fractions (see Subheading 3.3). Subsequently, fractions identified as having RNase contamination can be
excluded from downstream purification steps.
3.8 RNA Binding
Assays
Proteins interact with RNA through electrostatic interactions,
hydrophobic interactions, hydrogen bonding, and base stacking
in a similar fashion to protein–DNA interactions. Protein–RNA
interactions are also influenced by the tertiary structure of the
RNA. Therefore, both the RNA and protein(s) must be correctly
folded in order to facilitate the correct RNA–protein interactions.
RNA is prone to degradation and care must be taken not to
introduce RNases into the reaction (see Note 1 and Subheading
3.3). The most common methods for confirming and studying
RNA–protein interactions are described below.
3.8.1 EMSA
EMSA (also known as the gel retardation assay or band shift assay)
is an in vitro technique that can be used to detect RNA–protein
interactions by monitoring the electrophoretic mobility of RNA
and to estimate kinetic parameters, e.g., dissociation constants, for
RNA–protein complexes [17]. RNA–protein complexes usually
remain intact when fractionated by gel electrophoresis, provided
that the affinity of the complex is high enough, and migrate with
reduced mobility on non-denaturing gels compared with free
(unbound) RNA (see Fig. 4; for a comprehensive method for running electrophoretic mobility shift assays, see ref. 14 and
Chapter 14). RNAs can be labeled with radioisotopes, covalent or
non-covalent fluorophores, or biotin and detected using
430
Andrew P. Turnbull and Xiaoqiu Wu