Potential RNA-binding sites of PlzA
The recently solved crystal structure of holo -PlzA reveals a
bilobed protein of small N - and C -terminal β-barrels
connected by a short linker region (58). The antiparallel seven-stranded
β-barrels in the N - and C -terminal domains are under 100
amino acids each and have multiple aromatic and basic side chains (Fig.
S5). Small β-barrel domains (SBB) are characterized by their
β-strand-rich secondary structure and their small size, less than
approximately 100 amino acids. Proteins containing SBB domains are
functionally diverse and can bind to DNA, RNA and proteins (90). TheE. coli major cold shock protein CspA is an SBB protein that
binds RNA along one surface and is proposed to destabilize mRNA
secondary structures at low temperatures (91). The RNA chaperone
activity of CspA is dependent on three aromatic side chains protruding
from the surface of the β-barrel, which disrupt the base pairs in an RNA
hairpin via stacking interactions (92). The surrounding basic amino
acids are also thought to contribute to the RNA chaperone activity of
CspA by compensating for the negative charges on the RNA backbone (92).
Other RNA-binding proteins also utilize the combination of aromatic and
basic amino acid side chains to bind RNA (93). PlzA has numerous
aromatic and basic amino acid side chains (Fig. 4 and Fig. S5). We
propose that the protruding aromatic amino acids are necessary for
RNA-binding and chaperone activity. The N -terminal F92 and F95
and C -terminal F168 and F218 amino acids are positioned to
interact with RNA (Fig. 4A and Fig. S5) and were targeted for
mutagenesis to investigate RNA-binding and unwinding activity (Fig. 5).
Wild-type PlzA has modest RNA antitermination activity in E.
coli , while the PlzA R145D mutant has strong RNA antitermination
activity (Fig. 3), consequently we mutated the aromatic amino acids in
an R145D background. F92 and F95 in the N -terminal domain and
F168 and F218 in the C -terminal β-barrel were mutated to alanine,
resulting in two triple-mutant plzA alleles (F92A/F95A/R145D and
F168A/F218A/R145D). We measured the circular dichroism (CD) spectra of
wild-type PlzA and the mutants to determine if the mutations affected
the structure of the protein. The CD spectra of wild type and all the
mutants are characteristic of well-folded proteins (Fig. 4B). There are
small deviations between the wild-type and mutant CD spectra, but these
differences have small effects on the secondary structure content of the
variants relative to wild-type PlzA determined from the CD spectra
(Table S4). The secondary structure content differs somewhat from that
observed for the X-ray structure of PlzA, which was obtained in the
presence of c-di-GMP. The increase in coil and the decrease in β-sheet
observed with CD data for wild type and all mutants of PlzA in the
absence of c-di-GMP are consistent with the structure of apo -PlzA
being more flexible than holo -PlzA.
The in vivo antitermination assay was performed in the RL211
strain with plasmids expressing the PlzA triple mutant proteins. We
found that E. coli expressing PlzA containing the mutated
aromatic residues were unable to grow in Cm, suggesting that these amino
acids are important for unwinding RNA and the antitermination activity
of PlzA (Fig. 5). We hypothesized that the triple mutants were unable to
antiterminate in vivo because of a lower affinity for the RNA. To
examine the role of the aromatic residues in RNA binding, we performed
filter binding assays with ssRNA, dsRNA and the trpL terminator
RNA (Fig. 5B). The data demonstrate that the triple mutants have
diminished (PlzA F92A/F95A/R145D) or abolished (PlzA F168A/F218A/R145D)
RNA binding to all the RNA substrates. Apparent disassociation constants
could not be calculated for all RNAs tested for PlzA F168A/F218A/R145D
and ssRNA and dsRNA for PlzA F92A/F95A/R145D. TheKD for PlzA F92A/F95A/R145D could be calculated
but was over sevenfold higher than wild-type PlzA for the trpLRNA (Fig. S5). Again, the KD for StpA was in the
low µM range, as expected (Fig. S5). The R145D mutant PlzA protein also
had a lower affinity for all RNAs tested compared to the wild type, but
still efficiently bound RNA (Fig. 5B and Table S5). Regardless, PlzA
R145D efficiently antiterminates transcription in vivo in the
RL211 strain. The data suggest that the reduced affinity for thetrpL RNA in vitro does not affect the antitermination
activity of the PlzA R145D mutant in vivo . Surprisingly, the data
show that PlzA binding to RNA does not necessarily result in RNA
unwinding and antitermination. Wild-type PlzA can bind RNA, but in the
presence of c-di-GMP cannot antiterminate. PlzA R145D binds RNA, albeit
with a reduced affinity, but cannot bind c-di-GMP and has
antitermination activity. However, the mutation of several key aromatic
residues in the R145D background results in abolished or diminished RNA
binding and loss of antitermination activity. Taken together, the data
suggest that PlzA must be able to bind RNA and be in its apo-form to
unwind RNA and antiterminate transcription in vivo . To our
knowledge this is the first example of a small molecule regulating the
activity of an RNA binding protein without affecting its ability to bind
RNA.
RNA chaperones use multiple RNA-binding domains or multiple copies of
the same protein for their various activities, including refolding RNA
and annealing two RNAs as a matchmaker (16). The multiple RNA-binding
sites of PlzA may facilitate binding to multiple RNAs, thus allowing
them to interact. In addition, multiple interactions with the same RNA
may allow PlzA to rearrange contacts with the RNA without releasing it.Apo -PlzA could not be crystalized and has more coil structure
when analyzed, which indicates the protein structure may be dynamic in
the absence of c-di-GMP and suggests that PlzA becomes less flexible
upon c-di-GMP binding, possibly serving as a mechanism to inhibit the
RNA unfolding activity.
RNA-binding proteins and DNA-binding proteins both rely on positively
charged and polar amino acids for interacting with RNA and DNA. However,
the chemical and structural differences of RNA and DNA result in
distinct types of interactions with these residues (94-99). Despite the
dissimilar mechanisms of RNA binding and DNA binding, numerous proteins
bind both DNA and RNA, including Hfq, StpA and H-NS (100-102). The
domains, or specific residues, required to bind RNA and DNA may be
different in a protein. For example, human ADAR1 binds both RNA and
Z-form DNA via two separate domains. The electrostatic surface of PlzA
(Fig. 4A) reveals several electropositive surfaces including a deep,
electropositive groove where c-di-GMP binds. The groove is larger than
the c-di-GMP-binding site. Perhaps this surface interacts with DNA
and/or RNA. Further experimentation is required to examine the role of
these surfaces in nucleic acid binding. Notably, our data demonstrate
that the electropositive surfaces of PlzA are not sufficient to bind RNA
as mutating two aromatic amino acids diminished or abolished RNA
binding.