Susan Gottesman (NCI)
Small Regulatory RNAs as Switches for Bacterial Stress Responses
The sequencing of the genome of many organisms gave us a reasonable estimate
of the number of proteins it encodes. However, only recently have we realized some of
the additional important regulators hidden in the DNA. In Escherichia coli, more than 50
small RNAs, each about 100 nucleotides in size, have now been found. These noncoding RNAs are encoded in the spaces between protein coding genes. They are
generally conserved in related bacteria. Most importantly, the subset that have been
studied in some detail play unsuspected and important regulatory roles, turning sets of
genes on and off in response to stress signals and environmental changes. These small
regulatory RNAs resemble in biological action the microRNAs of eukaryotes. Thus, both
bacteria and eukaryotes use small non-coding RNAs as critical regulatory molecules.
The bacterial regulatory RNAs are made from their own promoters, and, unlike
the eukaryotic microRNAs, generally are not processed. A large family of these
regulatory RNAs act by pairing to target mRNAs, aided by an RNA chaperone called
Hfq. I will describe the action of three different small RNAs of this family that provide
examples of some of the critical regulatory events these small RNAs carry out and how
they do it.
The first two small RNAs, called DsrA and RprA, positively regulate the
translational of a major developmental switch in bacteria, the RpoS or sigma 38 protein, a
sigma factor. RpoS protein is made when cells enter stationary phase or when they are
subjected to many different stresses; the protein combines with the core RNA polymerase
and redirects it to the genes the cells needs to survive and recover from stationary phase
and/or stress. The cell regulates the abundance of RpoS in many ways, at the level of
synthesis and at the level of protein stability. Here, I will focus on regulation at the level
of synthesis. Translation of RpoS is normally occluded by an inhibitory hairpin that folds
back onto the ribosome-binding site. It was known that translation increased in response
to stress, but it was unclear how. This is where the small RNAs provided an unexpected
answer. Both DsrA and RprA are complementary to the inhibitory arm of the hairpin of
the rpoS upstream mRNA. If one of these small RNAs is synthesized at a high level, it
pairs with the inhibitory arm of the hairpin, removing it from action. This frees the
ribosome binding site, allowing high level of RpoS translation.
Why are there two (and probably more) small RNAs that regulate the same
target? One part of the answer is that each of these small RNAs is made under a different
stress condition. DsrA is made at low temperatures, and is necessary for RpoS to be
made at low temperature. This response is important for allowing the cell to survive at
these low temperatures. The second small RNA, RprA, is made in response to cell
surface stress. This activates a phosphorelay signaling system, resulting in activation of
synthesis of a number of genes, including RprA. When RprA is made, that results in
RpoS synthesis. Thus, the cell can sense and integrate many different stress signals by
using a different small RNA to coordinate the response to each signal. In addition, the
RpoS example demonstrates that small RNAs can not only interfere with gene expression
but can stimulate it.
The third example is a small RNA identified by its high conservation in
Salmonella, Klebsiella, Vibrio, and Yersinia; it was named RyhB. It is regulated in
response to iron depletion. Bacterial cells, and pathogens in particular, are known to
carefully regulate their iron levels. Too much iron can lead to oxygen-induced damage to
cellular components; too little iron is lethal. During infections, bacteria must obtain iron
from their hosts, not always an easy task. A repressor, Fur, that helps the cell to regulate
iron has been studied for many years. Fur represses genes involved in iron assimilation
when iron is abundant; when iron is depleted, Fur is inactive as a repressor and the cell
makes the iron transport proteins it needs. We have found that RyhB controls another
level of iron homeostasis. RyhB is repressed by Fur in the classic way. Thus RyhB is
only made when cells are devoid of iron. When this small RNA is made, it pairs with and
causes the rapid degradation of the mRNAs for genes that encode non-essential ironbinding proteins. Thus, the cell shuts down synthesis of some iron-binding proteins,
sparing any iron that is available for the essential enzymes. At least 10 different operons
are regulated in this fashion by RyhB. This RNA can significantly change the iron usage
of the cell very rapidly. It is present in many pathogens, and similarly regulated small
RNAs with parallel functions can be found in yet other pathogens, suggesting an
important role for this small RNA in pathogenesis.
Every regulatory switch needs to be turned off as well as on. Studies of RyhB
have led to an insight into how these small RNAs are turned off; this mechanism may
apply in eukaryotes as well. We find that RyhB, normally a relatively stable RNA, is
rapidly degraded when it can pair with its target mRNA. Therefore, we believe that the
small RNAs in this family are destroyed as they are used.
Studies in E. coli have provided new insights into how to find small RNAs, how
abundant they are, and the range of important regulation they are responsible for. We
can currently estimate that the number of small regulatory RNAs is about 2% of that of
protein coding genes. Most probably every important stress response will include at least
one small RNA, and developmental switches may utilize multiple such RNAs. Clearly
this layer of regulation may be as significant and as complex as the transcriptional
regulation we are more familiar with and small RNAs may provide new antibiotic targets
if they prove to have the expected central roles in pathogenesis.
One-paragraph bio:
Susan Gottesman received her BA and Ph.D. at Harvard University and has been
an investigator in the Laboratory of Molecular Biology at the National Cancer Institute
since 1976. Her investigations on the regulation of gene expression by energy-dependent
proteases in bacteria led to her election to the National Academy of Sciences in 1998.
More recently, her lab has focused on the role of small RNAs in bacterial regulatory
circuits and approaches to identifying small RNAs in genomes.