Download Prokaryotic Regulatory RNAs Cole Franks Proteins have been

Prokaryotic Regulatory RNAs
Cole Franks
Proteins have been known to have regulatory actions for many years.
Prokaryotic regulatory proteins are particularly well understood; allosteric
enzymes have been known since the 1960’s to carry out negative feedback. It seems,
however, that proteins are far from the whole regulatory story. Evidence has been
compiling for regulation by RNA itself. Most are familiar with the idea that DNA is
transcribed to a single strand of mRNA, which is then translated to proteins by
ribosomes. RNA structures such as small RNA, riboswitches, T boxes, and
translation mediated transcription attenuation mechanisms have recently been
shown to have an undeniable contribution to regulation of the prokaryotic genome.
They also have some analogs in the eukaryotic genome. These RNA structures
suggest that researchers have been overlooking a great deal of the picture when
seeking to understand regulation, since they behave quite differently than most
regulatory proteins. They function post-transcriptionally or by attenuating
transcription, and are often attached to the mRNAs they regulate. They also provide
further support for the RNA world hypothesis - the idea that RNA carried out more
complex functions in organisms before protein evolved. This paper will provide an
overview of some of these RNA structures in prokaryotes.
The impact of post-transcriptional regulation is much more widespread than
originally supposed. Nearly fifty-four percent of the proteins in the Salmonella
proteome are indirectly or directly affected by small RNA regulation (Ansong et al.
2009). The aforementioned study was conducted by proteomic analysis after
knocking out (disabling) the Hfq protein. Hfq is a protein known as an RNA
chaperone, and is crucial for the function of some small RNAs. Most small RNAs
(sRNAs) function by base pairing with mRNAs, causing the mRNAs to be degraded
and/or preventing their translation. Hfq is so crucial because it binds single
stranded RNA that is rich in AU (adjacent adenine and uracil nucleotides); it
therefore binds with sRNA and aids in its intracellular stability and its binding to
target mRNA (reviewed in Vogel, 2009). Hfq, a hexameric ring protein, works in
several ways. It remodels sRNAs to remove structures which would inhibit binding
with mRNA, modulates sRNA levels, protects sRNAs from degradation until they
manage to base pair with an mRNA, and may even serve to recruit RNA degradation
machinery to break down sRNA-mRNA complexes (reviewed in Waters and Storz,
2009). The hfq knockout shows the sheer importance of RNA regulation.
The mechanisms of sRNA action are quite variable. Some even bind proteins
rather than mRNA. CsrA is an RNA binding protein that binds to mRNA to regulate
the translation of proteins involved in carbon usage. Two sRNAs, CsrB and CsrC,
bind this protein to prevent it from contacting the 5’ untranslated region of the
mRNA so it cannot affect translation (reviewed in Babitzke and Romeo, 2007). Most
sRNAs, however, bind a complementary or partly complementary mRNA. There are
two major varieties of such base pairing sRNA. Cis encoded antisense sRNA
(encoded from the same region of DNA) is often encoded from the complementary
DNA strand opposite the target RNA, and has long stretches of complementary base
pairs (reviewed in Brantl, 2007). Cis-encoded sRNA does not usually need hfq to
help it anneal with the target mRNA; it anneals more easily because of how
thoroughly complementary it is to the target. Though the sRNA and its target mRNA
are encoded from the same stretch of DNA, they act as two separate molecules in the
cell. In plasmids and transposons, they function to maintain the appropriate number
of copies of the mobile element. The cis-encoded sRNAs use several mechanisms to
prevent the mobile element, which is a piece of DNA that has been transferred from
another part of the organism’s chromosome or from another organism, from being
copied too many times. Two of these mechanisms are the inhibition of replication
primer formation and transposase translation. Primers are short sequences
required to replicate regions of DNA, and transposase “cut and pastes” transposons
to other parts of the genome. Cis encoded sRNAs from the other parts of the
bacterial chromosome are not well understood. Some of these regulate proteins that
are necessary, but are toxic in high amounts. Others affect the expression of genes
involved in operons (reviewed in Waters and Storz, 2009)
Trans encoded sRNA action is better understood than that of cis encoded
sRNA. These sRNA have only limited complementarity, and are functionally
analogous to miRNA in eukaryotes. Trans encoded sRNAs often bind to the 5’
untranslated region (the region of the mRNA upstream from the coding region) and
cover up the ribosome binding site, which is a sequence of base pairs known as the
Shine-Dalgarno sequence. After they bind to the mRNA, RNase-E degrades the
sRNA-mRNA complex. Another mechanism is activation of translation by an antiantisense mechanism, in which the binding of the sRNA to the mRNA disrupts a
secondary inhibitory structure on the mRNA and allows it to be expressed. Because
these have limited complementarity, they only require a small core of base pairings
to function, and can have multiple interactions. While usually these sRNAs require
Hfq to anneal, they may also function when there are high concentrations of the
sRNA. Since these sRNA are trans encoded, there is little correlation between their
location in the genome and the location of their target gene. They are often
synthesized by prokaryotes under very strict growth conditions (reviewed in
Waters and Storz, 2009).
Since mainly trans encoded sRNAs requires Hfq, it is likely that in knockout
studies on hfq, the main type of sRNA tested is trans-encoded sRNA. Salmonella is a
well understood gram negative bacteria which has recently been studied as a model
organism for RNA mediated regulation. When hfq was knocked out in Salmonella
under varying growth conditions, the bacteria had a decreased growth rate, had
decreased virulence, and decreased ability to replicate inside macrophages, a type of
white blood cell (reviewed in Ansong et al. 2009). Hfq knockout altered expression
in about fifty percent of Salmonella’s proteins when tested across several media
conditions. The impacts were widespread; Hfq was crucial in dealing with stressful
growth conditions, multiple kinds of metabolism, lipopolysaccharide synthesis
(essential for virulence in Gram negative bacteria), carbon usage, and other
processes. Since transcripts of mRNA for all tested proteins remained the same in
hfq knockouts, trans-encoded sRNAs must have a huge impact on posttranscriptional regulation (Ansong et al. 2009).
There are several other noteworthy mechanisms of RNA post-transcriptional
and transcription attenuation regulation. One was discovered in examinations of the
Btub and Cob operons in E. Coli and Salmonella, respectively. These operons are
involved in the synthesis of B12 coenzyme, which is needed to use vitamin B12. It
was previously known that these operons have feedback relationships with
coenzyme B12, and that the 5’ untranslated regions of their mRNAs were required
for this feedback to work. However, no protein was implicated in interactions with
coenzyme B12 and the mRNA. It was also noteworthy that the 5’ untranslated
regions of the mRNAs from the operons reorganized themselves by spontaneous
cleavage in the presence of coenzyme B12. Analysis showed that there was actually
direct binding of coenzyme B12 by these mRNAs (Mandal and Breaker, 2004). This
mechanism is known as a riboswitch – a structure in the 5’ untranslated region of
mRNA that detects the metabolite coded for by the mRNA and alters the structure of
the 5’ untranslated region to affect translation or transcription of the RNA.
Riboswitches typically have two domains (Figure 1). The aptamer domain
recognizes the target molecule, and the expression platform reorganizes the 5’
untranslated region of the mRNA in response to the binding of the metabolite. There
are two main mechanisms used by the expression platform. The first regulates RNA
transcription by forming an intrinsic terminator stem. An intrinsic terminator is a
stem loop structure that is usually followed by six or more U residues. This
structure causes RNA polymerase to abort transcription before the coding portion
has been made. Put simply, the aptamer domain is transcribed, and if the metabolite
is present the riboswitch binds the molecule and rearranges its structure to prevent
the coding region of the mRNA from being transcribed. When there are low
concentrations of metabolite, the unbound aptamer domain allows the formation of
an antiterminator stem. The formation of this stem prevents the terminator stem
from forming, and allows the mRNA to be fully transcribed. The other mechanism
operates by altering translation initiation. When the metabolite binds to the
aptamer domain, the expression platform alters the 5’ untranslated region to keep
the ribosome from accessing the ribosome binding site and prevents translation.
There is also a possibility that if the terminator stem were formed by base pairing
with the ribosome binding site, transcription and translation could be controlled
simultaneously – the terminator stem would stop any more mRNA from being
transcribed, and any that had passed the point of transcription termination could
not be translated (Mandal and Breaker, 2004) These mechanisms are common to
the coenzyme B12 riboswitch, but evidence exists that other riboswitches share
these mechanisms (reviewed in Mandal and Breaker, 2004). Riboswitches appear to
have diverse structures and mechanisms; in some cases they may even act as ON
switches instead of OFF switches (Mandal and Breaker, 2004).
Two other mechanisms of transcription-attenuation regulation by
prokaryotes are translation mediated transcription attenuation mechanisms and T-
boxes. Translation mediated transcription attenuation mechanisms use the speed of
translation, which is determined by the concentration of aminoacylated tRNA, to
prevent or facilitate the formation of a terminator stem. They usually affect amino
acid operons because in the leader regions of the operon being regulated there are
several adjacent codons that code for the pertinent amino acid (Figure 2). If the
ribosome races down these codons, indicating a high concentration of
aminoacylated tRNA, the terminator forms in the leader transcript. If the ribosome
stalls, the nascent mRNA that was transcribed during the stalling folds to form an
antiterminator stem, which prevents the formation of a terminator stem. This is the
mechanism used by many Gram negative prokaryotes, with some variation. In the
tryptophan operon in E. Coli, there is also a pause hairpin. This pause hairpin is
crucial – it allows the stalled ribosome to catch up with transcription. The pause
hairpin, like the antiterminator, prevents the terminator stem from forming. It
forms while the ribosome stalls and prevents further transcription (reviewed in
Henkin and Yanofsky, 2002).
T-boxes are hairpin structures in the 5’ untranslated region of mRNA (Figure
3). They regulate amino acid operons in Gram positive bacteria. This structure has a
specifier hairpin, which has an anti-anti codon. This is a codon complementary to
the anticodon in the tRNA of the amino acid for the particular operon. This anti-anti
codon binds to the tRNA, which in turn causes the mRNA to form an antiterminator
stem by interaction of a 5’UGGN3’ sequence in the 5’ untranslated region and the
complementary sequence on the 3’ end of the tRNA (Putzer et al. 1995, Yousef et al.
2005). Since the tRNA is unbound, this occurs when there are low concentrations of
the required amino acid. The binding of the tRNA can also allow an antisequester of
the ribosome binding site to form. An antisequester is a structure that forms to
prevent another structure (a sequester) from folding the ribosome binding
sequence in a fashion that would be inaccessible to the ribosome. Put in simple
geometric terms, without tRNA binding two hairpin structures would already form
in the T-box upon transcription. When the tRNA binds to the specifier hairpin,
however, it “reaches” over and forms either an antiterminator or an antisequester
that prevents a terminator or a sequester from forming (Vitreschak et al. 2008).
These relatively newfound RNA structures show that RNA regulation
deserves at least as much, if not more, attention than protein regulation. Not only
can RNA attach to transcribed mRNA to cause degradation and prevent
transcription, but it can also form built-in structures in the leading ends of
transcripts to halt transcription and/or translation. These structures are also clearly
important pieces of evidence in the argument for RNA world. Complex folded RNA
structures suggest that at some point in time RNA could have had many more
functions. How has non-coding RNA changed over time? What other functions can
RNA have? What are the full impacts of these functions? Small RNA, riboswitches, Tboxes, and translation mediated transcription attenuation mechanisms raise many
such questions. While many researchers continue to provide answers, what we still
do not understand about RNA may be crucial to our understanding of life.
Figure 1 (Mandal and Breaker, 2004)
A)
This is the transcription attenuation mechanism of a riboswitch. The
terminators and antiterminator are in red, and one can see how the
antiterminator binds with one of the stretches of terminator sequence when
nothing binds with the aptamer domain. This prevents the terminator stem
from forming, hereby maintaining transcription.
B)
This is the mechanism in which the ribosome binding site, or ShineDalgarno sequence, is sequestered in high concentrations of the metabolite.
The RBS is in green, and the sequester and antisequester are in red.
Figure 2 (Henkin and Yanofsky, 2002)
Termination: High concentration of aminoacylated tRNA allows normal translation
to occur, so a terminator forms.
Antitermination: The ribosome stalls because of the low aminoacylated tRNA
concentration while transcription continues. The nascent mRNA folds to
form an antiterminator.
Figure 3 (Vitreschak et al. 2008)
A) In A and B, variants of the transcription attenuation mechanism
of a T-box are shown. The uncharged tRNA allows an
antiterminator to form, preventing termination of transcription.
B) In C, the uncharged tRNA binds with the specifier hairpin and
forms an antisequester, allowing translation.
References:
Mandal, M., & Breaker, R.R. (2004). Gene regulation by riboswitches.
Nature Reviews:Molecular Cell Biology , 5. Retrieved from
http://rnaworld.bio.ku.edu/reprints/main/Mandal(2004c)Nature_
Rev_Molec_Cell_Biol _5,451.pdf
V itreschak, A.G. et al. (2008). Comparative genomic analysis of t-box
regulatory systems in bacteria. RNA, 14. Retrieved from
http://rnajournal.cshlp.org/content/14/4/717.full
Henkin, T. M., & Yanofsky, C. (2002). Regulation by transcription
attenuation in bacteria : how rna provides instructions for
transcription termination/antitermination decisions. BioEssays,
24(8), Retrieved from
http://rnaworld.bio.ku.edu/reprints/incoming/_misc/+Henkin()B
ioEssays_24,700.%5BYanofsky,attenuation%5Dpdf.pdf
Ansong, C. et al. (2009). Global systems -level analysis of hfq and smpb
deletion mutants in salmonella : implications for virulence and
global protein translation. Plos one, Retrieved from
http://www.ncbi.nlm.nih.gov/pubmed/19277208
Vogel, J. (2009). A rough guide to the non-coding rna world of
salmonella. Molecular Microbiology, Retrieved from
http://www.ncbi.nlm.nih.gov/pubmed/19277208
Waters, L. S., & Storz, G. (2009). Regulatory rnas in bacteria. Cell,
136(4), Retrieved from http://www.cell.com/abstract/S0092 8674(09)00125-1
Babitzke, P., and Romeo, T. (2007). CsrB sRNA family: Sequestration of RNAbinding regulatory proteins. Curr. Opin. Microbiol. 10, 156–163.
Brantl, S. (2007). Regulatory mechanisms employed by cis-encoded antisense
RNAs. Curr. Opin. Microbiol. 10, 102–109.
Yousef, M.R., Grundy, F.J., and Henkin, T.M. 2005. Structural
transitions induced by the interaction between tRNAGly and the
Bacillus subtilis glyQS T box leader RNA. J. Mol. Biol. 349: 273–
287.
Putzer, H., Laalami, S., Brakhage, A.A., Condon, C., and GrunbergManago, M. 1995. Aminoacyl-tRNA synthetase gene regulation in
Bacillus subtilis: Induction, repression and growth-rate regulation.
Mol. Microbiol. 16: 709–718.