Frontiers in Translation
(Post-Transcriptional Regulatory Mechanisms)
David M. Bedwell, PhD
dbedwell@uab.edu
a)
Different mRNA structures that
enable different modes of
regulation
Riboswitches: Depending on the type of
riboswitch, the binding of a ligand to the
aptamer provokes activation or
repression of the downstream gene at
transcriptional or/and translational level.
b)
Thermosensors: The structural
arrangement of a thermosensor that
blocks the access of the 30S ribosomal
subunit to the translation initiation site
at low temperature. An increase of the
temperature melts the structure and
translation can occur.
c)
Non-coding RNA (ncRNA) Activation:
Trans-acting ncRNAs can activate
translation by preventing the formation
of an inhibitory structure that sequesters
the ribosome binding site (RBS).
d)
RNA binding Sites by Repressor Proteins: Proteins can interfere with translation by competing with the 30S
ribosomal subunit for binding or by trapping the 30S onto to the mRNA.
e)
Non-coding RNA Repression: Unpaired regions (in blue) of ncRNAs bind to complementary nucleotides of
target mRNAs (forming perfect or imperfect duplexes) and regulate gene expression at multiple levels:
transcriptional attenuation, translation initiation, mRNA decay and mRNA processing. Many ncRNAs bind to
the ribosome binding site (RBS) but some of them can bind mRNA far upstream of the RBS, within the coding
region, or at the 3' end of the mRNA. The mRNA structure was not schematized although it plays an essential
role in these regulations.
Geissmann et al., RNA Biol 6: 153-160 (2009)
Thermosensors
Model of the Mechanism Underlying
Thermoregulated Expression of PrfA
The prfA-UTR forms a secondary structure at low temperatures (≤30°C) masking the
ribosomal region of prfA, thus preventing the binding of the ribosome. PrfA is not
translated and virulence genes are not expressed. At high temperatures (≥37°C) the
prfA-UTR partially melts, and thereby permits binding of the ribosome to the ShineDalgarno sequence. Translation of prfA allows virulence gene expression.
Johansson et al., Cell 110: 551-561 (2002)
Non-Coding RNAs
Factors that Influence ncRNA Function in Gene Control
• RNA structure
• Base pairing
• RNA localization
• Association with the Sm-like protein Hfq
– Protects ncRNAs from RNase attack
– Enhances the association rate constant of ncRNA-mRNA complexes
– Acts as an RNA chaperone
• Other proteins?
Structures of the S. aureus Hfq and an Hfq–RNA complex
A. Structure of the Hfq hexamer with each subunit colored differently.
B. Ribbon diagram of an Hfq subunit. The Sm1 motif is colored blue and the Sm2 motif is green.
Regions outside the two motifs, i.e. the N-terminal α-helix and the variable region, are colored
yellow. Hfq residue Gly-34, the sole conserved residue among Hfq and the Sm proteins, is blocked
in red.
C. Electrostatic surface representation of the RNA binding site of the Hfq hexamer. Blue is
electropositive and red is electronegative. The RNA is shown as a stick model, with oxygen,
nitrogen, carbon and phosphorus atoms colored red, blue, white and yellow respectively. The
opposite side of the Hfq hexamer is predominantly non-polar.
Valentin-Hansen et al., Mol. Micro. 51: 1525-1533 (2004)
Mechanisms of target gene activation by sRNAs
In some mRNAs, translation efficiency of a transcript is markedly reduced due to the formation of a
secondary structure within the 5´-UTR covering the Ribosome Binding Site (RBS). Competitive binding
of a trans-encoded sRNA can induce structural rearrangements to unmask the RBS and thus increase
target translation.
Frohlich and Vogel, Curr. Opin. In Microbiol. 12: 674-682 (2009)
Mechanisms of target gene activation by sRNAs
GadY sRNA encoded on the opposite strand in the gadX–gadW intergenic
region (IGR) interacts with the 3´-UTR of gadX to stabilize this transcript.
Frohlich and Vogel, Curr. Opin. In Microbiol. 12: 674-682 (2009)
Mechanisms of target gene activation by sRNAs
While being highly similar regarding
both sequence and structure, GlmZ
(but not GlmY) contains a sequence
stretch to facilitate glmS translation
by direct base-pairing via the antiantisense mechanism.
By competing for factors promoting
GlmZ inactivation, GlmY sRNA
contributes to the maintenance of the
activating effect of GlmZ RNA on glmS
mRNA translation.
Frohlich and Vogel, Curr. Opin. In Microbiol. 12: 674-682 (2009)
Sequence conservation of glmS mRNA and GlmZ sRNA binding sites
 Sequence alignments reveal the conservation of an inhibitory stem-loop structure
within glmS mRNA among different enterobacteria.
 Activation of glmS translation by GlmZ sRNA via the anti-antisense mechanism
depends on the interaction at two sites, S1 and S2 with S1´ and S2´ respectively.
 The S1–S1´ interaction appears to be highly conserved, while the S2–S2´ pairing
seems be more heterogeneous in both sequence and position, with least
conservation in Photorhabdus.
Frohlich and Vogel, Curr. Opin. In Microbiol. 12: 674-682 (2009)
Mechanisms of target gene repression by sRNAs
In the absence of chitosugars:
The constitutively expressed ybfMN
mRNA is bound by the highly
abundant MicM sRNA.
While the interaction results in
transcript decay, MicM is recycled
and remains functional.
In the presence of inducing
chitosugars:
The inhibitory effect of MicM on
ybfMN is abrogated by the
expression of the chitobiose
operon.
An intergenic region of the
chitobiose operon (chbBCAFRG)
mRNA shares substantial
complementarity with MicM and
functions as a molecular trap for
the sRNA, leading to the
degradation of MicM and
consequent expression of ybfMN.
Frohlich and Vogel, Curr. Opin. In Microbiol. 12: 674-682 (2009)
Mechanisms of target gene repression by sRNAs
Hairpin 13 of S. aureus RNAIII forms an extended duplex with spa mRNA encoding for
protein A that most probably initiates via a loop-loop interaction. Inhibition of
translation is followed by a rapid degradation by the double-strand specific RNase III.
Degradation is probably coupled to translation repression since both events are
required for efficient regulation in vivo.
Geissmann et al., RNA Biol 6: 153-160 (2009)
Mechanisms of target gene repression by sRNAs
Hairpin 7 and 14 of S. aureus RNAIII block translation of rot mRNA by the formation of a
two loop-loop interaction. Inhibition of translation is followed by a rapid degradation
by the double-strand specific RNase III. As in the example shown in A), degradation is
probably coupled to translation repression since both events are required for efficient
regulation in vivo.
Geissmann et al., RNA Biol 6: 153-160 (2009)
Mechanisms of target gene repression by sRNAs
The 5' tail of E. coli MicA RNA binds to the single-stranded Shine-Dalgarno sequence (SD)
of ompA mRNA coding for an outer membrane protein. This process is assisted by the
chaperone protein Hfq38. The ncRNA-mRNA complexes are degraded by the RNase E
degradosome complex.
Geissmann et al., RNA Biol 6: 153-160 (2009)
Mechanisms of target gene repression by sRNAs
RyhB binds to the translational initiation region after a structural rearrangement of the
sodB mRNA coding for a superoxide dismutase by the chaperone protein Hfq38. The
ncRNA-mRNA complexes are degraded by the RNase E degradosome complex.
Geissmann et al., RNA Biol 6: 153-160 (2009)
Riboswitches
Atomic-resolution structures for representatives of eight
riboswitch aptamer classes conserved among diverse species
Riboswitches are RNA elements that
control expression of their
downstream genes in cis through a
metabolite-induced alteration of their
secondary structure. Many conserved
class of riboswitches have been
found, including:
a) The purine riboswitch aptamer.
b) The TPP riboswitch aptamer.
c) The S-adenosylmethionine (SAM)I riboswitch aptamer.
d) The SAM-II riboswitch aptamer.
e) The SAM-III/SMK riboswitch
aptamer.
f)
The lysine riboswitch aptamer.
g) The GlcN6P-responsive glmS
ribozyme.
h) The Mg2+-responsive M-box
riboswitch aptamer.
Kinetic and thermodynamic factors
that influence riboswitch function
Riboswitches consist of an aptamer which
binds the ligand, and an expression platform,
which regulates gene expression through
alternative RNA structures that affect
transcription or translation. Upon ligand
binding, the riboswitch changes conformation.
a) Schematic representation of a riboswitch that
represses gene expression upon ligand binding
by controlling transcription termination.
Factors that affect riboswitch function include:
(1) rate constants for aptamer folding and
unfolding, (2) rate constants for ligand
association and dissociation, (3) rate constants
for expression platform folding and unfolding,
and (4) speed of transcription elongation by
RNA polymerase (RNAP).
b) Schematic representation of a riboswitch that
represses gene expression upon ligand binding
by controlling translation initiation. Numbers 1
through 4 are as described in panel (a); (5) is
the speed of Rho-dependent transcription
termination.
Gene control by a eukaryotic thiamine
pyrophosphate (TPP) riboswitch
a) Secondary structure model of the TPP aptamer
residing in the 5´ untranslated region (UTR) of
Neurospora crassa NMT1 (involved in thiamine
metabolism). Sequences of the aptamer P4/P5
domain are complementary (orange shading) to a
region adjoining a key splice site, illustrating one way
in which the availability of TPP influences splice site
selection. Similar types of base-pairing interactions
between aptamer domains and expression platforms
are exploited by other eukaryotic TPP riboswitches.
b) Control of alternative splicing by TPP riboswitches
regulates NMT1 gene expression in fungi. The main
features are depicted of the riboswitch from N. crassa
NMT1, including splice sites, pairing elements of the
TPP aptamer (P1 through P5), and upstream open
reading frames (uORFs) that compete with translation
of the primary ORF to reduce gene expression. Green
arrows and red inhibition lines refer to splicing
determinants that are activated or inhibited,
respectively, depending on the occupancy state of the
aptamer domain.
Function of the SAM Riboswitch
The absence of SAM (low nutrient levels) enables the riboswitch element to form an
anti- termination structure that allows transcription of the downstream genes. Binding
of SAM (rich nutrient conditions) to the riboswitch element alters its formation and a
terminator structure is formed (lollipop). As a result, downstream genes are not
synthesized.
Conserved sequences and secondary structures corresponding to
different classes of S-adenosylmethionine (SAM) riboswitch aptamers
SAM riboswitches are found upstream of a
number of genes that encode proteins
involved in Methionine or cysteine
biosynthesis in Gram-positive bacteria.
Two SAM riboswitches in B. subtilis act at
the level of transcription termination.
However, others may regulate gene
expression at the level of translation
initiation.
The SAM-I, SAM-II, and SAM-III classes have
distinct architectures, whereas the SAM-IV
aptamer is related in sequence and
secondary structure to the SAM-I class.
Nucleotides highlighted in yellow are
observed (SAM-I, SAM-II, and SAM-III) (4446) or predicted (SAM-IV) (42) to contact
the SAM ligand directly.
[Abbreviation: K-turn, kink-turn motif.]
Conserved sequences and secondary structure models
corresponding to molybdenum cofactor (Moco) and
tungsten cofactor (Wco) RNAs
These RNAs exhibit characteristics of
riboswitches, including a complex
aptamer-like structure and control
genes involved in cofactor
biosynthesis, metal transporters, and
apoenzymes that utilize the metal
cofactors.
Many representatives reside
immediately upstream of an AUG
start codon and are predicted to
function by sequestering the ShineDalgarno (SD) sequence (blue
shading).
The phylogenetic distributions and
specific gene associations of these
motifs suggest that RNAs containing
the P3 element recognize Moco and
that those lacking this stem bind the
related coenzyme Wco.
Tandem arrangements of aptamers and riboswitches
Riboswitches or their
component aptamers can be
combined in distinct ways to
effect more sophisticated
genetic control. The circuits
shown repress gene
expression via transcription
termination.
Abbreviations: AdoCbl:
adenosylcobalamin (coenzyme
B12); Gly, glycine; SAM, Sadenosylmethionine; TPP,
thiamine pyrophosphate.
[NOR Logic: Circuit is on only
if both metabolites are low]
RNA Control by Repressor Proteins
mRNA-protein induced-fit allows
translation regulation via different
mechanisms: ribosomal protein S15
autoregulation in different bacteria.
A.
Protein sequence alignment showing the high
degree of conservation of r-protein S15 in three
evolutionary distant bacteria (E. coli, T.
thermophilus and B. stearothermophilus). Their
structures are also very similar (see top of C–E).
B.
S15 binds to a conserved three-way junction of
the 16S rRNA in the 30S subunit. The structures
of the T. thermophilus and E. coli 30S ribosome
show substantially no difference in this region.
C.
Autoregulation of E. coli S15. The 5' UTR of S15
mRNA (rpsO gene) folds into a pseudoknot structure bearing two distinct determinants for S15 recognition which
partially mimic the 16S rRNA binding site. Stabilizing the pseudoknot on the ribosome S15 represses its own translation
through an entrapment mechanism.
D.
Autoregulation of T. thermophilus S15. A structure mimicking the 16S rRNA three-way junction characterizes the 5'
UTR. Tt S15 regulates translation via a direct competition mechanism.
E.
Autoregulation of B. stearothermophilus S15. The predicted structure for the 5' UTR is a three-way junction that
partially mimics the 16S rRNA binding site. In all cases, both S15 and the RNA targets show the ability to change their
structure upon complex formation.

Sites 1 and 2 in 16S rRNA (see panel B) are universally conserved and contain the specific determinant for S15 binding.
For regulation, S15 recognizes the three regulatory mRNA regions in a way which partially mimics the 16S rRNA
binding sites. Site 2 is found in E. coli mRNAs and is recognized by S15 in a way similar to that found in 16S rRNA
whereas site 1 with its bulged A is mRNA-specific and significantly differ from 16S site 1. In T. thermophilus, site 2 is
mRNA-specific while S15 recognizes site 1 like its the 16S rRNA counterpart.
Geissmann et al., RNA Biol 6: 153-160 (2009)