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Frontiers in Translation (Post-Transcriptional Regulatory Mechanisms) David M. Bedwell, PhD

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1 Frontiers in Translation (Post-Transcriptional Regulatory Mechanisms) David M. Bedwell, PhD

2 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. Different mRNA structures that enable different modes of regulation a)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). Geissmann et al., RNA Biol 6: (2009)

3 Thermosensors

4 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 Shine- Dalgarno sequence. Translation of prfA allows virulence gene expression. Model of the Mechanism Underlying Thermoregulated Expression of PrfA Johansson et al., Cell 110: (2002)

5 Non-Coding RNAs

6 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?

7 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. Structures of the S. aureus Hfq and an Hfq–RNA complex Valentin-Hansen et al., Mol. Micro. 51: (2004)

8 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: (2009)

9 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: (2009)

10 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 anti- antisense 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: (2009)

11  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. Sequence conservation of glmS mRNA and GlmZ sRNA binding sites Frohlich and Vogel, Curr. Opin. In Microbiol. 12: (2009)

12 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: (2009)

13 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: (2009) Mechanisms of target gene repression by sRNAs

14 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: (2009) Mechanisms of target gene repression by sRNAs

15 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: (2009) Mechanisms of target gene repression by sRNAs

16 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: (2009) Mechanisms of target gene repression by sRNAs

17 Riboswitches

18 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 Mg 2+ -responsive M-box riboswitch aptamer. 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:

19 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. Kinetic and thermodynamic factors that influence riboswitch function

20 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. Gene control by a eukaryotic thiamine pyrophosphate (TPP) riboswitch

21 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. Function of the SAM Riboswitch

22 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) (44- 46) or predicted (SAM-IV) (42) to contact the SAM ligand directly. [Abbreviation: K-turn, kink-turn motif.] Conserved sequences and secondary structures corresponding to different classes of S-adenosylmethionine (SAM) riboswitch aptamers

23 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 Shine- Dalgarno (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. Conserved sequences and secondary structure models corresponding to molybdenum cofactor (Moco) and tungsten cofactor (Wco) RNAs

24 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, S- adenosylmethionine; TPP, thiamine pyrophosphate. Tandem arrangements of aptamers and riboswitches [NOR Logic: Circuit is on only if both metabolites are low]

25 RNA Control by Repressor Proteins

26 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: (2009)

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