scholarly journals Translational regulation by bacterial small RNAs via an unusual Hfq-dependent mechanism

2017 ◽  
Author(s):  
Muhammad S. Azam ◽  
Carin K. Vanderpool
Keyword(s):  
Binding Site ◽  
Small Rnas ◽  
Translational Regulation ◽  
Regulatory Mechanism ◽  
Role Reversal ◽  
Translational Repression ◽  
Ribosome Binding ◽  
Reversal Mechanism ◽  
Direct Regulator ◽  
Dependent Mechanism

ABSTRACTIn bacteria, the canonical mechanism of translational repression by small RNAs (sRNAs) involves sRNA-mRNA base pairing that occludes the ribosome binding site (RBS), directly preventing translation. In this mechanism, the sRNA is the direct regulator, while the RNA chaperone Hfq plays a supporting role by stabilizing the sRNA. There are a few examples where the sRNA does not directly interfere with ribosome binding, yet translation of the target mRNA is still inhibited. Mechanistically, this non-canonical regulation by sRNAs is poorly understood. Our previous work demonstrated repression of the mannose transporter manX mRNA by the sRNA SgrS, but the regulatory mechanism was unknown. Here, we report that manX translation is controlled by a molecular role-reversal mechanism where Hfq, not the sRNA, is the direct repressor. Hfq binding adjacent to the manX RBS is required for sRNA-mediated translational repression. Translation of manX is also regulated by another sRNA, DicF, via the same non-canonical Hfq-dependent mechanism. Our results suggest that the sRNAs recruit Hfq to its binding site or stabilize the mRNA-Hfq complex. This work adds to the growing number of examples of diverse mechanisms of translational regulation by sRNAs in bacteria.

scholarly journals Crystal structure and ligand-induced folding of the SAM/SAH riboswitch

2020 ◽  
Author(s):  
Lin Huang ◽  
Ting-Wei Liao ◽  
Jia Wang ◽  
Taekjip Ha ◽  
David M J Lilley
Keyword(s):  
Binding Site ◽  
Single Molecule ◽  
Translational Regulation ◽  
Stable State ◽  
Helical Structure ◽  
Ribosome Binding Site ◽  
Stem Loop ◽  
Ribosome Binding ◽  
Induced Folding ◽  
Initiation Of Translation

Abstract While most SAM riboswitches strongly discriminate between SAM and SAH, the SAM/SAH riboswitch responds to both ligands with similar apparent affinities. We have determined crystal structures of the SAM/SAH riboswitch bound to SAH, SAM and other variant ligands at high resolution. The riboswitch forms an H-type pseudoknot structure with coaxial alignment of the stem–loop helix (P1) and the pseudoknot helix (PK). An additional three base pairs form at the non-open end of P1, and the ligand is bound at the interface between the P1 extension and the PK helix. The adenine nucleobase is stacked into the helix and forms a trans Hoogsteen–Watson–Crick base pair with a uridine, thus becoming an integral part of the helical structure. The majority of the specific interactions are formed with the adenosine. The methionine or homocysteine chain lies in the groove making a single hydrogen bond, and there is no discrimination between the sulfonium of SAM or the thioether of SAH. Single-molecule FRET analysis reveals that the riboswitch exists in two distinct conformations, and that addition of SAM or SAH shifts the population into a stable state that likely corresponds to the form observed in the crystal. A model for translational regulation is presented whereby in the absence of ligand the riboswitch is largely unfolded, lacking the PK helix so that translation can be initiated at the ribosome binding site. But the presence of ligand stabilizes the folded conformation that includes the PK helix, so occluding the ribosome binding site and thus preventing the initiation of translation.

scholarly journals An RNA Hairpin Sequesters the Ribosome Binding Site of the Homing Endonuclease mobE Gene

2009 ◽  
Vol 191 (7) ◽  
pp. 2409-2413 ◽  
Author(s):  
Ewan A. Gibb ◽  
David R. Edgell
Keyword(s):  
Binding Site ◽  
Translational Regulation ◽  
Homing Endonuclease ◽  
Ribosome Binding Site ◽  
Galactosidase Activity ◽  
Transcript Mapping ◽  
Ribosome Binding ◽  
Rna Hairpin

ABSTRACT Previous transcript mapping of the bacteriophage Aeh1 nrd operon revealed a predicted RNA hairpin upstream of the homing endonuclease mobE gene. We enzymatically mapped the hairpin, showing that the mobE ribosome binding site is sequestered. Cloning of the hairpin upstream of lacZ resulted in reduced β-galactosidase activity, consistent with translational regulation.

scholarly journals Modeling slow-processing of toxin messenger RNAs in type-I Toxin-Antitoxin systems: post-segregational killing and noise filtering

2018 ◽  
Author(s):  
Yusuke Himeoka ◽  
Namiko Mitarai
Keyword(s):  
Small Rnas ◽  
Present Model ◽  
Regulatory Mechanism ◽  
Type I ◽  
Messenger Rnas ◽  
Ribosome Binding ◽  
Plasmid Loss ◽  
Zero Number ◽  
Growth Inhibiting

AbstractIn type-I toxin-antitoxin (TA) systems, the action of growth-inhibiting toxin proteins is counteracted by the antitoxin small RNAs (sRNAs) that prevent the translation of toxin messenger RNAs (mRNAs). When a TA module is encoded on a plasmid, the short lifetime of antitoxin sRNA compared to toxin mRNAs mediates post-segregational killing (PSK) that contribute the plasmid maintenance, while some of the chromosomal encoded TA loci have been reported to contribute to persister formation in response to a specific upstream signal. Some of the well studied type-I TA systems such ashok/sokare known to have a rather complex regulatory mechanism. Transcribed full-length toxin mRNAs fold such that the ribosome binding site is not accessible and hence cannot be translated. The mRNAs are slowly processed by RNases, and the truncated mRNAs can be either translated or bound by antitoxin sRNA to be quickly degraded. We analyze the role of this extra processing by a mathematical model. We first consider the PSK scenario, and demonstrate that the extra processing compatibly ensures the high toxin expression upon complete plasmid loss, without inducing toxin expression upon acquisition of a plasmid or decrease of plasmid number to a non-zero number. We further show that the extra processing help filtering the transcription noise, avoiding random activation of toxins in transcriptionally regulated TA systems as seen in chromosomal ones. The present model highlights impacts of the slow processing reaction, offering insights on why the slow processing reactions are commonly identified in multiple type-I TA systems.

scholarly journals Recognition of cyclic-di-GMP by a riboswitch conducts translational repression through masking the ribosome-binding site distant from the aptamer domain

2018 ◽  
Vol 23 (6) ◽  
pp. 435-447 ◽  
Author(s):  
Saki Inuzuka ◽  
Hitoshi Kakizawa ◽  
Kei-ichiro Nishimura ◽  
Takuto Naito ◽  
Katsushi Miyazaki ◽  
...  
Keyword(s):  
Binding Site ◽  
Ribosome Binding Site ◽  
Translational Repression ◽  
Ribosome Binding ◽  
Aptamer Domain

scholarly journals Regulation of pyr Gene Expression in Mycobacterium smegmatis by PyrR-Dependent Translational Repression

2007 ◽  
Vol 189 (17) ◽  
pp. 6236-6245 ◽  
Author(s):  
Christopher J. Fields ◽  
Robert L. Switzer
Keyword(s):  
Binding Site ◽  
Mycobacterium Smegmatis ◽  
Regulatory Protein ◽  
Ribosome Binding Site ◽  
Translational Repression ◽  
Ribosome Binding ◽  
Gram Positive Bacteria ◽  
Attenuation Mechanism ◽  
Operon Expression ◽  
Mrna Binding

ABSTRACT Regulation of pyrimidine biosynthetic (pyr) genes by a transcription attenuation mechanism that is mediated by the PyrR mRNA-binding regulatory protein has been demonstrated for numerous gram-positive bacteria. Mycobacterial genomes specify pyrR genes and contain obvious PyrR-binding sequences in the initially transcribed regions of their pyr operons, but transcription antiterminator and attenuation terminator sequences are absent from their pyr 5′ leader regions. This work demonstrates that repression of pyr operon expression in Mycobacterium smegmatis by exogenous uracil requires the pyrR gene and the pyr leader RNA sequence for binding of PyrR. Plasmids containing the M. smegmatis pyr promoter-leader region translationally fused to lacZ also displayed pyrR-dependent repression, but transcriptional fusions of the same sequences to a lacZ gene that retained the lacZ ribosome-binding site were not regulated by PyrR plus uracil. We propose that PyrR regulates pyr expression in M. smegmatis, other mycobacteria, and probably in numerous other bacteria by a translational repression mechanism in which nucleotide-regulated binding of PyrR occludes the first ribosome-binding site of the pyr operon.

pGR71 plasmid promotor sequence temporally regulated in Bacillus subtilis

1998 ◽  
Vol 44 (12) ◽  
pp. 1186-1192
Author(s):  
Guy Daxhelet ◽  
Philippe Gilot ◽  
Etienne Nyssen ◽  
Philippe Hoet
Keyword(s):  
Bacillus Subtilis ◽  
Binding Site ◽  
Transcription Initiation ◽  
Promoter Sequence ◽  
Initiation Site ◽  
Chloramphenicol Acetyltransferase ◽  
Ribosome Binding Site ◽  
Chloramphenicol Resistance ◽  
E Coli ◽  
Ribosome Binding

pGR71, a composite of plasmids pUB110 and pBR322, replicates in Escherichia coli and in Bacillus subtilis. It carries the chloramphenicol resistance gene (cat) from Tn9, which is not transcribed in either host by lack of a promoter. The cat gene is preceded by a Shine-Dalgarno sequence functional in E. coli but not in B. subtilis. Deleted pGR71 plasmids were obtained in B. subtilis when cloning foreign viral DNA upstream of this cat sequence, as well as by BAL31 exonuclease deletions extending upstream from the cat into the pUB110 moiety. These mutant plasmids expressed chloramphenicol acetyltransferase (CAT), conferring on B. subtilis resistance to high chloramphenicol concentrations. CAT expression peaked at the early postexponential phase of B. subtilis growth. The transcription initiation site of cat, determined by primer extension, was located downstream of a putative promoter sequence within the pUB110 moiety. N-terminal amino acid sequencing showed that native CAT was produced by these mutant plasmids. The cat ribosome-binding site, functional in E. coli, was repositioned within the pUB110 moiety and had consequently an extended homology with B. subtilis 16S rRNA, explaining the production of native enzyme.Key words: chloramphenicol acetyltransferase, Bacillus subtilis, postexponential gene expression, plasmid pUB110, ribosome-binding site, transcriptional promoter.

scholarly journals P-bodies and the miRNA pathway regulate translational repression of bicoid mRNA during Drosophila melanogaster oogenesis

2018 ◽  
Author(s):  
John M. McLaughlin ◽  
Daniel F.Q. Smith ◽  
Irina E. Catrina ◽  
Diana P. Bratu
Keyword(s):  
Drosophila Melanogaster ◽  
Small Rna ◽  
Translational Regulation ◽  
Translational Repression ◽  
Temporal Regulation ◽  
P Bodies ◽  
Maternal Transcripts ◽  
Spatio Temporal ◽  
Mirna Pathway ◽  
Rna Pathways

ABSTRACTEmbryonic axis patterning in Drosophila melanogaster is partly achieved by mRNAs that are maternally localized to the oocyte; the spatio-temporal regulation of these transcripts’ stability and translation is a characteristic feature of oogenesis. While protein regulatory factors are necessary for the translational regulation of some maternal transcripts (e.g. oskar and gurken), small RNA pathways are also known to regulate mRNA stability and translation in eukaryotes. MicroRNAs (miRNAs) are small RNA regulators of gene expression, widely conserved throughout eukaryotic genomes and essential for animal development. The main D. melanogaster anterior determinant, bicoid, is maternally transcribed, but it is not translated until early embryogenesis. We investigated the possibility that its translational repression during oogenesis is mediated by miRNA activity. We found that the bicoid 3’UTR contains a highly conserved, predicted binding site for miR-305. Our studies reveal that miR-305 regulates the translation of a reporter gene containing the bicoid 3’UTR in cell culture, and that miR-305 only partially contributes to bicoid mRNA translational repression during oogenesis. We also found that Processing bodies (P-bodies) in the egg chamber may play a role in stabilizing bicoid and other maternal transcripts. Here, we offer insights into the possible role of P-bodies and the miRNA pathway in the translational repression of bicoid mRNA during oogenesis.

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