T box riboswitches in Actinobacteria: Translational regulation via novel tRNA interactions

The T box riboswitch regulates many amino acid-related genes in Gram-positive bacteria. T box riboswitch-mediated gene regulation was shown previously to occur at the level of transcription attenuation via structural rearrangements in the 5′ untranslated (leader) region of the mRNA in response to binding of a specific uncharged tRNA. In this study, a novel group of isoleucyl-tRNA synthetase gene (ileS) T box leader sequences found in organisms of the phylum Actinobacteria was investigated. The Stem I domains of these RNAs lack several highly conserved elements that are essential for interaction with the tRNA ligand in other T box RNAs. Many of these RNAs were predicted to regulate gene expression at the level of translation initiation through tRNA-dependent stabilization of a helix that sequesters a sequence complementary to the Shine–Dalgarno (SD) sequence, thus freeing the SD sequence for ribosome binding and translation initiation. We demonstrated specific binding to the cognate tRNAIle and tRNAIle-dependent structural rearrangements consistent with regulation at the level of translation initiation, providing the first biochemical demonstration, to our knowledge, of translational regulation in a T box riboswitch.

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TBDB – A database of structurally annotated T-box riboswitch:tRNA pairs

10.1101/2020.06.17.157016 ◽

2020 ◽

Author(s):

Jorge A. Marchand ◽

Merrick D. Pierson Smela ◽

Thomas H. H. Jordan ◽

Kamesh Narasimhan ◽

George M. Church

Keyword(s):

De Novo ◽

Bacterial Species ◽

Large Family ◽

T Box ◽

Gram Positive Bacteria ◽

Binding Partners ◽

Open Access Database ◽

Feature Sequence ◽

Cognate Trna ◽

Trna Binding

AbstractT-box riboswitches constitute a large family of tRNA-binding leader sequences that play a central role in gene regulation in many gram-positive bacteria. Accurate inference of the tRNA binding to T-boxes is critical to predict their cis-regulatory activity. However, there is no central repository of information on the tRNA binding specificities of T-box riboswitches and de novo prediction of binding specificities requires advance knowledge of computational tools to annotate riboswitch secondary structure features. Here we present T-box annotation Database (TBDB,https://tbdb.io), an open-access database with a collection of 23,497 T-box sequences, spanning the major phyla of 3,621 bacterial species. Among structural predictions, the TBDB also identifies specifier sequences, cognate tRNA binding partners, and downstream regulatory target. To our knowledge, the TBDB presents the largest collection of feature, sequence, and structural annotations carried out on this important family of regulatory RNA.

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The translational regulation of threonyl-tRNA synthetase. Functional relationship between the enzyme, the cognate tRNA and the ribosome

Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression ◽

10.1016/0167-4781(90)90192-5 ◽

1990 ◽

Vol 1050 (1-3) ◽

pp. 343-350 ◽

Cited By ~ 7

Author(s):

Hervé Moine ◽

Bernard Ehresmann ◽

Pascale Romby ◽

Jean Pierre Ebel ◽

Marianne Grunberg-Manago ◽

...

Keyword(s):

Translational Regulation ◽

Functional Relationship ◽

Trna Synthetase ◽

Cognate Trna

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Binding and recognition of aminoacyl-tRNA synthetase by cognate tRNA

Seibutsu Butsuri ◽

10.2142/biophys.13.172 ◽

1973 ◽

Vol 13 (4) ◽

pp. 172-186

Author(s):

Takeshi SENO

Keyword(s):

Trna Synthetase ◽

Aminoacyl Trna Synthetase ◽

Cognate Trna

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Conformation change of tRNAGlu in the complex with glutamyl-tRNA synthetase is required for the specific binding of L-glutamate

Biochemistry ◽

10.1021/bi00370a041 ◽

1986 ◽

Vol 25 (22) ◽

pp. 7031-7036 ◽

Cited By ~ 19

Author(s):

Miki Hara-Yokoyama ◽

Shigeyuki Yokoyama ◽

Tatsuo Miyazawa

Keyword(s):

Specific Binding ◽

Trna Synthetase ◽

Conformation Change

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Major structural rearrangements of the canonical eukaryotic translation initiation complex

Current Opinion in Structural Biology ◽

10.1016/j.sbi.2018.08.006 ◽

2018 ◽

Vol 53 ◽

pp. 151-158 ◽

Cited By ~ 4

Author(s):

Ewelina Guca ◽

Yaser Hashem

Keyword(s):

Translation Initiation ◽

Structural Rearrangements ◽

Initiation Complex ◽

Eukaryotic Translation ◽

Eukaryotic Translation Initiation

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Rapid purification of ribosomal particles assembled on histone H4 mRNA: a new method based on mRNA–DNA chimaeras

Biochemical Journal ◽

10.1042/bj20121211 ◽

2013 ◽

Vol 449 (3) ◽

pp. 719-728 ◽

Cited By ~ 9

Author(s):

Lydia Prongidi-Fix ◽

Laure Schaeffer ◽

Angelita Simonetti ◽

Sharief Barends ◽

Jean-François Ménétret ◽

...

Keyword(s):

Translation Initiation ◽

Rna Binding ◽

Rna Binding Proteins ◽

Specific Binding ◽

Full Length ◽

Molar Ratio ◽

Detailed Knowledge ◽

Histone H4 ◽

Purification Method

Detailed knowledge of the structure of the ribosomal particles during their assembly on mRNA is a prerequisite for understanding the intricate translation initiation process. In vitro preparation of eukaryotic translation initiation complexes is limited by the rather tricky assembly from individually purified ribosomal subunits, initiation factors and initiator tRNA. In order to directly isolate functional complexes from living cells, methods based on affinity tags have been developed which, however, often suffer from non-specific binding of proteins and/or RNAs. In the present study we present a novel method designed for the purification of high-quality ribosome/mRNA particles assembled in RRL (rabbit reticulocyte lysate). Chimaerical mRNA–DNA molecules, consisting of the full-length mRNA ligated to a biotinylated desoxy-oligonucleotide, are immobilized on streptavidin-coated beads and incubated with RRL to form initiation complexes. After a washing step, the complexes are eluted by specific DNase I digestion of the DNA moiety of the chimaera, releasing initiation complexes in native conditions. Using this simple and robust purification setup, 80S particles properly programmed with full-length histone H4 mRNA were isolated with the expected ribosome/mRNA molar ratio of close to 1. We show that by using this novel approach purified ribosomal particles can be obtained that are suitable for biochemical and structural studies, in particular single-particle cryo-EM (cryo-electron microscopy). This purification method thus is a versatile tool for the isolation of fully functional RNA-binding proteins and macromolecular RNPs.

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Inhibition of Methionyl-tRNA Synthetase by REP8839 and Effects of Resistance Mutations on Enzyme Activity

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00275-08 ◽

2008 ◽

Vol 53 (1) ◽

pp. 86-94 ◽

Cited By ~ 30

Author(s):

Louis S. Green ◽

James M. Bullard ◽

Wendy Ribble ◽

Frank Dean ◽

David F. Ayers ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Mechanistic Explanation ◽

Trna Synthetase ◽

Turnover Rates ◽

Wild Type ◽

Resistance Mutations ◽

Gram Positive ◽

Gram Positive Bacteria ◽

Human Orthologs ◽

Methionyl Trna Synthetase

ABSTRACT REP8839 is a selective inhibitor of methionyl-tRNA synthetase (MetRS) with antibacterial activity against a variety of gram-positive organisms. We determined REP8839 potency against Staphylococcus aureus MetRS and assessed its selectivity for bacterial versus human orthologs of MetRS. The inhibition constant (Ki ) of REP8839 was 10 pM for Staphylococcus aureus MetRS. Inhibition of MetRS by REP8839 was competitive with methionine and uncompetitive with ATP. Thus, high physiological ATP levels would actually facilitate optimal binding of the inhibitor. While many gram-positive bacteria, such as Staphylococcus aureus, express exclusively the MetRS1 subtype, many gram-negative bacteria express an alternative homolog called MetRS2. Some gram-positive bacteria, such as Streptococcus pneumoniae and Bacillus anthracis, express both MetRS1 and MetRS2. MetRS2 orthologs were considerably less susceptible to REP8839 inhibition. REP8839 inhibition of human mitochondrial MetRS was 1,000-fold weaker than inhibition of Staphylococcus aureus MetRS; inhibition of human cytoplasmic MetRS was not detectable, corresponding to >1,000,000-fold selectivity for the bacterial target relative to its cytoplasmic counterpart. Mutations in MetRS that confer reduced susceptibility to REP8839 were examined. The mutant MetRS enzymes generally exhibited substantially impaired catalytic activity, particularly in aminoacylation turnover rates. REP8839 Ki values ranged from 4- to 190,000-fold higher for the mutant enzymes than for wild-type MetRS. These observations provide a potential mechanistic explanation for the reduced growth fitness observed with MetRS mutant strains relative to that with wild-type Staphylococcus aureus.

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Sequence and structural similarities between the leucine-specific binding protein and leucyl-tRNA synthetase of Escherichia coli.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.87.12.4561 ◽

1990 ◽

Vol 87 (12) ◽

pp. 4561-4565 ◽

Cited By ~ 6

Author(s):

R. M. Williamson ◽

D. L. Oxender

Keyword(s):

Escherichia Coli ◽

Specific Binding ◽

Binding Protein ◽

Trna Synthetase ◽

Specific Binding Protein

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Novel Translation Initiation Regulation Mechanism in Escherichia coli ptrB Mediated by a 5′-Terminal AUG

Journal of Bacteriology ◽

10.1128/jb.00091-17 ◽

2017 ◽

Vol 199 (14) ◽

Cited By ~ 5

Author(s):

Heather J. Beck ◽

Gary R. Janssen

Keyword(s):

Escherichia Coli ◽

Translation Initiation ◽

Translational Regulation ◽

Current Knowledge ◽

Upstream Open Reading Frame ◽

Model Organisms ◽

Regulation Mechanism ◽

Rna Regulation ◽

Content Type ◽

E Coli

ABSTRACT Alternative translation initiation mechanisms, distinct from the Shine-Dalgarno (SD) sequence-dependent mechanism, are more prevalent in bacteria than once anticipated. Translation of Escherichia coli ptrB instead requires an AUG triplet at the 5′ terminus of its mRNA. The 5′-terminal AUG (5′-uAUG) acts as a ribosomal recognition signal to attract ribosomes to the ptrB mRNA rather than functioning as an initiation codon to support translation of an upstream open reading frame. ptrB expression exhibits a stronger dependence on the 5′-uAUG than the predicted SD sequence; however, strengthening the predicted ptrB SD sequence relieves the necessity for the 5′-uAUG. Additional sequences within the ptrB 5′ untranslated region (5′-UTR) work cumulatively with the 5′-uAUG to control expression of the downstream ptrB coding sequence (CDS), thereby compensating for the weak SD sequence. Replacement of 5′-UTRs from other mRNAs with the ptrB 5′-UTR sequence showed a similar dependence on the 5′-uAUG for CDS expression, suggesting that the regulatory features contained within the ptrB 5′-UTR are sufficient to control the expression of other E. coli CDSs. Demonstration that the 5′-uAUG present on the ptrB leader mRNA is involved in ribosome binding and expression of the downstream ptrB CDS revealed a novel form of translational regulation. Due to the abundance of AUG triplets at the 5′ termini of E. coli mRNAs and the ability of ptrB 5′-UTR regulation to function independently of gene context, the regulatory effects of 5′-uAUGs on downstream CDSs may be widespread throughout the E. coli genome. IMPORTANCE As the field of synthetic biology continues to grow, a complete understanding of basic biological principles will be necessary. The increasing complexity of the synthetic systems highlights the gaps in our current knowledge of RNA regulation. This study demonstrates that there are novel ways to regulate canonical Shine-Dalgarno-led mRNAs in Escherichia coli, illustrating that our understanding of the fundamental processes of translation and RNA regulation is still incomplete. Even for E. coli, one of the most-studied model organisms, genes with translation initiation mechanisms that do not fit the canonical Shine-Dalgarno sequence paradigm are being revealed. Uncovering diverse mechanisms that control translational expression will allow synthetic biologists to finely tune protein production of desired gene products.

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