Fluorescence Anisotropy: Analysis of tRNA Binding to the T Box Riboswitch Antiterminator RNA

2014 ◽  
pp. 143-152 ◽  
Author(s):  
S. Zhou ◽  
R. Anupam ◽  
J. V. Hines
Keyword(s):  
Fluorescence Anisotropy ◽  
T Box ◽  
Trna Binding

scholarly journals Two-step binding kinetics of tRNAGly by the glyQS T-box riboswitch and its regulation by T-box structural elements

2018 ◽  
Author(s):  
Jiacheng Zhang ◽  
Bhaskar Chetnani ◽  
Eric D. Cormack ◽  
Wei Liu ◽  
Alfonso Mondragón ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Regulatory Rna ◽  
Structural Elements ◽  
T Box ◽  
Loop Region ◽  
Rna Elements ◽  
Set Up ◽  
Trna Binding

ABSTRACTT-box riboswitches are cis-regulatory RNA elements that regulate mRNAs encoding for aminoacyl tRNA synthetases or proteins involved in amino acid biosynthesis and transport. Rather than using small molecules as their ligands, as do most riboswitches, T-box riboswitches uniquely bind tRNA and sense their aminoacylated state. Whereas the anticodon and elbow regions of the tRNA interact with Stem I, located in the 5’ portion of the T-box, sensing of the aminoacylation state involves direct binding of the NCCA sequence at the tRNA 3’ end to the anti-terminator sequence located in the 3’ portion of the T-box. However, the kinetic trajectory that describes how each of these interactions are established temporally during tRNA binding remains unclear. Using singlemolecule fluorescence resonance energy transfer (smFRET), we demonstrate that tRNA binds to the riboswitch in a two-step process, first with anticodon recognition followed by NCCA binding, with the second step accompanied by an inward motion of the 3’ portion of the T-box riboswitch relative to Stem I. By using site-specific mutants, we further show that the T-loop region of the T-box significantly contributes to the first binding step, and that the K-turn region of the T-box influences both binding steps, but with a more dramatic effect on the second binding step. Our results set up a kinetic framework describing tRNA binding by T-box riboswitches and highlight the important roles of several T-box structural elements in regulating each binding step.SIGNIFICANCEBacteria commonly use riboswitches, cis-regulatory RNA elements, to regulate the transcription or translation of the mRNAs upon sensing signals. Unlike small molecule binding riboswitches, T-box riboswitches bind tRNA and sense their aminoacylated state. T-box modular structural elements that recognize different parts of a tRNA have been identified, however, how each of these interactions is established temporally during tRNA binding remains unclear. Our study reveals that tRNA binds to the riboswitch in a two-step mechanism, with anticodon recognition first, followed by binding to the NCCA sequence at the 3’ end of the tRNA with concomitant conformational changes in the T-box. Our results also highlight the importance of the modular structural elements of the T-box in each of the binding steps.

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

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.

scholarly journals TBDB: a database of structurally annotated T-box riboswitch:tRNA pairs

2020 ◽  
Vol 49 (D1) ◽  
pp. D229-D235
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 ◽  
Annotation Database ◽  
T Box ◽  
Gram Positive Bacteria ◽  
Open Access Database ◽  
Feature Sequence ◽  
Advanced Knowledge ◽  
Trna Binding

Abstract T-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-box riboswitches 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 advanced knowledge of computational tools to annotate riboswitch secondary structure features. Here, we present the T-box Riboswitch Annotation Database (TBDB, https://tbdb.io), an open-access database with a collection of 23,535 T-box riboswitch sequences, spanning the major phyla of 3,632 bacterial species. Among structural predictions, the TBDB also identifies specifier sequences, cognate tRNA binding partners, and downstream regulatory targets. To our knowledge, the TBDB presents the largest collection of feature, sequence, and structural annotations carried out on this important family of regulatory RNA.

scholarly journals Specific structural elements of the T-box riboswitch drive the two-step binding of the tRNA ligand

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Jiacheng Zhang ◽  
Bhaskar Chetnani ◽  
Eric D Cormack ◽  
Dulce Alonso ◽  
Wei Liu ◽  
...  
Keyword(s):  
Second Step ◽  
Regulatory Rna ◽  
Structural Elements ◽  
Acid Biosynthesis ◽  
Site Specific ◽  
T Box ◽  
Set Up ◽  
Cognate Trna ◽  
Trna Binding ◽  
Different Parts

T-box riboswitches are cis-regulatory RNA elements that regulate the expression of proteins involved in amino acid biosynthesis and transport by binding to specific tRNAs and sensing their aminoacylation state. While the T-box modular structural elements that recognize different parts of a tRNA have been identified, the kinetic trajectory describing how these interactions are established temporally remains unclear. Using smFRET, we demonstrate that tRNA binds to the riboswitch in two steps, first anticodon recognition followed by the sensing of the 3’ NCCA end, with the second step accompanied by a T-box riboswitch conformational change. Studies on site-specific mutants highlight that specific T-box structural elements drive the two-step binding process in a modular fashion. Our results set up a kinetic framework describing tRNA binding by T-box riboswitches, and suggest such binding mechanism is kinetically beneficial for efficient, co-transcriptional recognition of the cognate tRNA ligand.

Table Substitution Box Method for Increasing Security in Interval Splitting Arithmetic Coding

2017 ◽  
Vol 13 (10) ◽  
pp. 6552-6557
Author(s):  
E.Wiselin Kiruba ◽  
Ramar K.
Keyword(s):  
Compression Ratio ◽  
Arithmetic Coding ◽  
Substitution Box ◽  
Plain Text ◽  
T Box ◽  
Novel Approach ◽  
Permutation Methods ◽  
Message Digest ◽  
Box Method ◽  
Arithmetic Coder

Amalgamation of compression and security is indispensable in the field of multimedia applications. A novel approach to enhance security with compression is discussed in this  research paper. In secure arithmetic coder (SAC), security is provided by input and output permutation methods and compression is done by interval splitting arithmetic coding. Permutation in SAC is susceptible to attacks. Encryption issues associated with SAC is dealt in this research method. The aim of this proposed method is to encrypt the data first by Table Substitution Box (T-box) and then to compress by Interval Splitting Arithmetic Coder (ISAC). This method incorporates dynamic T-box in order to provide better security. T-box is a method, constituting elements based on the random output of Pseudo Random Generator (PRNG), which gets the input from Secure Hash Algorithm-256 (SHA-256) message digest. The current scheme is created, based on the key, which is known to the encoder and decoder. Further, T-boxes are created by using the previous message digest as a key.  Existing interval splitting arithmetic coding of SAC is applied for compression of text data. Interval splitting finds a relative position to split the intervals and this in turn brings out compression. The result divulges that permutation replaced by T-box method provides enhanced security than SAC. Data is not revealed when permutation is replaced by T-box method. Security exploration reveals that the data remains secure to cipher text attacks, known plain text attacks and chosen plain text attacks. This approach results in increased security to Interval ISAC. Additionally the compression ratio  is compared by transferring the outcome of T-box  to traditional  arithmetic coding. The comparison proved that there is a minor reduction in compression ratio in ISAC than arithmetic coding. However the security provided by ISAC overcomes the issues of compression ratio in  arithmetic coding. 

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