scholarly journals The methyltransferase TrmA facilitates tRNA folding through interaction with its RNA-binding domain

2020 ◽  
Vol 48 (14) ◽  
pp. 7981-7990
Author(s):  
Laura Carole Keffer-Wilkes ◽  
Emily F Soon ◽  
Ute Kothe
Keyword(s):  
Catalytic Activity ◽  
Rna Binding ◽  
Binding Domain ◽  
Rna Modification ◽  
Functional Link ◽  
Pseudouridine Synthase ◽  
Rna Binding Domain ◽  
Mechanistic Basis ◽  
Trna Folding

Abstract tRNAs are the most highly modified RNAs in all cells, and formation of 5-methyluridine (m5U) at position 54 in the T arm is a common RNA modification found in all tRNAs. The m5U modification is generated by the methyltransferase TrmA. Here, we test and prove the hypothesis that Escherichia coli TrmA has dual functions, acting both as a methyltransferase and as a tRNA chaperone. We identify two conserved residues, F106 and H125, in the RNA-binding domain of TrmA, which interact with the tRNA elbow and are critical for tRNA binding. Co-culture competition assays reveal that the catalytic activity of TrmA is important for cellular fitness, and that substitutions of F106 or H125 impair cellular fitness. We directly show that TrmA enhances tRNA folding in vitro independent of its catalytic activity. In conclusion, our study suggests that F106 and H125 in the RNA-binding domain of TrmA act as a wedge disrupting tertiary interactions between tRNA’s D arm and T arm; this tRNA unfolding is the mechanistic basis for TrmA’s tRNA chaperone activity. TrmA is the second tRNA modifying enzyme next to the pseudouridine synthase TruB shown to act as a tRNA chaperone supporting a functional link between RNA modification and folding.

scholarly journals RNA modification enzyme TruB is a tRNA chaperone

2016 ◽  
Vol 113 (50) ◽  
pp. 14306-14311 ◽  
Author(s):  
Laura Carole Keffer-Wilkes ◽  
Govardhan Reddy Veerareddygari ◽  
Ute Kothe
Keyword(s):  
Molecular Mechanism ◽  
Flow Analysis ◽  
Chaperone Activity ◽  
Rna Modification ◽  
E Coli ◽  
Pseudouridine Synthase ◽  
Bacterial Fitness ◽  
Rna Chaperones ◽  
Trna Folding

Cellular RNAs are chemically modified by many RNA modification enzymes; however, often the functions of modifications remain unclear, such as for pseudouridine formation in the tRNA TΨC arm by the bacterial tRNA pseudouridine synthase TruB. Here we test the hypothesis that RNA modification enzymes also act as RNA chaperones. Using TruB as a model, we demonstrate that TruB folds tRNA independent of its catalytic activity, thus increasing the fraction of tRNA that can be aminoacylated. By rapid kinetic stopped-flow analysis, we identified the molecular mechanism of TruB’s RNA chaperone activity: TruB binds and unfolds both misfolded and folded tRNAs thereby providing misfolded tRNAs a second chance at folding. Previously, it has been shown that a catalytically inactive TruB variant has no phenotype when expressed in an Escherichia coli truB KO strain [Gutgsell N, et al. (2000) RNA 6(12):1870–1881]. However, here we uncover that E. coli strains expressing a TruB variant impaired in tRNA binding and in in vitro tRNA folding cannot compete with WT E. coli. Consequently, the tRNA chaperone activity of TruB is critical for bacterial fitness. In conclusion, we prove the tRNA chaperone activity of the pseudouridine synthase TruB, reveal its molecular mechanism, and demonstrate its importance for cellular fitness. We discuss the likelihood that other RNA modification enzymes are also RNA chaperones.

scholarly journals The RNA Binding Domain of Jerky Consists of Tandemly Arranged Helix-Turn-Helix/Homeodomain-Like Motifs and Binds Specific Sets of mRNAs

2003 ◽  
Vol 23 (12) ◽  
pp. 4083-4093 ◽  
Author(s):  
Wencheng Liu ◽  
Jeremy Seto ◽  
Etienne Sibille ◽  
Miklos Toth
Keyword(s):  
Dna Binding ◽  
Ribosome Biogenesis ◽  
Rna Binding ◽  
Binding Domain ◽  
Cellular Stress Response ◽  
Rna Binding Domain ◽  
Necessary And Sufficient ◽  
Mrna Binding

ABSTRACT A deficit in the Jerky protein in mice causes recurrent seizures reminiscent of temporal lobe epilepsy. Jerky is present in mRNA particles in neurons. We show that the N-terminal 168 amino acids of Jerky are necessary and sufficient for mRNA binding. The binding domain is similar to the two tandemly arranged homeodomain-like helix-turn-helix DNA binding motifs of centromere binding protein B. The putative helix-turn-helix motifs of Jerky can also bind double-stranded DNA and represent a novel mammalian RNA/DNA binding domain. Microarray analysis identified mRNAs encoding proteins involved in ribosome assembly and cellular stress response that specifically bound to the RNA binding domain of Jerky both in vitro and in vivo. These data suggest that epileptogenesis in Jerky-deficient mice most likely involves pathways associated with ribosome biogenesis and neuronal survival and/or apoptosis.

scholarly journals The 54-kD protein of signal recognition particle contains a methionine-rich RNA binding domain.

1990 ◽  
Vol 111 (5) ◽  
pp. 1793-1802 ◽  
Author(s):  
K Römisch ◽  
J Webb ◽  
K Lingelbach ◽  
H Gausepohl ◽  
B Dobberstein
Keyword(s):  
Amino Acids ◽  
Rna Binding ◽  
Signal Sequence ◽  
Signal Recognition Particle ◽  
Binding Domain ◽  
Secretory Proteins ◽  
Signal Recognition ◽  
Rna Binding Domain ◽  
Necessary And Sufficient

Signal recognition particle (SRP) plays the key role in targeting secretory proteins to the membrane of the endoplasmic reticulum (Walter, P., and V. R. Lingappa. 1986. Annu. Rev. Cell Biol. 2:499-516). It consists of SRP7S RNA and six proteins. The 54-kD protein of SRP (SRP54) recognizes the signal sequence of nascent polypeptides. The 19-kD protein of SRP (SRP19) binds to SRP7S RNA directly and is required for the binding of SRP54 to the particle. We used deletion mutants of SRP19 and SRP54 and an in vitro assembly assay in the presence of SRP7S RNA to define the regions in both proteins which are required to form a ribonucleoprotein particle. Deletion of the 21 COOH-terminal amino acids of SRP19 does not interfere with its binding to SRP7S RNA. Further deletions abolish SRP19 binding to SRP7S RNA. The COOH-terminal 207 amino acids of SRP54 (M domain) were found to be necessary and sufficient for binding to the SRP19/7S RNA complex in vitro. Limited protease digestion of purified SRP confirmed our results for SRP54 from the in vitro binding assay. The SRP54M domain could also bind to Escherichia coli 4.5S RNA that is homologous to part of SRP7S RNA. We suggest that the methionine-rich COOH terminus of SRP54 is a RNA binding domain and that SRP19 serves to establish a binding site for SRP54 on the SRP7S RNA.

scholarly journals Crystal structure of SARS-CoV-2 nucleocapsid protein RNA binding domain reveals potential unique drug targeting sites

2020 ◽  
Author(s):  
Sisi Kang ◽  
Mei Yang ◽  
Zhongsi Hong ◽  
Liping Zhang ◽  
Zhaoxia Huang ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Nucleocapsid Protein ◽  
Rna Binding ◽  
Structural Information ◽  
Antiviral Drug ◽  
Binding Domain ◽  
Binding Studies ◽  
Terminal Domain ◽  
Rna Binding Domain

AbstractThe outbreak of coronavirus disease (COVID-19) in China caused by SARS-CoV-2 virus continually lead to worldwide human infections and deaths. It is currently no specific viral protein targeted therapeutics yet. Viral nucleocapsid protein is a potential antiviral drug target, serving multiple critical functions during the viral life cycle. However, the structural information of SARS-CoV-2 nucleocapsid protein is yet to be clear. Herein, we have determined the 2.7 Å crystal structure of the N-terminal RNA binding domain of SARS-CoV-2 nucleocapsid protein. Although overall structure is similar with other reported coronavirus nucleocapsid protein N-terminal domain, the surface electrostatic potential characteristics between them are distinct. Further comparison with mild virus type HCoV-OC43 equivalent domain demonstrates a unique potential RNA binding pocket alongside the β-sheet core. Complemented by in vitro binding studies, our data provide several atomic resolution features of SARS-CoV-2 nucleocapsid protein N-terminal domain, guiding the design of novel antiviral agents specific targeting to SARS-CoV-2.

scholarly journals A Novel Conserved RNA-binding Domain Protein, RBD-1, Is Essential For Ribosome Biogenesis

2002 ◽  
Vol 13 (10) ◽  
pp. 3683-3695 ◽  
Author(s):  
Petra Björk ◽  
Göran Baurén ◽  
ShaoBo Jin ◽  
Yong-Guang Tong ◽  
Thomas R. Bürglin ◽  
...  
Keyword(s):  
Ribosome Biogenesis ◽  
Rna Binding ◽  
Ribosomal Subunit ◽  
Binding Domain ◽  
Dependent Manner ◽  
Binding Domains ◽  
40S Ribosomal Subunit ◽  
Rna Binding Domain

Synthesis of the ribosomal subunits from pre-rRNA requires a large number of trans-acting proteins and small nucleolar ribonucleoprotein particles to execute base modifications, RNA cleavages, and structural rearrangements. We have characterized a novel protein, RNA-binding domain-1 (RBD-1), that is involved in ribosome biogenesis. This protein contains six consensus RNA-binding domains and is conserved as to sequence, domain organization, and cellular location from yeast to human. RBD-1 is essential in Caenorhabditis elegans. In the dipteran Chironomus tentans, RBD-1 (Ct-RBD-1) binds pre-rRNA in vitro and anti-Ct-RBD-1 antibodies repress pre-rRNA processing in vivo. Ct-RBD-1 is mainly located in the nucleolus in an RNA polymerase I transcription-dependent manner, but it is also present in discrete foci in the interchromatin and in the cytoplasm. In cytoplasmic extracts, 20–30% of Ct-RBD-1 is associated with ribosomes and, preferentially, with the 40S ribosomal subunit. Our data suggest that RBD-1 plays a role in structurally coordinating pre-rRNA during ribosome biogenesis and that this function is conserved in all eukaryotes.

scholarly journals RNA Binding Domain of Telomerase Reverse Transcriptase

2001 ◽  
Vol 21 (4) ◽  
pp. 990-1000 ◽  
Author(s):  
Cary K. Lai ◽  
James R. Mitchell ◽  
Kathleen Collins
Keyword(s):  
Catalytic Activity ◽  
Reverse Transcriptase ◽  
Rna Binding ◽  
Binding Domain ◽  
Amino Terminus ◽  
Telomerase Reverse Transcriptase ◽  
Telomerase Rna ◽  
Sequence Motifs ◽  
Rna Binding Domain ◽  
Rna Interaction

ABSTRACT Telomerase is a ribonucleoprotein reverse transcriptase that extends the ends of chromosomes. The two telomerase subunits essential for catalysis in vitro are the telomerase reverse transcriptase (TERT) and the telomerase RNA. Using truncations and site-specific mutations, we identified sequence elements of TERT and telomerase RNA required for catalytic activity and protein-RNA interaction for Tetrahymena thermophila telomerase. We found that the TERT amino and carboxyl termini, although evolutionarily poorly conserved, are nonetheless important for catalytic activity. In contrast, high-affinity telomerase RNA binding requires only a small region in the amino terminus of TERT. Surprisingly, the TERT region necessary and sufficient for telomerase RNA binding is completely separable from the reverse transcriptase motifs. The minimalTetrahymena TERT RNA binding domain contains two sequence motifs with ciliate-specific conservation and one TERT motif with conservation across all species. With human TERT, we demonstrate that a similar region within the TERT amino terminus is essential for human telomerase RNA binding as well. Finally, we defined theTetrahymena telomerase RNA sequences that are essential for TERT interaction. We found that a four-nucleotide region 5′ of the template is critical for TERT binding and that the 5′ end of telomerase RNA is sufficient for TERT binding. Our results reveal at least one evolutionarily conserved molecular mechanism by which the telomerase reverse transcriptase is functionally specialized for obligate use of an internal RNA template.

Bioactive Compounds from Medicinal Plants and Their Possible Effect as Therapeutics Agents against COVID-19: A Review

2021 ◽  
Vol 17 ◽  
Author(s):  
Khairan Khairan ◽  
Rinaldi Idroes ◽  
Trina E. Tallei ◽  
Muhammad J. Nasim ◽  
Claus Jacob
Keyword(s):  
Medicinal Plants ◽  
Bioactive Compounds ◽  
Rna Binding ◽  
Docking Study ◽  
Binding Domain ◽  
Molecular Docking Study ◽  
Target Proteins ◽  
Rna Binding Domain

Background: SARS-CoV-2 has caused more than fifty three million people worldwide infected and almost one million and four hundred thousand deaths. Currently, the appropriate therapeutic drugs are not yet available to treat diseases caused by this coronaviruses (CoVs) infection. It is due to the fact that discoveries and developments of new medication require a relatively long time. The alternative solutions for this viral infection is by utilizing medicinal plants-based bioactive compounds as therapeutic agents against COVID-19. Methods: In this review, a molecular docking study was a method that used to determine the potential of some bioactive compounds from medicinal plants as therapeutics agents against COVID-19. The results of this review still require further investigation to clinically validate either in vitro or in vivo, to find the effective antiviral drugs from medicinal plants for COVID-19 treatment. Results: From a total of 60 identified of medicinal plants, 50 of them have possible effects as therapeutics agents against particular target proteins encoded by the CoVs genes such as Nsp1, Nsp3 (Nsp3b, Nsp3c, PLpro and Nsp3e), Nsp7_Nsp8, Nsp9-Nsp10, Nsp14-Nsp16 complexes, 3CLpro, E protein, ORF7a, Spike (S) glycoprotein, C-terminal RNA binding domain (CRBD), N-terminal RNA binding domain (NRBD), helicase and RdRp. The most common of the bioactive compounds from the medicinal plants as therapeutics agents for COVID-19 treatment were flavonoids compounds. Conclusion: The medicinal plants can serve as starting points for therapeutics agent development against some target proteins of SARS-CoV-2. Nevertheless, the results are in need for clinical validation, either through in vitro or in vivo in COVID-19 treatment.

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