scholarly journals Distinct ways of G:U recognition by conserved tRNA binding motifs

2018 ◽  
Vol 115 (29) ◽  
pp. 7527-7532 ◽  
Author(s):  
Yeeting E. Chong ◽  
Min Guo ◽  
Xiang-Lei Yang ◽  
Bernhard Kuhle ◽  
Masahiro Naganuma ◽  
...  
Keyword(s):  
Base Pair ◽  
Homo Sapiens ◽  
Binding Motifs ◽  
Trna Synthetases ◽  
E Coli ◽  
Human Enzyme ◽  
Domains Of Life ◽  
Direct Recognition ◽  
Dyad Symmetry ◽  
Trna Binding

Throughout three domains of life, alanyl-tRNA synthetases (AlaRSs) recognize a G3:U70 base pair in the acceptor stem of tRNAAla as the major identity determinant of tRNAAla. The crystal structure of the archaeon Archaeoglobus fulgidus AlaRS in complex with tRNAAla provided the basis for G3:U70 recognition with residues (Asp and Asn) that are conserved in the three domains [Naganuma M, et al. (2014) Nature 510:507–511]. The recognition mode is unprecedented, with specific accommodation of the dyad asymmetry of the G:U wobble pair and exclusion of the dyad symmetry of a Watson–Crick pair. With this conserved mode, specificity is based more on “fit” than on direct recognition of specific atomic groups. Here, we show that, in contrast to the archaeal complex, the Escherichia coli enzyme uses direct positive (energetically favorable) minor groove recognition of the unpaired 2-amino of G3 by Asp and repulsion of a competing base pair by Asn. Strikingly, mutations that disrupted positive recognition by the E. coli enzyme had little or no effect on G:U recognition by the human enzyme. Alternatively, Homo sapiens AlaRS selects G:U without positive recognition and uses Asp instead to repel a competitor. Thus, the widely conserved Asp-plus-Asn architecture of AlaRSs can select G:U in a straightforward (bacteria) or two different unconventional (eukarya/archaea) ways. The adoption of different modes for recognition of a widely conserved G:U pair in alanine tRNAs suggests an early and insistent role for G:U in the development of the genetic code.

scholarly journals Lysine Acetylation Regulates Alanyl-tRNA Synthetase Activity in Escherichia coli

Genes ◽  
2018 ◽  
Vol 9 (10) ◽  
pp. 473 ◽  
Author(s):  
Takuya Umehara ◽  
Saori Kosono ◽  
Dieter Söll ◽  
Koji Tamura
Keyword(s):  
Escherichia Coli ◽  
Trna Synthetase ◽  
Synthetase Activity ◽  
Lysine Acetylation ◽  
Trna Synthetases ◽  
E Coli ◽  
Domains Of Life ◽  
The Impact

Protein lysine acetylation is a widely conserved posttranslational modification in all three domains of life. Lysine acetylation frequently occurs in aminoacyl-tRNA synthetases (aaRSs) from many organisms. In this study, we determined the impact of the naturally occurring acetylation at lysine-73 (K73) in Escherichia coli class II alanyl-tRNA synthetase (AlaRS) on its alanylation activity. We prepared an AlaRS K73Ac variant in which Nε-acetyl-l-lysine was incorporated at position 73 using an expanded genetic code system in E. coli. The AlaRS K73Ac variant showed low activity compared to the AlaRS wild type (WT). Nicotinamide treatment or CobB-deletion in an E. coli led to elevated acetylation levels of AlaRS K73Ac and strongly reduced alanylation activities. We assumed that alanylation by AlaRS is affected by K73 acetylation, and the modification is sensitive to CobB deacetylase in vivo. We also showed that E. coli expresses two CobB isoforms (CobB-L and CobB-S) in vivo. CobB-S displayed the deacetylase activity of the AlaRS K73Ac variant in vitro. Our results imply a potential regulatory role for lysine acetylation in controlling the activity of aaRSs and protein synthesis.

scholarly journals Functional expression of the eukaryotic proton pump rhodopsin OmR2 in Escherichia coli and its photochemical characterization

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Masuzu Kikuchi ◽  
Keiichi Kojima ◽  
Shin Nakao ◽  
Susumu Yoshizawa ◽  
Shiho Kawanishi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Proton Pump ◽  
Expression System ◽  
Spectroscopic Characterization ◽  
Functional Expression ◽  
Proton Pumping ◽  
E Coli ◽  
Oxyrrhis Marina ◽  
Domains Of Life

AbstractMicrobial rhodopsins are photoswitchable seven-transmembrane proteins that are widely distributed in three domains of life, archaea, bacteria and eukarya. Rhodopsins allow the transport of protons outwardly across the membrane and are indispensable for light-energy conversion in microorganisms. Archaeal and bacterial proton pump rhodopsins have been characterized using an Escherichia coli expression system because that enables the rapid production of large amounts of recombinant proteins, whereas no success has been reported for eukaryotic rhodopsins. Here, we report a phylogenetically distinct eukaryotic rhodopsin from the dinoflagellate Oxyrrhis marina (O. marina rhodopsin-2, OmR2) that can be expressed in E. coli cells. E. coli cells harboring the OmR2 gene showed an outward proton-pumping activity, indicating its functional expression. Spectroscopic characterization of the purified OmR2 protein revealed several features as follows: (1) an absorption maximum at 533 nm with all-trans retinal chromophore, (2) the possession of the deprotonated counterion (pKa = 3.0) of the protonated Schiff base and (3) a rapid photocycle through several distinct photointermediates. Those features are similar to those of known eukaryotic proton pump rhodopsins. Our successful characterization of OmR2 expressed in E. coli cells could build a basis for understanding and utilizing eukaryotic rhodopsins.

scholarly journals Inhibitory mechanism of reveromycin A at the tRNA binding site of a class I synthetase

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Bingyi Chen ◽  
Siting Luo ◽  
Songxuan Zhang ◽  
Yingchen Ju ◽  
Qiong Gu ◽  
...  
Keyword(s):  
Binding Site ◽  
Catalytic Domain ◽  
Trna Synthetase ◽  
Rational Drug Design ◽  
Class I ◽  
Binding Assays ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases ◽  
Rational Drug ◽  
Trna Binding

AbstractThe polyketide natural product reveromycin A (RM-A) exhibits antifungal, anticancer, anti-bone metastasis, anti-periodontitis and anti-osteoporosis activities by selectively inhibiting eukaryotic cytoplasmic isoleucyl-tRNA synthetase (IleRS). Herein, a co-crystal structure suggests that the RM-A molecule occupies the substrate tRNAIle binding site of Saccharomyces cerevisiae IleRS (ScIleRS), by partially mimicking the binding of tRNAIle. RM-A binding is facilitated by the copurified intermediate product isoleucyl-adenylate (Ile-AMP). The binding assays confirm that RM-A competes with tRNAIle while binding synergistically with l-isoleucine or intermediate analogue Ile-AMS to the aminoacylation pocket of ScIleRS. This study highlights that the vast tRNA binding site of the Rossmann-fold catalytic domain of class I aminoacyl-tRNA synthetases could be targeted by a small molecule. This finding will inform future rational drug design.

scholarly journals Lysyl-tRNA synthetase from Escherichia coli K12. Chromatographic heterogeneity and the lysU-gene product

1987 ◽  
Vol 248 (1) ◽  
pp. 43-51 ◽  
Author(s):  
J Charlier ◽  
R Sanchez
Keyword(s):  
Escherichia Coli ◽  
Gene Product ◽  
Activity Ratio ◽  
Deae Cellulose ◽  
Trna Synthetase ◽  
Trna Synthetases ◽  
E Coli ◽  
Aminoacyl Trna Synthetases

In contrast with most aminoacyl-tRNA synthetases, the lysyl-tRNA synthetase of Escherichia coli is coded for by two genes, the normal lysS gene and the inducible lysU gene. During its purification from E. coli K12, lysyl-tRNA synthetase was monitored by its aminoacylation and adenosine(5′)tetraphospho(5′)adenosine (Ap4A) synthesis activities. Ap4A synthesis was measured by a new assay using DEAE-cellulose filters. The heterogeneity of lysyl-tRNA synthetase (LysRS) was revealed on hydroxyapatite; we focused on the first peak, LysRS1, because of its higher Ap4A/lysyl-tRNA activity ratio at that stage. Additional differences between LysRS1 and LysRS2 (major peak on hydroxyapatite) were collected. LysRS1 was eluted from phosphocellulose in the presence of the substrates, whereas LysRS2 was not. Phosphocellulose chromatography was used to show the increase of LysRS1 in cells submitted to heat shock. Also, the Mg2+ optimum in the Ap4A-synthesis reaction is much higher for LysRS1. LysRS1 showed a higher thermostability, which was specifically enhanced by Zn2+. These results in vivo and in vitro strongly suggest that LysRS1 is the heat-inducible lysU-gene product.

scholarly journals Demonstration and origin of six tertiary base pair resonances in the NMR spectrum of E. coli tRNAVal1

Nature ◽  
1975 ◽  
Vol 257 (5524) ◽  
pp. 287-291 ◽  
Author(s):  
Brian R. Reid ◽  
George T. Robillard
Keyword(s):  
Base Pair ◽  
Nmr Spectrum ◽  
E Coli

Evolutionary divergence of the Citrobacter freundii tryptophan operon regulatory region: comparison with other enteric bacteria

1982 ◽  
Vol 152 (1) ◽  
pp. 57-62
Author(s):  
M Blumenberg ◽  
C Yanofsky
Keyword(s):  
Regulatory Region ◽  
Enteric Bacteria ◽  
Evolutionary Divergence ◽  
Citrobacter Freundii ◽  
Initial Portion ◽  
Trp Repressor ◽  
E Coli ◽  
Weak Expression ◽  
Dyad Symmetry ◽  
Trp Operon

The regulatory region of the trp operon of Citrobacter freundii was sequenced and compared with the corresponding regions of other enteric bacteria. Significant differences were noted in the promoter region. These differences are presumably responsible for the weak expression of the cloned trp operon in Escherichia coli. The presumed operator region, although nonfunctional in E. coli, has dyad symmetry, but the sequence of the symmetrical region differs appreciably from those of operators that can be regulated by the E. coli trp repressor. The sequence of the trp leader region of C. freundii resembles that of other enteric bacteria, suggesting that the C. freundii operon is also regulated by attenuation. Comparison of the sequence of the initial portion of trpE with the homologous regions of E. coli and Salmonella typhimurium indicates that the three organisms probably are evolutionary equidistant.

Application of RtcB ligase to monitor self-cleaving ribozyme activity by RNA-seq

2022 ◽  
Vol 0 (0) ◽  
Author(s):  
V. Janett Olzog ◽  
Lena I. Freist ◽  
Robin Goldmann ◽  
Jörg Fallmann ◽  
Christina E. Weinberg
Keyword(s):  
Rna Seq ◽  
Site Specific ◽  
Pyrococcus Horikoshii ◽  
Total Rna ◽  
E Coli ◽  
Catalytic Rnas ◽  
Adapter Ligation ◽  
Domains Of Life ◽  
Cyclic Phosphate ◽  
A Site

Abstract Self-cleaving ribozymes are catalytic RNAs and can be found in all domains of life. They catalyze a site-specific cleavage that results in a 5′ fragment with a 2′,3′ cyclic phosphate (2′,3′ cP) and a 3′ fragment with a 5′ hydroxyl (5′ OH) end. Recently, several strategies to enrich self-cleaving ribozymes by targeted biochemical methods have been introduced by us and others. Here, we develop an alternative strategy in which 5ʹ OH RNAs are specifically ligated by RtcB ligase, which first guanylates the 3′ phosphate of the adapter and then ligates it directly to RNAs with 5′ OH ends. Our results demonstrate that adapter ligation to highly structured ribozyme fragments is much more efficient using the thermostable RtcB ligase from Pyrococcus horikoshii than the broadly applied Escherichia coli enzyme. Moreover, we investigated DNA, RNA and modified RNA adapters for their suitability in RtcB ligation reactions. We used the optimized RtcB-mediated ligation to produce RNA-seq libraries and captured a spiked 3ʹ twister ribozyme fragment from E. coli total RNA. This RNA-seq-based method is applicable to detect ribozyme fragments as well as other cellular RNAs with 5ʹ OH termini from total RNA.

Synthetic Riboswitches for the Analysis of tRNA Processing by eukaryotic RNase P Enzymes

RNA ◽  
2022 ◽  
pp. rna.078814.121
Author(s):  
Anna Ender ◽  
Nadine Grafl ◽  
Tim Kolberg ◽  
Sven Findeiss ◽  
Peter F. Stadler ◽  
...  
Keyword(s):  
Homo Sapiens ◽  
Rnase P ◽  
Single Stranded Region ◽  
Domains Of Life ◽  
The Individual ◽  
The Impact ◽  
Individual Enzyme ◽  
Trna Precursor

Removal of the 5' leader region is an essential step in the maturation of tRNA molecules in all domains of life. This reaction is catalyzed by various RNase P activities, ranging from ribonucleoproteins with ribozyme activity to protein-only forms. In Escherichia coli, the efficiency of RNase P mediated cleavage can be controlled by computationally designed riboswitch elements in a ligand-dependent way, where the 5' leader sequence of a tRNA precursor is either sequestered in a hairpin structure or presented as a single-stranded region accessible for maturation. In the presented work, the regulatory potential of such artificial constructs is tested on different forms of eukaryotic RNase P enzymes – two protein-only RNase P enzymes (PRORP1 and PRORP2) from Arabidopsis thaliana and the ribonucleoprotein of Homo sapiens. The PRORP enzymes were analyzed in vitro as well as in vivo in a bacterial RNase P complementation system. We also tested in HEK293T cells whether the riboswitches remain functional with human nuclear RNase P. While the regulatory principle of the synthetic riboswitches applies for all tested RNase P enzymes, the results also show differences in the substrate requirements of the individual enzyme versions. Hence, such designed RNase P riboswitches represent a novel tool to investigate the impact of the structural composition of the 5'-leader on substrate recognition by different types of RNase P enzymes.

scholarly journals Measuring the tolerance of the genetic code to altered codon size

2021 ◽  
Author(s):  
E. DeBenedictis ◽  
D. Söll ◽  
K. Esvelt
Keyword(s):  
Genetic Code ◽  
Protein Translation ◽  
Single Amino Acid ◽  
Trna Synthetases ◽  
E Coli ◽  
Aminoacyl Trna Synthetases ◽  
Triplet Codon ◽  
Orthogonal Set ◽  
Expanded Genetic Code ◽  
Primordial Earth

SummaryProtein translation using four-base codons occurs in both natural and synthetic systems. What constraints contributed to the universal adoption of a triplet-codon, rather than quadruplet-codon, genetic code? Here, we investigate the tolerance of the E. coli genetic code to tRNA mutations that increase codon size. We found that tRNAs from all twenty canonical isoacceptor classes can be converted to functional quadruplet tRNAs (qtRNAs), many of which selectively incorporate a single amino acid in response to a specified four-base codon. However, efficient quadruplet codon translation often requires multiple tRNA mutations, potentially constraining evolution. Moreover, while tRNAs were largely amenable to quadruplet conversion, only nine of the twenty aminoacyl tRNA synthetases tolerate quadruplet anticodons. These constitute a functional and mutually orthogonal set, but one that sharply limits the chemical alphabet available to a nascent all-quadruplet code. Our results illuminate factors that led to selection and maintenance of triplet codons in primordial Earth and provide a blueprint for synthetic biologists to deliberately engineer an all-quadruplet expanded genetic code.

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