Synthesis and Aminoacyl-tRNA Synthetase Inhibitory Activity of Prolyl Adenylate Analogs

Heacock, Donald H.; Forsyth, Craig J.; Shiba, Kiyotaka; Musier‐Forsyth, Karin

doi:10.1006/bioo.1996.0025

Cited by 112 publications

(122 citation statements)

References 35 publications

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“…WT Sc ProRS, ⌬N183 Sc ProRS, WT Deinococcus radiodurans ProRS, WT Ec AlaRS, and the Ec ProRS INS domain were purified using a Talon cobalt affinity resin (Clontech, Mountain View, CA) as described (9,43). Plasmids encoding the Ec/Sc chimera and the K279A Ec/Sc chimera were transformed into Ec SG13009 [pREP4] (Qiagen) competent cells and protein expression was induced with 0.1 mM isopropyl ␤-D-thiogalactoside for 22 h at room temperature.…”

Section: Methodsmentioning

confidence: 99%

Restoring species-specific posttransfer editing activity to a synthetase with a defunct editing domain

SternJohn

Hati

Siliciano

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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In contrast, the isolated INS domain displays only weak editing activity and lacks tRNA sequence specificity. These results emphasize the modular nature of synthetase editing active sites and demonstrate how in evolution, a weak editing activity can be converted to a more robust state through fusion to the body of a synthetase. In this manner, a single editing module can be distributed to different synthetases, and simultaneously acquire specificity and enhanced activity.Prolyl-tRNA synthetase ͉ Saccharomyces cerevisiae

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Section: Methodsmentioning

confidence: 99%

Restoring species-specific posttransfer editing activity to a synthetase with a defunct editing domain

SternJohn

Hati

Siliciano

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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“…Enzyme Preparation-Wild-type (WT) E. coli ProRS and AlaRS, and WT Methanococcus jannaschii ProRS were purified using the Talon cobalt affinity technique (Clontech) as previously described (33). WT H. influenzae YbaK protein was purified using the IMPACT TM I system (New England Biolabs) as described (16,34).…”

Section: Methodsmentioning

confidence: 99%

Cys-tRNAPro Editing by Haemophilus influenzae YbaK via a Novel Synthetase·YbaK·tRNA Ternary Complex

Musier‐Forsyth

2005

Journal of Biological Chemistry

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Aminoacyl-tRNA synthetases are multidomain enzymes that often possess two activities to ensure translational accuracy. A synthetic active site catalyzes tRNA aminoacylation, while an editing active site hydrolyzes mischarged tRNAs. Prolyl-tRNA synthetases (ProRS) have been shown to misacylate Cys onto tRNA Pro , but lack a Cys-specific editing function. The synthetase-like Haemophilus influenzae YbaK protein was recently shown to hydrolyze misacylated Cys-tRNA Pro in trans. However, the mechanism of specific substrate selection by this single domain hydrolase is unknown. Here, we demonstrate that YbaK alone appears to lack specific tRNA recognition capabilities. Moreover, YbaK cannot compete for aminoacyl-tRNAs in the presence of elongation factor Tu, suggesting that YbaK acts before release of the aminoacyl-tRNA from the synthetase. In support of this idea, cross-linking studies reveal the formation of binary (ProRS⅐YbaK) and ternary (ProRS⅐YbaK⅐tRNA) complexes. The binding constants for the interaction between ProRS and YbaK are 550 nM and 45 nM in the absence and presence of tRNA Pro , respectively. These results suggest that the specificity of trans-editing by YbaK is ensured through formation of a novel ProRS⅐YbaK⅐tRNA complex.Aminoacyl-tRNA synthetases maintain the high fidelity of protein synthesis by activating specific amino acids and transferring them to cognate tRNAs in a process known as aminoacylation. However, some synthetases are known to misactivate noncognate amino acids, transferring them onto their cognate tRNAs. Therefore, to ensure accurate translation of the genetic code, these enzymes, which include members of both classes of synthetases, have evolved proofreading or editing functions (1-17). Pretransfer editing involves hydrolysis of the misactivated aminoacyl-adenylate, whereas post-transfer editing refers to specific hydrolysis of misacylated tRNA.To clear noncognate amino acids, a double-sieve mechanism was proposed, wherein larger amino acids are rejected by the aminoacylation active site (i.e. coarse sieve) while smaller amino acids are cleared in a second editing active site (i.e. fine sieve) (18,19). This hypothesis was confirmed by more recent biochemical and x-ray crystallographic studies (20). Although the exact site of pretransfer editing is still an open question in most systems (21), class I editing enzymes, including isoleucyl-tRNA synthetase, valyl-tRNA synthetase, and leucyl-tRNA synthetase, use the highly conserved connective polypeptide 1 domain to edit misacylated tRNAs (1, 2, 4 -8, 12). In contrast, the post-transfer editing domains used by class II synthetases are much more diverse. The internal editing domain of alanyl-tRNA synthetase (AlaRS) 2 and the N-terminal editing domain of threonyl-tRNA synthetase (with the exception of the archaebacterial enzymes) share sequence similarity (9, 22), while the editing domains of bacterial prolyl-tRNA synthetase (ProRS) (13) and phenylalanyl-tRNA synthetase (17) appear to be unique, sharing no sequence homology to any of the ot...

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“…Human LysRS (Shiba et al 1997), LysRSΔN65 (Shiba et al 1997), TrpRS (Wakasugi et al 2002), and ProRS (Heacock et al 1996) were prepared as previously described. Each protein contains a His 6 tag and was purified using Ni 2+ -NTA resin (Qiagen), Co…”

Section: Preparation Of Proteins and Nucleic Acidsmentioning

confidence: 99%

“…Active site titrations were used for assaying TrpRS and ProRS enzyme activities (Fersht et al 1975). Enzyme activity was additionally tested using aminoacylation assays with in vitro transcribed human tRNA Lys3 in the case of hLysRS and hLysRSΔN65 and human tRNA Pro in the case of ProRS (Heacock et al 1996;Shiba et al 1997 , 180-nt RSV RNA, and 98-nt MAL123-218 RNA-were prepared by in vitro transcription using T7 RNA polymerase (Milligan et al 1987) . The 76-nt tRNA Lys3 used in this study was prepared as described previously (Shiba et al 1997).…”

Section: +mentioning

confidence: 99%

Molecular mimicry of human tRNA^Lys anti-codon domain by HIV-1 RNA genome facilitates tRNA primer annealing

Jones

Saadatmand

Kleiman

et al. 2012

RNA

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The primer for initiating reverse transcription in human immunodeficiency virus type 1 (HIV-1) is tRNA Lys3. Host cell tRNA Lys is selectively packaged into HIV-1 through a specific interaction between the major tRNA Lys -binding protein, human lysyl-tRNA synthetase (hLysRS), and the viral proteins Gag and GagPol. Annealing of the tRNA primer onto the complementary primerbinding site (PBS) in viral RNA is mediated by the nucleocapsid domain of Gag. The mechanism by which tRNA Lys3 is targeted to the PBS and released from hLysRS prior to annealing is unknown. Here, we show that hLysRS specifically binds to a tRNA anti-codon-like element (TLE) in the HIV-1 genome, which mimics the anti-codon loop of tRNA Lys and is located proximal to the PBS. Mutation of the U-rich sequence within the TLE attenuates binding of hLysRS in vitro and reduces the amount of annealed tRNA Lys3 in virions. Thus, LysRS binds specifically to the TLE, which is part of a larger LysRS binding domain in the viral RNA that includes elements of the Psi packaging signal. Our results suggest that HIV-1 uses molecular mimicry of the anti-codon of tRNA Lys to increase the efficiency of tRNA Lys3 annealing to viral RNA.

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Synthesis and Aminoacyl-tRNA Synthetase Inhibitory Activity of Prolyl Adenylate Analogs

Cited by 112 publications

References 35 publications

Restoring species-specific posttransfer editing activity to a synthetase with a defunct editing domain

Restoring species-specific posttransfer editing activity to a synthetase with a defunct editing domain

Cys-tRNAPro Editing by Haemophilus influenzae YbaK via a Novel Synthetase·YbaK·tRNA Ternary Complex

Molecular mimicry of human tRNA^Lys anti-codon domain by HIV-1 RNA genome facilitates tRNA primer annealing

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Synthesis and Aminoacyl-tRNA Synthetase Inhibitory Activity of Prolyl Adenylate Analogs

Cited by 112 publications

References 35 publications

Restoring species-specific posttransfer editing activity to a synthetase with a defunct editing domain

Restoring species-specific posttransfer editing activity to a synthetase with a defunct editing domain

Cys-tRNAPro Editing by Haemophilus influenzae YbaK via a Novel Synthetase·YbaK·tRNA Ternary Complex

Molecular mimicry of human tRNALys anti-codon domain by HIV-1 RNA genome facilitates tRNA primer annealing

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Molecular mimicry of human tRNA^Lys anti-codon domain by HIV-1 RNA genome facilitates tRNA primer annealing