Coordination of tRNA Synthetase Active Sites for Chemical Fidelity

Boniecki, Michal T.; Martinis, Susan A.

doi:10.1074/jbc.c111.325795

Cited by 11 publications

(17 citation statements)

References 41 publications

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“…Our recent work revealed that fusion of the CP1 domains to M. mobile LeuRS can improve amino acid selectivity for aminoacylation (30 conformation more suitable for pretransfer editing and amino acid selection. In many cases, this activity seems to be enhanced in the presence of tRNA (20,21,23).…”

Section: Discussionmentioning

confidence: 99%

“…As such, the CP1 domain from E. coli LeuRS was fused to M. mobile LeuRS (MmLeuRS/CP1 Leu ) (30). We also constructed hybrids using the CP1 domains from IleRS (MmLeuRS/CP1 Ile ) and ValRS (MmLeuRS/CP1 Val ) (30). Each of the hybrid LeuRSs displayed leucylation activities comparable to the wild-type M. mobile enzyme (30) (Fig.…”

Section: X-ray Crystal Structure Of M Mobile Leurs-leuams Is Similar Tomentioning

confidence: 99%

“…We asked whether this increased fidelity could be attributed to increases in amino acid discrimination in the aminoacylation site (30) or enhanced pretransfer editing activity, or a combination of both mechanisms. Whereas the k cat was similar for leucine (11 ± 2 s −1 ) and isoleucine (9 ± 2 s −1 ), PP i exchange assays showed that the K m was, respectively, 0.03 ± 0.01 mM and 2.7 ± 0.7 mM, resulting in a discrimination factor of just 1/111.…”

Section: X-ray Crystal Structure Of M Mobile Leurs-leuams Is Similar Tomentioning

confidence: 99%

“…Thus, we hypothesized that we can use M. mobile LeuRS as a scaffold to reconstruct evolutionary events that adapted the aaRS for increased accuracy. As such, the CP1 domain from E. coli LeuRS was fused to M. mobile LeuRS (MmLeuRS/CP1 Leu ) (30). We also constructed hybrids using the CP1 domains from IleRS (MmLeuRS/CP1 Ile ) and ValRS (MmLeuRS/CP1 Val ) (30).…”

Section: X-ray Crystal Structure Of M Mobile Leurs-leuams Is Similar Tomentioning

confidence: 99%

“…Using this M. mobile LeuRS core as a scaffold, we created a series of LeuRS hybrids with CP1 domains from different origins as well as from IleRS and ValRS (30). Remarkably, we determined that the addition of the CP1 domain enhances pretransfer editing activity and fidelity for each of these diverse hybrid models.…”

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confidence: 99%

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Leucyl-tRNA synthetase editing domain functions as a molecular rheostat to control codon ambiguity in Mycoplasma pathogens

Palencia

Lukk³

et al. 2013

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

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Mycoplasma leucyl-tRNA synthetases (LeuRSs) have been identified in which the connective polypeptide 1 (CP1) amino acid editing domain that clears mischarged tRNAs are missing (Mycoplasma mobile) or highly degenerate (Mycoplasma synoviae). Thus, these enzymes rely on a clearance pathway called pretransfer editing, which hydrolyzes misactivated aminoacyl-adenylate intermediate via a nebulous mechanism that has been controversial for decades. Even as the sole fidelity pathway for clearing amino acid selection errors in the pathogenic M. mobile, pretransfer editing is not robust enough to completely block mischarging of tRNA Leu , resulting in codon ambiguity and statistical proteins. A high-resolution X-ray crystal structure shows that M. mobile LeuRS structurally overlaps with other LeuRS cores. However, when CP1 domains from different aminoacyl-tRNA synthetases and origins were fused to this common LeuRS core, surprisingly, pretransfer editing was enhanced. It is hypothesized that the CP1 domain evolved as a molecular rheostat to balance multiple functions. These include distal control of specificity and enzyme activity in the ancient canonical core, as well as providing a separate hydrolytic active site for clearing mischarged tRNA.protein evolution | quality control | statistical proteins | aminoacylation | translation

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Section: Discussionmentioning

confidence: 99%

Section: X-ray Crystal Structure Of M Mobile Leurs-leuams Is Similar Tomentioning

confidence: 99%

Section: X-ray Crystal Structure Of M Mobile Leurs-leuams Is Similar Tomentioning

confidence: 99%

Section: X-ray Crystal Structure Of M Mobile Leurs-leuams Is Similar Tomentioning

confidence: 99%

mentioning

confidence: 99%

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Leucyl-tRNA synthetase editing domain functions as a molecular rheostat to control codon ambiguity in Mycoplasma pathogens

Palencia

Lukk³

et al. 2013

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

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Exploring the Substrate Range of Wild‐Type Aminoacyl‐tRNA Synthetases

Fan

Chirathivat

et al. 2014

ChemBioChem

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We tested the substrate range of four wild-type Escherichia coli aminoacyl-tRNA synthetases (AARSs) with a library of non-standard amino acids (nsAAs). While these AARSs could discriminate efficiently against the other canonical amino acids, they were able to use many nsAAs as substrates. Our results also showed E. coli tryptophanyl-tRNA synthetase (TrpRS) and tyrosyl-tRNA synthetase to have overlapping substrate ranges. In addition, we found that the nature of the anticodon sequence of tRNATrp altered the nsAA substrate range of TrpRS; this implies that the sequence of the anticodon affects the TrpRS amino acid binding pocket. These results highlight again that inherent AARS polyspecificity will be a major challenge to the goal of incorporating multiple different amino acids site-specifically into proteins.

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Altering the Enantioselectivity of Tyrosyl-tRNA Synthetase by Insertion of a Stereospecific Editing Domain

Richardson

First

2016

Biochemistry

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Translation of mRNAs by the ribosome is stereospecific, with only l-amino acids being incorporated into the nascent polypeptide chain. This stereospecificity results from the exclusion of d-amino acids at three steps during protein synthesis: (1) the aminoacylation of tRNA by aminoacyl-tRNA synthetases, (2) binding of aminoacyl-tRNAs to EF-Tu, and (3) recognition of aminoacyl-tRNAs by the ribosome. As a first step toward incorporating d-amino acids during protein synthesis, we have altered the enantioselectivity of tyrosyl-tRNA synthetase. This enzyme is unusual among aminoacyl-tRNA synthetases, as it can aminoacylate tRNA with d-tyrosine (albeit at a reduced rate compared to l-tyrosine). To change the enantioselectivity of tyrosyl-tRNA synthetase, we introduced the post-transfer editing domain from Pyrococcus horikoshii phenylalanyl-tRNA synthetase into the connective polypeptide 1 (CP1) domain of Geobacillus stearothermophilus tyrosyl-tRNA synthetase (henceforth designated TyrRS-FRSed). We show that the phenylalanyl-tRNA synthetase editing domain is stereospecific, hydrolyzing l-Tyr-tRNA(Tyr), but not d-Tyr-tRNA(Tyr). We further show that inserting the phenylalanyl-tRNA synthetase editing domain into the CP1 domain of tyrosyl-tRNA synthetase decreases the activity of the synthetic site in tyrosyl-tRNA synthetase. This decrease in activity is critical, as it prevents the rate of synthesis from overwhelming the ability of the editing domain to hydrolyze the l-Tyr-tRNA(Tyr) product. Overall, inserting the phenylalanyl-tRNA synthetase editing domain results in a 2-fold shift in the enantioselectivity of tyrosyl-tRNA synthetase toward the d-Tyr-tRNA(Tyr) product. When a 4-fold excess of d-tyrosine is used, approximately 40% of the tRNA(Tyr) is aminoacylated with d-tyrosine.

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Coordination of tRNA Synthetase Active Sites for Chemical Fidelity

Cited by 11 publications

References 41 publications

Leucyl-tRNA synthetase editing domain functions as a molecular rheostat to control codon ambiguity in Mycoplasma pathogens

Leucyl-tRNA synthetase editing domain functions as a molecular rheostat to control codon ambiguity in Mycoplasma pathogens

Exploring the Substrate Range of Wild‐Type Aminoacyl‐tRNA Synthetases

Altering the Enantioselectivity of Tyrosyl-tRNA Synthetase by Insertion of a Stereospecific Editing Domain

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