Translating protein enzymes without aminoacyl-tRNA synthetases

Ji, Chen; Chen, Mengyin; Zhu, Ting

doi:10.1016/j.chempr.2021.01.017

Cited by 10 publications

(12 citation statements)

References 50 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Toward realizing mirror-image translation, we previously used a mirror-image version of the 46-nt dinitro-flexizyme (dFx) ( 28 ) to charge three d -amino acids ( d -lysine, d -alanine, and d -phenylalanine) and the achiral glycine onto the corresponding mirror-image transfer RNAs (tRNAs) transcribed by d -Dpo4-5m-Y12S ( 10 ). However, d -Dpo4-5m-Y12S was unable to transcribe certain mirror-image tRNA sequences, such as the 90-nt mirror-image serine tRNA (tRNA Ser ) (fig.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Mirror-image T7 transcription of chirally inverted ribosomal and functional RNAs

Zhu

2022

Science

View full text Add to dashboard Cite

To synthesize a chirally inverted ribosome with the goal of building mirror-image biology systems requires the preparation of kilobase-long mirror-image ribosomal RNAs that make up the structural and catalytic core and about two-thirds of the molecular mass of the mirror-image ribosome. Here, we chemically synthesized a 100-kilodalton mirror-image T7 RNA polymerase, which enabled efficient and faithful transcription of the full-length mirror-image 5 S , 16 S , and 23 S ribosomal RNAs from enzymatically assembled long mirror-image genes. We further exploited the versatile mirror-image T7 transcription system for practical applications such as biostable mirror-image riboswitch sensor, long-term storage of unprotected kilobase-long l -RNA in water, and l -ribozyme–catalyzed l -RNA polymerization to serve as a model system for basic RNA research.

show abstract

Section: Resultsmentioning

confidence: 99%

“…After mirror-image DNA replication, transcription, and reverse transcription (2,(4)(5)(6)(7)(8), the next step is to realize mirror-image translation (Fig. 1A) (3,9,10). This requires high-quality long L-RNAs such as the kilobase-long mirrorimage ribosomal RNAs (rRNAs), which make up the structural and catalytic core of the >2-MDa mirror-image ribosome.…”

mentioning

confidence: 99%

Mirror-image T7 transcription of chirally inverted ribosomal and functional RNAs

Zhu

2022

Science

View full text Add to dashboard Cite

show abstract

“…38,40 Another possibility is the addition of self-cleaving ribozymes, such as hammerhead ribozymes, at the 5′-end of tRNAs. 29,41 Further modifications of the sequence and experimental conditions would be required for these methods to be used in the tfPURE system.…”

Section: ■ Discussionmentioning

confidence: 99%

“…For this challenge, the six tRNAs starting with non-guanine nucleotides must be expressed through in vitro transcription. A possible method is to use RNase P, which cuts a certain position of RNA. , Another possibility is the addition of self-cleaving ribozymes, such as hammerhead ribozymes, at the 5′-end of tRNAs. , Further modifications of the sequence and experimental conditions would be required for these methods to be used in the tfPURE system.…”

Section: Discussionmentioning

confidence: 99%

“…In Escherichia coli, a set of tRNAs with 41 different anticodons serves this function, although, theoretically, 21 tRNAs are sufficient for encoding the 20 amino acids and formylmethionine. In previous studies, in vitro translation using 21 chemically synthesized or in-vitro-transcribed − tRNA sets has been demonstrated. However, in these studies, tRNAs were synthesized and purified before being used for translation.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Transfer RNA Synthesis-Coupled Translation and DNA Replication in a Reconstituted Transcription/Translation System

2022

View full text Add to dashboard Cite

Transfer RNAs (tRNAs) are key molecules involved in translation. In vitro synthesis of tRNAs and their coupled translation are important challenges in the construction of a self-regenerative molecular system. Here, we first purified EF-Tu and ribosome components in a reconstituted translation system of Escherichia coli to remove residual tRNAs. Next, we expressed 15 types of tRNAs in the repurified translation system and performed translation of the reporter luciferase gene depending on the expression. Furthermore, we demonstrated DNA replication through expression of a tRNA encoded by DNA, mimicking information processing within the cell. Our findings highlight the feasibility of an in vitro selfreproductive system, in which tRNAs can be synthesized from replicating DNA.

show abstract

Strategies for Detecting Aminoacylation and Aminoacyl‐tRNA Editing In Vitro and In Cells

Watkins,

Kavoor,

Musier‐Forsyth

2024

Israel Journal of Chemistry

View full text Add to dashboard Cite

Aminoacyl‐tRNA synthetases (aaRSs) maintain translational fidelity by ensuring the formation of correct aminoacyl‐tRNA pairs. Numerous point mutations in human aaRSs have been linked to disease phenotypes. Structural studies of aaRSs from human pathogens encoding unique domains support these enzymes as potential candidates for therapeutics. Studies have shown that the identity of tRNA pools in cells changes between different cell types and under stress conditions. While traditional radioactive aminoacylation analyses can determine the effect of disease‐causing mutations on aaRS function, these assays are not amenable to drug discovery campaigns and do not take into account the variability of the intracellular tRNA pools. Here, we review modern techniques to characterize aaRS activity in vitro and in cells. The cell‐based approaches analyse the aminoacyl‐tRNA pool to observe trends in aaRS activity and fidelity. Taken together, these approaches allow high‐throughput drug screening of aaRS inhibitors and systems‐level analyses of the dynamic tRNA population under a variety of conditions and disease states.

show abstract

Translating protein enzymes without aminoacyl-tRNA synthetases

Cited by 10 publications

References 50 publications

Mirror-image T7 transcription of chirally inverted ribosomal and functional RNAs

Mirror-image T7 transcription of chirally inverted ribosomal and functional RNAs

Transfer RNA Synthesis-Coupled Translation and DNA Replication in a Reconstituted Transcription/Translation System

Strategies for Detecting Aminoacylation and Aminoacyl‐tRNA Editing In Vitro and In Cells

Contact Info

Product

Resources

About