Part:BBa_K4171008

tRNA (Sec)

Background

This part is the native tRNA^Sec (BBa_K4171008) sequence of E. coli. Since selenocysteine (Sec) does not have its own aminoacyl-tRNA synthetase, it uses seryl-tRNA synthetase (SerRS) to acylate tRNA^Sec with serine (Ser) in native Sec synthesis pathway. Then, L-seryl-tRNA^Sec selenium transferase (SelA) will transform seryl-tRNA^Sec into selenocysteinyl-tRNA^Sec[1].

Usage

tRNA^Sec (BBa_K4171008) is used for aminoacylation with Ser, which becomes seryl-tRNA^Sec, the main substrate of SelA (BBa_K4171002). Nevertheless, according to research^[2], tRNA^Sec was proved to have poorer reaction efficiency with SerRS (BBa_K4171001) than tRNA^Ser (BBa_K4171007).

Fig. 1. In vitro Sec synthesis pathway

Characterization

Fig. 2. Confirmation of in vitro tRNA^Sec (BBa_K4171008) synthesis by agarose gel electrophoresis. M: Marker; Lane1: tRNA^Sec (95bp)

The bands of seryl-tRNA^Sec have upshifted compared to the band of the tRNA^Sec (Fig. 3). The result shows that the amino acid was successfully charged to the tRNA.

Fig. 2. Confirmation of the result after aminoacylation by Urea PAGE. Lane1: tRNA^Sec (BBa_K4171008); Lane2: seryl-tRNA^Sec

Reference

[1] Fu X, Crnković A, Sevostyanova A, Söll D. Designing seryl‐ tRNA synthetase for improved serylation of selenocysteine tRNA s. FEBS Letters. 2018;592(22):3759-3768. doi:10.1002/1873-3468.13271

[2] Fu X, Söll D, Sevostyanova A. Challenges of site-specific selenocysteine incorporation into proteins by Escherichia coli. RNA Biology. 2018;15(4-5):461-470. doi:10.1080/15476286.2018.1440876

[3] Escherichia sp. E10V4 tRNA-Sec | URS00002F9CDD. rnacentral.org. Accessed September 26, 2022. https://rnacentral.org/rna/URS00002F9CDD/2478971

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]