Part:BBa_K4171007

tRNA (Ser)

Background

This is the native tRNA^Ser sequence (BBa_K4171007) of E. coli which can bind with serine (Ser) by the help of enzyme SerRS (BBa_K4171001).

Usage

tRNA^Ser is used for aminoacylation with Ser, which becomes seryl-tRNA^Ser, the main substrate of SelA (BBa_K4171002). In addition, because of its structure, research has proved that tRNA^Ser has better aminoacylation efficiency with SerRS than tRNA^Sec and tRNA^{UTuX [1]}.

Fig. 1. In vitro Sec synthesis pathway

Characterization

Fig. 2. Confirmation of in vitro tRNA^Ser (BBa_K4171007) synthesis by agarose gel electrophoresis. M: Marker; Lane1: tRNA^Ser (88bp)

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

Fig. 3. Confirmation of the result after aminoacylation by Urea PAGE. Lane1: tRNA^Ser (BBa_K4171007) ; Lane2: seryl-tRNA^Ser

After successful aminoacylation, serine on the tRNA was transformed into Sec. In the HPLC results, the retention time of the Sec standard is 10.5 minutes (the peaks at 3.5 minutes and 4.5 minutes are solvent peaks), which is the same as that of Sec synthesized via the in vitro pathway, indicating that both tRNA and enzymes (BBa_K4171002 and BBa_K4171003) function as expected.

Fig. 4. Confirmation of Sec by HPLC. This is the result of Sec synthesized by tRNA^Ser (BBa_K4171007).

Reference

[1]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

[2]Asahara H, Himeno H, Tamura K, Nameki N, Hasegawa T, Shimizu M. Escherichia coli Seryl-tRNA Synthetase Recognizes tRNASer by Its Characteristics Tertiary Structure. Journal of Molecular Biology. 1994;236(3):738-748. doi:10.1006/jmbi.1994.1186

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
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 27
  
• 1000
  COMPATIBLE WITH RFC[1000]