Part:BBa_K2012000

c-di-GMP tandem riboswitche bc3-5

Three complete c-di-GMP riboswitches (Bc3, Bc4 and Bc5 RNA) with similar structures, which are arranged in tandem to constitute a triple-tandem (Bc3-5 RNA) riboswitch in the 5′-UTR of the cspABCDE mRNA in Bacillus thuringiensis subsp. chinensis CT-43. They are c-di-GMP riboswitches (termed c-di-GMP-I), c-di-GMP-I binds the second messenger c-di-GMP through hydrogen bonding or base stacking of the two guanosines of c-di-GMP with five specific bases in the aptamer, resulting in transcriptional changes of downstream genes correlating with c-di-GMP function.

Figure 1. Illustration of riboswitch mechanism. As shown in the illustration, transcript of riboswitches region would form a hairpin and terminate transcription under low concentration of c-di-GMP.

Figure 2. Schematic of riboswitches. (a) Comparison of Bc3, Bc4, Bc5 terminators. Red rectangle shows the GC rich region of three terminators, respectively. (b) Multiply local sequence blast of riboswitches’ terminators. Blue rectangle shows U region of riboswitches.

Figure 3. Characterization of Riboswitch. PC: pET-28-pleD\J23106+sfGFP 64F: pET-28-pleD\ J23106+Bc3+sfGFP 65F: pET-28-pleD\ J23106+Bc5+sfGFP 644F: pET-28-pleD\ J23106+Bc4+Bc4+sfGFP 635F: pET-28-pleD\ J23106+Bc3-5+sfGFP 74F: pET-28-pleD\ J23117+Bc3+sfGFP 75F: pET-28-pleD\ J23117+Bc5sfGFP 735F: pET-28-pleD\ J23117+Bc3-5+sfGFP.

Figure 4. Characterization of Riboswitch. We use “read-through rate” (RTR) of the downstream gene to measure the terminator forming efficiency of a riboswitch. The read-through rate of each riboswitch in liquid medium was assessed by relative fluorescence intensity, which is the ratio of specific activity of a test strain to specific activity of the control strain (pET-28-pleD/J23117+sfGFP) with the same promoter of test circuits (such as J23117+Bc3-5+sfGFP).

Usage and Biology

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]