Part:BBa_K5377100

Congregibacter litoralis KT71 SAM-I/IV variant riboswitch

Description

This SAM-I/IV variant riboswitch in Congregibacter litoralis KT71^[1] is a unique RNA regulatory element that controls genes involved in sulfur metabolism by binding S-adenosylmethionine (SAM), a vital metabolite in methylation and sulfur transfer reactions. Riboswitches are structured RNA molecules that can directly bind metabolites and modulate gene expression without the need for proteins.

Fig.1 The secondary structure of the SAM-I/IV variant riboswitch. (From Wikipedia)

The SAM-I/IV riboswitch is a hybrid of two distinct riboswitch classes: SAM-I and SAM-IV. SAM-I riboswitches, one of the most studied riboswitch classes, regulate genes responsible for SAM biosynthesis and sulfur metabolism in many bacteria. SAM-IV riboswitches are a more recently discovered class with some structural differences but similar function.^[2] They combine features of SAM-I and SAM-IV riboswitches. For example, they have the P4 outside of the multistem junction, like SAM-IV riboswitches, but also often have a SAM-I–like P4 within the multistem junction. However, unlike SAM-I and SAM-IV riboswitches, the variant riboswitches entirely lack an internal loop in P2. As noted above, this internal loop in SAM-I riboswitches forms a kink turn motif that allows the RNA to form a pseudoknot. The variant riboswitches lack an internal loop that might allow such a turn, and also show no signs of forming the SAM-I–like pseudoknot.

Usage and Biology

It is SAM-I/IV variant riboswitch from Congregibacter litoralis KT71, capable of regulating methionine synthesis. SAM (S-adenosyl methionine) can bind to the aptamer domain of the riboswitch as a ligand, causing a local conformational change that inhibits downstream gene expression.
In the absence of SAM, the riboswitch adopts a structure that allows the transcription of the downstream genes, promoting SAM biosynthesis. When SAM is abundant, it binds to the riboswitch, triggering a structural rearrangement that halts the production of enzymes required for SAM biosynthesis. This regulatory mechanism is highly conserved and plays a crucial role in maintaining proper cellular SAM levels.
By replacing the downstream genes of the riboswitch sequence with the lacI gene, the riboswitch directly regulates the expression of the repressor protein instead of controlling SAM biosynthesis. Our team use this method to design the SAM self-induced production system. For more details, please refer to the BBa_K5377810page.

Characterization

Introduction

To validate the function of the riboswitch, we set up different SAM concentration gradients and added them manually.

Methods

We constructed the riboswitch 'OFF system' plasmid by using Gibson Assembly to insert the riboswitch fragment and sfgfp fragment into the pHG101 plasmid. This was then transformed into the E.coli MG1655 strain for fermentation. We took 200 μL of overnight culture solution, incubated it for 4 hours, and added different concentrations of SAM. Wild-type strains were used as negative controls, and strains with a dysfunctional riboswitch were used as positive controls. 4 hours later, we collected samples, centrifuged them at 13,000 rpm and 4℃ for 2 minutes, resuspended them in PBS buffer, and repeated this process three times. We then measured the optical density (OD) and fluorescence intensity. (excitation at 488 nm and emission at 509 nm)

Results

Fig.3 Downregulation of sfgfp expression by the SAM-I/IV variant riboswitch at different SAM concentrations.

We successfully validated the OFF function of the riboswitch. For both the positive and negative controls, there were no significant differences in the fluorescence intensity of the fermentation cultures when different concentrations of SAM were added. However, for the strain containing the complete riboswitch, the addition of SAM resulted in a significant decrease in the fluorescence intensity of the fermentation cultures. Moreover, within a certain concentration range, higher ligand concentrations led to more pronounced decreases in fluorescence intensity. For the SAM-I/IV variant riboswitch, the fluorescence intensity decreased at SAM concentrations of 0.1 mM, 0.2 mM, 0.3 mM, and 0.4 mM, with a maximum reduction of 93% at 0.4mM. (The results are shown in Fig. 3.)

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]