Part:BBa_K598001

Theophylline Responsive Riboswitch 1G1 with Engineered RBS+GFP generator

This is a GFP generator regulated by theophylline responsive riboswitch. mRNA with a theophylline riboswitch in it responds to theophylline concerntration, producing different fluorescence strength of GFP. Regulated by different promoters, it would be used to demonstrate theophylline responsive curve, providing data for modeling.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 71
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 730
  

Background

Riboswitches are structured non-coding RNA domains performing as genetic control elements, regulating gene expression through ligand-RNA interactions without assistance of proteins. Several natural riboswitches have been reported, sensing small molecules including vitamin B1 and B12, FMN, SAM, lysine, guanine, adenine, TPP and theophylline [1]. Various mechanisms are utilized to regulate gene expression, ranging from transcription initiation to translation attenuation.

Figure.1 Growth curves of E.coli under treatments of different theophylline concentrations. Under 10mM theophylline concentration there is negligible growth inhibition.

Theophylline responsive riboswitches have been widely studied in recent years. Theophylline is small molecule which is passable through bacterial cytomembrane, but not an essential nutrient of bacteria. Although theophylline has moderate toxicity leading to growth inhibition in logarithmic phase with 15mM theophylline in LB medium, this theophylline responsive riboswitch shows an approving regulation curve under 10mM theophylline concentration with negligible growth inhibition in M9 liquid medium (Fig.1).

Experimental Data

Parental 1G1

We adopted the original theophylline responsive riboswitch, named Parental 1G1 (P1G1) in our project, designed by Beatrix Suess on Nucleic Acids Research, as a candidate of our RNA controller toolbox. Stereo representation of the three-dimensional structure of theophylline aptamer is schematized in Fig.2.

Theophylline responsive riboswitch P1G1 integrates the theophylline aptamer with a helical communication module based on a ligand–dependent one-nucleotide slipping mechanism (Fig.3) [2]. This structural element lies at a position just interfering with translation in the non ligand-binding form. Addition of ligand shifts the inhibitory secondary structure element that is originally closed to RBS to a distance that permits efficient translation. This slippage mechanism regulating gene expression makes it different from natural riboswtiches those are based on sequenstration or antitermination [3].

Figure.2 Schematic of the theophylline aptamer structure.Red: theophylline; yellow: theophylline binding site. .

Figure.3 Predicted secondary structure of P1G1 in the absence and presence of theophylline. Theophylline aptamer is boxed. 1nt slipping mechanism is shown in yellow. UGGA is shown in red as RBS sequence. AUG initiation codon is in black. .

We constructed a device via PCR and standard assembly. It composes of RNA controller P1G1 which contains original RBS sequence and GFP as reporter gene (Fig.4). A graph of predicted secondary structure (by mFold) of translation initiation region with introduced regulatory element P1G1 containing theophylline aptamer (boxed) and stem-loop structure is shown in Fig.4. The proposed theophylline-mediated slipping mechanism of the bridge domain is shown schematically as well.

Figure.4 Scheme of constructs designed to demonstrate the modularity of theophylline riboswitches.P1G1 composes of pBAD promoter, theophylline responsive riboswitch, GFP BBa_E0040 and Terminator BBa_B0015 with original RBS (UGGA). 1G1 composes of pBAD promoter, theophylline responsive riboswitch, GFP BBa_E0040 and Terminator BBa_B0015 with optimized RBS (AGGAGGU). .

Figure.5 Dose responses of P1G1 riboswitch and 1G1 riboswitch.(A) Photo shows GFP fluorescence intensity induced by gradients of theophylline concentration. The left most well denotes induction in the absence of theophylline. GFP fluorescence intensity increases with the concentration of theophylline. (B) Dose responsive curves of 1G1 and P1G1 with a theophylline concentration gradient in the presence of arabinose. RBS sequences and corresponding dG are also presented in the graph. Arrow indicates the global increase of translation strength variation range.

GFP fluorescence intensity is measured to present the ability of P1G1 riboswitches to modulate downstream gene expression. GFP fluorescence intensity measured and with increasing concentrations of theophylline is shown in Fig.5. P1G1 displays a 6-fold activation ratio of the absence to presence of 10mM theophylline (Fig.6). Inspection of the highest translation rate reveals that although high concentration of ligand fully motivates translation, the GFP fluorescence intensity is still low, indicating relatively low translation strength. However, we expect wider translation strength available for theophylline riboswitch, thus the performance of P1G1 needs to be genetically optimized.

Figure.6The ability of P1G1 and 1G1 to activate translation strength under induction by a theophylline concentration gradient. .

Engineered Theophylline riboswitch—Theo 1G1

On account of the circumscribed property of RNA controllers, insertion of RNA controller probably makes even the highest expression level lower than sole RBS sequence upstream AUG start codon. Insertion of RNA controller could expediently down-regulate downstream gene translation, but it is impossible to up-regulate downstream gene translation rate exceeding that of sole RBS. A strong RBS is thought to proportionally shift the modulating range/activation ratio of RNA controllers without changing the width to a higher level, as they are turned gradually from completely OFF state to fully ON state in the presence of ligand. Therefore, we optimized original RBS sequence of P1G1 to AGGAGGU, the consensus RBS sequence predicted to initiate high rate of translation according to the RBS calculator. Simultaneously, as we positioned RNA controller 1G1 upstream of GFP coding sequence by standard assembly, the spacing between RBS and AUG strat codon is ACUAG (scar generated during standard assembly). The modification of RBS sequence and spacing between RBS and AUG start codon had inappreciable influence on the core secondary stem-loop structure of functional original riboswitch P1G1 and almost preserves the quondam base-pairing region. Stem-loop structure of 1G1 is schematized in Fig.7. 1G1 shares the same slipping mechanism with P1G1.

Figure.7 Predicted secondary structure of 1G1 in the absence and presence of theophylline. Theophylline aptamer is boxed. 1nt slipping mechanism is shown in yellow. UGGA is shown in red as RBS sequence. AUG start codon is in black. .

Dose response curve is shown in Fig.5. 1G1 displays a 10-fold activation ratio of the absence to presence of 10mM theophylline (Fig.6). However, under a concentration of 5mM theophylline, 1G1 shares similar activation ratio with P1G1, consistent with the prediction that alteration of RBS provides more flexible absolute translation strength with a fixed regulative range. Inspection of these two version of theophylline riboswitch reveals that 1G1 with the highest level of expression in the presence of theophylline not only possesses a longer RBS sequence (AGGAGGU) than P1G1, but also a more optimal spacing (5nt upstream to AUG start codon). Comparison of basal level of P1G1 and 1G1 suggests that 1G1 holds a more significant leakage in the absence of theophylline than P1G1, coincident with the holistic high expression of 1G1. According to our experiment data, engineered riboswitch possesses a more optimal absolute translation rate range, consistent with the prediction that a stronger RBS provides the RNA controller with a stronger regulation capability.

In summary, our semi-rational method to modify RNA controller (in our case, theophylline riboswitch) offers a promising clue for optimizing theperformance of RBS-related riboswitches.

Reference： [1]Nahvi,A., Sudarsan,N., Ebert,M.S., Zou,X., Brown,K.L. and Breaker ,R.R. (2002) Genetic control by a metabolite binding mRNA. Chem. Biol., 9, 1043-1049. [2]Beatrix Suess, Barbara Fink, Christian Berens, Régis Stentz and Wolfgang Hillen. (2004) A theophylline responsive riboswitch based on helix slipping controls gene expression in vivo. Nucleic Acids Research, 32, 1610-1614. [3]Sean A. Lynch, Shawn K. Desai, Hari Krishna Sajja, and Justin P. Gallivan. (2006) A High-Throughput Screen for Synthetic Riboswitches Reveals Mechanistic Insights into Their Function. Chem. Biol., 14, 173-184.