Part:BBa_K598000

TPP Down-regulated Hammerhead Ribozyme 2.5 with Native RBS+E0040+B0015

Background

Hammerhead ribozymes are small self-cleaving RNAs, first discovered in satellite RNAs of plant viruses that catalyze a specific phosphodiester bond isomerization reaction in the course of rolling-circle replication [1]. More recently a full-length hammerhead ribozyme from Schistosoma mansoni is being more frequently utilized for application. As shown in Fig.1A, this hammerhead ribozyme can be truncated to a minimal, catalytically active motif consisting of three base-pairing stems (marked in colors) flanking a central core of 15 mostly invariant nucleotides (marked in frame). And the conserved central bases are essential for the hammerhead ribozyme’s catalytic activity [1]. The tertiary structure shown in Fig.1B indicates that the secondary structure of the Schistosoma hammerhead ribozyme can be distorted into a uridine turn because of distant loop/bulge interaction which induces changes in stem II while simultaneously unwinding stem I. For the basic catalytic function of hammerhead ribozyme, the active site for self-cleaving of Schistosoma hammerhead ribozyme resides between stem III and stem I, as shown in Fig.1A.

It has been reported previously that mRNAs encoding enzymes involved in thiamine (vitamin B1) biosynthesis in Escherichia coli can bind thiamine or its pyrophosphate derivative without the assistance from protein cofactors [2]. These ligand-binding mRNAs actually possess thiamine or pyrophosphate binding domain, called aptamer, in which the binding event can bring about a conformational change which is important for genetic control. This natural thiamine pyrophosphate (TPP) aptamer can bind to TPP specifically and a defined structure is stabilized. As shown in Fig.1C, upon addition of TPP, TPP can bind loop in green through non-covalent bond. Fig.1D shows the tertiary structure of natural TPP aptamer binding to TPP.

Figure 1 The schematic structures of Schistosoma hammerhead ribozyme and natural TPP aptamer. A) The secondary structure of Schistosoma hammerhead ribozyme. Three base-pairing stems are shaded in colors. The part in yellow represents stem III, which is later modified to be the linker between hammerhead ribozyme and aptamer. The part in blue represents stem II, and the purple and red ones stand for two parts of stem I. The sequence in frame represents for the conserved nucleotides. The red arrow points to the scissile bond. B) The tertiary structure of Schistosoma hammerhead ribozyme. The cyan part indicates the fragment of mRNA after cleavage. The red one indicates the active site for self-cleavage. The linker between hammerhead ribozyme and aptamer is shown in yellow. C) The secondary structure of natural TPP aptamer. TPP can bind to loop in green through non-covalent bond and the part marked in yellow indicates the linker between hammerhead ribozyme and apatamer. D) The tertiary structure of natural TPP aptamer. The three-dimensional segment in blue is TPP, and the yellow part represents the linker between Schistosoma hammerhead ribozyme and natural TPP aptamer. Nucleotides that bind to TPP are shown in green.

Original Design of TPP Ligand Responsive Hammerhead Ribozyme

In fact, the natural aptamer domain of the TPP riboswitch can be exploited to construct very efficient ribozyme-based artificial switches that regulate gene expression, demonstrated by Markus Wieland et. al.[3]. To couple the natural TPP aptamer riboswitch with Schistosoma hammerhead ribozyme, stem III of Schistosoma hammerhead ribozyme and yellow shaded stem of TPP aptamer in Fig.1-C were modified to construct linker between hammerhead ribozyme and aptamer. The resulting artificial ribozymes functioned with high performance, whose highest fold reached 1000.

Markus Wieland et. al. have created several mutants of the constructed ribozyme-based TPP-responsive artificial ribozyme switches (TPP ribozyme)[3]. We chose two of the mutants in our project, one of which can activate downstream gene expression upon adding TPP, numbered 1.20, and the other would inhibit downstream gene expression when TPP added, numbered 2.5. The secondary structure of TPP ribozyme 1.20 and 2.5 are shown in Fig.2. The lower part of the structure is natural TPP aptamer riboswitch, and the upper part is Schistosoma hammerhead ribozyme. Stem III in green indicates the linker between aptamer and hammerhead ribozyme, the pairing nucleotides of which is the only distinction between TPP ribozyme 1.20 and 2.5.

Figure 2Secondary structure of artificial thiamine pyrophosphate (TPP) ribozymes 2.5. The natural TPP aptamer domain (blue) is fused to stem III of the Schistosoma hammerhead ribozyme. The linker between aptamer and hammerhead ribozyme is shown in green. Stems are indicated by roman numerals; rate-enhancing interaction between stem I and stem II are shown as gray lines; the cleavage site is marked by a red arrow. RBS is shaded in pink and the translation start code (AUG) is shaded in black. The figure was modified from [3].

Figure 3Allosteric mechanism of TPP ribozymes. Upon self-cleavage the RBS would be released from pairing, thus ribosome could get access to RBS and initiate translation of the downstream gene .A) Mechanism for TPP ribozyme 1.20. Upon addition of TPP, TPP ribozyme 1.20 adopts a conformation that facilitates the self-cleavage of hammerhead domain. B) Mechanism for TPP ribozyme 2.5. When TPP added, TPP ribozyme 2.5 would change to a conformation that hinders the self-cleavage of hammerhead domain. Red: RBS sequence, blue: natural TPP aptamer, green: linker between aptamer and hammerhead domain, black: Schistosoma hammerhead ribozyme, red arrow: self-cleavage site. The figure is modified from [3].

The ribosomal binding site (RBS) of TPP ribozyme locates at the extended stem (shaded red in Fig.2). The Schistosoma hammerhead domain in TPP ribozyme could perform self-cleavage when posed in an appropriate conformation, and upon self-cleavage the RBS would be released from pairing, thus ribosome could get access to RBS and initiate translation of the downstream gene. Though similar in secondary structure, TPP ribozyme 1.20 and 2.5 undergo different mechanisms to regulate the translation of downstream gene. Upon addition of TPP, the aptamer domain would bind to TPP; while TPP ribozyme 1.20 would change to a conformation that is suitable for hammerhead domain to cleave itself, TPP ribozyme 2.5 would undergo a conformational change that would decelerate the self-cleaving rate of hammerhead domain. Therefore, upon adding TPP, TPP ribozyme 1.20 would facilitate the translation of downstream gene, whereas TPP ribozyme 2.5 would decrease the translation strength of downstream gene (Fig.3).

Manipulating and Experimental Data

We designed several experiments to demonstrate that TPP ribozymes are truly modular RNA controllers, independent of sequence context. The plasmids kindly provided by Prof. Hartig’s lab contain TPP ribozyme 1.20/2.5 with an upstream T7 promoter, and the downstream coding sequence is a copy of GFP gene, followed by a T7 terminator (Fig. 4-A). We termed these two constructs T7-1.20 and T7-2.5 respectively. To prove that the performance of TPP ribozymes is not relevant to the downstream coding sequence, we inserted first 36 base pairs of the coding sequence from Part BBa_E0040 ahead of the GFP coding sequence in the plasmid (Fig. 4-B). The two constructs were termed 36-1.20/36-2.5 respectively.

Figure 4 Construction of TPP Down-regulated Hammerhead Ribozyme 2.5 with Native RBS+E0040+B0015. This part consists of TPP ribozyme 2.5 with native RBS (AAGGAGAT), BBa_E0040 and BBa_B0015.It is obtained by PCR from inactive TPP-HHAZ 2.5 [3] which is kindly provided by Markus Wieland et. al., and then inserted into pSB1C3 through standard assembly.

After transforming T7-1.20, T7-2.5, 36-1.20, 36-2.5 into E. coli DH5αcells, the bacteria were characterized in M9 medium with TPP concentration gradient. The result turned out that the corresponding working curves were similar, despite of different downstream coding sequence (Fig.5 & Fig.6, grids and curves in black and in red).

To further investigate whether the performance of TPP ribozymes is influenced by upstream promoter and downsteam terminator, we constructed two plasmids that contain TPP ribozyme 1.20/2.5 with an upstream pBAD promoter, and the downstream coding sequence is a GFP gene from Part BBa_E0040, followed by a terminator (Part:BBa_B0015) (Fig. 4-C). These two constructs were named pBAD-1.20 and pBAD-2.5 respectively. Similarly, we characterized these two constructs, and the working curves were also similar to those of T7-1.20/ T7-2.5 (Fig.5 & Fig.6, grids and curves in black and in blue). Another two plasmids were constructed by inserting first 36 base pairs of CI gene ahead of GFP coding sequence in pBAD-1.20/ pBAD-2.5 (Fig. 4-D). These are named CI-1.20 and CI-2.5 respectively. The working curves of these two constructs overlapped to a large extent with those of other constructs (Fig.5 & Fig.6, grids and curves in green), further demonstrating that the performance of TPP ribozyme 1.20 and 2.5 was independent of their coding sequence context, rendering them truly modular RNA devices to regulate gene expression.

Figure 5 Scheme of constructs designed to demonstrate the modularity of TPP ribozymes. A) pBAD-1.20/pBAD-2.5 consists of pBAD promoter, TPP ribozyme, BBa_E0040 and BBa_B0015 with native RBS (AAGGAGAT). B) T7-1.20/T7-2.5 consists of T7 promoter, TPP ribozyme, GFP and T7 terminator with native RBS.

Additionally, to further confirm that the rise or drop of the working curves in Fig.5 or Fig.6 on different TPP concentrations was indeed the contribution of TPP ribozymes, we constructed a plasmid as control by substituting TPP ribozyme in pBAD-1.20/ pBAD-2.5 to the native RBS (AAGGAGAT) of TPP ribozyme, termed TPP-RBS (Fig.4-E). Similar characterization was performed, and the result showed that the fluorescence intensity produced by TPP-RBS fluctuated, yet not significant enough to show a trend to increase or decrease when TPP concentration went up, compared with the obvious fluorescence intensity change produced by pBAD-1.20/ pBAD-2.5 (Fig.7). Therefore, we can reach the conclusion that TPP ribozyme 1.20 and 2.5 functioned modularly to regulate downstream gene’s translation strength upon different concentrations of TPP.

Sequence and Features

Assembly Compatibility:

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

  Illegal BamHI site found at 121
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 794