Pseudoknots

Pseudoknots are secondary structures formed in mRNA that cause ribosome stalling and thus slow down the rate of expression at the translational level.

Design

Viruses use RNA pseudoknots as a mechanism to cause ribosome shifting, allowing expression of more than one protein from one mRNA transcript. They are found in the middle of viral open reading frames (ORFs) [ref]. We used these sequences, removed all start codons, and placed them in between ORFs. We ordered DNA synthesis of the parts, and aimed to characterise them and determine whether the effects of sequential pseudoknots would be an additive and linear increase in expression delay. For more insight into our pseudoknot story, see “Encounters of the IP kind”. [link] + insert IMAGES of pseudoknots from that paper!! Our synthesised parts include 20 base pairs on either side of the knot structures, derived from the surrounding sequence in the viral genomes.

Why?

Part of our biosafety strategy involved actually removing the invasin listeriolysin genes from endosymbionts once they had invaded one cell. This would limit further spread. To do so, we used a Cre-lox system, explained in more detail below [link]. Essentially, the system relies on expression of the Cre recombinase to recombine the lox sites and remove the gene segments encoded between. It is critical that enough invasin and listeriolysin proteins are expressed before the genes were removed, so we used RNA pseudoknots to delay expression of the Cre recombinase. [not great] Further, an interesting aspect of pseudoknots is the combinatorial approach that could be used to delay translation. Simply, do two pseudoknots in a row have a linear increase in the delay or will larger secondary structure? Additionally if a larger secondary structure forms will it cause an increase or a decrease based off the linear model or will it cause the ribosome to fall off the mRNA?

Background

RNA pseudoknots are RNA structures with diverse functions [1], usually formed by the interaction of the loop of an RNA stem-loop with nucleotides downstream of the stem. These are referred to as H-type pseudoknots. Alternatively, two RNA stem-loops can interact via their loops, a type of pseudoknot known as kissing stem-loops. Among their diverse functions, certain pseudoknots can induce ribosome pausing during translation. In viral mRNA molecules, this mechanism is often combined with a heptanucleotide sequence upstream of the pseudoknot to induce a translation frameshift, which is necessary for the proper expression of certain viral ORFs [2].

Our Project

For our project, we decided to exploit the ribosome-pausing capabilities of viral RNA pseudoknots to delay the translation of genes transcribed in a polycistronic mRNA molecule. To achieve this, we placed the pseudoknots in between genes rather than within the ORFs of our composite parts.

We have designed a number of BioBricks containing one or more pseudoknots. The rationale of the design was the following: the sequences of the chosen pseudoknots were synthesised, including the 20 bases upstream and downstream of the pseudoknot, based on the original viral genomes. This is to give enough space in between pseudoknots, should one wish to string several pseudoknots together to further delay the translation of a gene. We also included the standard BioBrick prefix and suffix to the parts. Since the goal is to delay the ribosome before it reaches the next gene of the polycistronic mRNA, it is important to avoid creating any new ORFs in the pseudoknot BioBricks. Thus, when necessary, we removed any potential START codons from the parts in our design. In the cases where potential ATG codons existed within the pseudoknot structure, we inverted any of the three base pairs in which the ATG triplet participated in order to keep the new sequence as close to the original as possible. We then conducted a few RNA pseudoknot predictions to verify that the new sequences yielded similar structures. The criteria chosen were to keep the estimated free energy of the new pseudoknots as close as possible to the original, while keeping the same stems and loops as the reported structures. Pseudoknot predictions were achieved with DotKnot [3, 4], while structure visualisation was carried out with PseudoViewer [5].

DotKnot was able to predict the correct stems and loops as reported in [2] for the wild type sequences. One exception was the kissing stem-loops of HCV229E, where DotKnot was unable to predict the full length of the second stem, probably due to a series of mismatches reported in this stem. However, the interaction of the loops was accurately predicted. This increased our confidence in the prediction of the new structures after removal of the START codons.

We chose to synthesise four known pseudoknots to test which, alone or in combination, could cause sufficient delay of the ribosome: three H-type pseudoknots, from the Mouse Mammary Tumor Virus (MMTV), the Infectious Bronchitis Virus (IBV) and the Transmissible Gastroenteritis Virus (TGV); and one kissing stem-loops type, from the Human Coronavirus 229E (HCV229E). These pseudoknots vary in length (34 nucleotides in the MMTV pseudoknot to over 200 nucleotides in the kissing stem-loops) and in the number of paired nucleotides. For example, for the H-type structures, the stem in the TGV pseudoknot is longer and contains no mismatches, whereas the IBV pseudoknot is shorter and contains one mismatch. As such, we expect these different structures to pause the ribosome with varying efficiencies.