Berkeley2006-RiboregulatorsMain

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[[Image:Berkeley2006Ribo3.GIF]]<br>
[[Image:Berkeley2006Ribo3.GIF]]<br>
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Initially focussing on the sequence of the key, we constructed 3 variants of key3 that either deleted the hairpin sequence, made the annealing region a perfect complementary sequence to the lock, and moved the position of the annealing region to the loop of a large, very stable RNA hairpin.  First and foremost, removing the point mutations between the key and the lock had no effect on the observed gain.  These mutations are therefore neither necessary for the stability of the duplex nor are they detrimental to unlocking.  All our further designs were therefore perfect complements of the lock sequences.  In contrast, deleting the hairpin element of the key entirely destroyed unlocking.  This secondary structure element is likely necessary to protect the RNA element from degradation within the cell.  Moving the annealing region to the middle of a large hairpin provided the largest gain in fluorescense of the first 3 key variants.  In addition to providing secondary structure to the key, placing the annealing region within the loop of the hairpin insulates the key from variation in the 5' and 3' ends of the key molecules.  These results led us to take secondary structure to another level--we constructed a key variant that placed the annealing region within the anticodon loop of a Ser2-derived tRNA sequence.  This structure had the highest activity of all our basic keys, an X-fold gain.
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We next examined the effect of fusing sequence to the 5' or 3' ends of the key.  When we placed an explicit terminator downstream of any of our functional keys, we observed a consistant 2-fold increase in unlocking activity.  When we placed an open reading frame sequence and terminator downstream of the key, unlocking was entirely eliminated.  These results suggest that shorter key transcripts lead to optimal unlocking.  Additionally, we examined the activity of a duplex key sequence.  When the key3C and key3D variants were fused in a single transcript, the observed gain was 2x over either key alone.  This suggests that increasing the gene dosage of the key can increase gain.  This increase would also be expected if the key were expressed from a stronger promoter.
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Next, we examined the spacing of the annealing region within the key's anticodon loop.  We increased and decreased the spacing outside of the annealing region within the loop in both the hairpin (key3D) and tRNA-like designs.  Unexpectedly, the highest activity is observed with smaller loops for both the tRNA and hairpin designs.  This most likely results from the periodicity of the RNA within the loop, or perhaps the rigidity imposed by a shorter loop in some way improves annealing with lock.  Further work will focus on fine-tuning the spacing within the loop to determine whether it is the size of the loop per se or rather the phasing of the RNA helix within the loop that is responsible for this effect.
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[[Image:Berkeley2006Ribo4.GIF]]<br>
[[Image:Berkeley2006Ribo4.GIF]]<br>
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Next section: [[Berkeley2006-ConjugationMain | Conjugation]]
Next section: [[Berkeley2006-ConjugationMain | Conjugation]]

Revision as of 20:41, 28 October 2006

Within our addressable conjugation system, every cell contains a unique nucleic acid "address" sequence. This address is encoded within an RNA riboregulator "lock" and is used to control the bacterium's ability to communicate with other cells. It was imperative to develop high-gain riboregulators that can be rationally modified to construct many orthogonal lock/key pairs. Here we show the evolution of the original riboregulator design into a high-performance regulatory system.

Riboregulators translationally control gene expression and are composed of two RNA parts--a lock and a key sequence. The lock sequence replaces the ribosome binding site of the controlled gene. Within the lock, a ribosome binding site is connected by a short linker sequence to its own reverse complement. This results in a hairpin structure that occludes the ribosome binding site thereby prevent access to the ribosome. Shown below are the initial lock and key sequences for our studies
Berkeley2006Ribo2.GIF
Genes under the control of the lock show very little protein biosynthesis. The key sequence is expressed from a separate gene (in trans) and encodes a sequence complementary to the lock. The identity of this sequence is the address in our addressable conjugation system. When the key anneals to the lock sequence, the ribosome binding site becomes exposed permitting expression of the downstream gene.
Berkeley2006Ribo1.GIF

The RNA sequence outside of the lock's ribosome binding site binding region is of arbitrary sequence. However, this sequence must still match the key sequence to obtain unlocking. It should therefore be possible to construct millions of non-crossreactive variants of the riboregulator system.

However, the original riboregulator design shows only a 1.7 fold unlocking of protein expression when the key is added. We therefore introduced a series of perturbations to the original design to determine the features of RNA locks and keys that would lead to a low background, high gain regulation system. First, we describe modifications to the lock sequence:

Berkeley2006Ribo3.GIF

Initially focussing on the sequence of the key, we constructed 3 variants of key3 that either deleted the hairpin sequence, made the annealing region a perfect complementary sequence to the lock, and moved the position of the annealing region to the loop of a large, very stable RNA hairpin. First and foremost, removing the point mutations between the key and the lock had no effect on the observed gain. These mutations are therefore neither necessary for the stability of the duplex nor are they detrimental to unlocking. All our further designs were therefore perfect complements of the lock sequences. In contrast, deleting the hairpin element of the key entirely destroyed unlocking. This secondary structure element is likely necessary to protect the RNA element from degradation within the cell. Moving the annealing region to the middle of a large hairpin provided the largest gain in fluorescense of the first 3 key variants. In addition to providing secondary structure to the key, placing the annealing region within the loop of the hairpin insulates the key from variation in the 5' and 3' ends of the key molecules. These results led us to take secondary structure to another level--we constructed a key variant that placed the annealing region within the anticodon loop of a Ser2-derived tRNA sequence. This structure had the highest activity of all our basic keys, an X-fold gain.

We next examined the effect of fusing sequence to the 5' or 3' ends of the key. When we placed an explicit terminator downstream of any of our functional keys, we observed a consistant 2-fold increase in unlocking activity. When we placed an open reading frame sequence and terminator downstream of the key, unlocking was entirely eliminated. These results suggest that shorter key transcripts lead to optimal unlocking. Additionally, we examined the activity of a duplex key sequence. When the key3C and key3D variants were fused in a single transcript, the observed gain was 2x over either key alone. This suggests that increasing the gene dosage of the key can increase gain. This increase would also be expected if the key were expressed from a stronger promoter.

Next, we examined the spacing of the annealing region within the key's anticodon loop. We increased and decreased the spacing outside of the annealing region within the loop in both the hairpin (key3D) and tRNA-like designs. Unexpectedly, the highest activity is observed with smaller loops for both the tRNA and hairpin designs. This most likely results from the periodicity of the RNA within the loop, or perhaps the rigidity imposed by a shorter loop in some way improves annealing with lock. Further work will focus on fine-tuning the spacing within the loop to determine whether it is the size of the loop per se or rather the phasing of the RNA helix within the loop that is responsible for this effect.

Berkeley2006Ribo4.GIF


Next section: Conjugation

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