Berkeley2006-RiboregulatorsMain

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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. <br>
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. <br>
[[Image:Berkeley2006Ribo1.GIF]]<br>
[[Image:Berkeley2006Ribo1.GIF]]<br>
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=== ===
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.<br>
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.<br>
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[[Image:Berkeley2006Ribo3.GIF]]<br>
[[Image:Berkeley2006Ribo3.GIF]]<br>
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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 20:39, 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

Berkeley2006Ribo4.GIF


Next section: Conjugation

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