A major goal of synthetic biology is to reprogram cells to perform complex tasks. Here we show how a combination of in vitro and in vivo selection rapidly identifies a synthetic riboswitch that activates protein translation in response to the herbicide atrazine. We further demonstrate that this riboswitch can reprogram bacteria to migrate in the presence of atrazine. Finally, we show that incorporating a gene from an atrazine catabolic pathway allows these cells to seek and destroy atrazine.
We previously reported that a group I intron-derived ribozyme can catalyze the excision of targeted sequences from within RNAs in vitro and that dissociation of the bridge-3' exon intermediate between the two reaction steps is a significant contributing factor to low product yields. We now analyze the effects of increasing the length, and thus the strength, of helices P9.0 and P10, which occur between the ribozyme and the bridge-3' exon region of the substrate, on this trans excision-splicing reaction. Using substrates where lengthy targeted regions are excised, these modifications can significantly increase product yields, specifically by enhancing the second reaction step. A threshold for product formation is obtained, however, at around five base pairs for P10 and eight base pairs for P9.0. Nevertheless, elongating P9.0 appears to be the more effective strategy, as both substrate binding and the rate of the second reaction step increase. In addition, P10 is required when P9.0 is not elongated. Also, a strong P9.0 helix cannot replace a weaker P10 helix, indicating that P9.0 and P10 play somewhat distinct roles in the reaction. We also show that second-step inhibition stems from the formation of an extended P1 helix (P1ex), consisting of as little as a single Watson-Crick base pair, as well as the mere presence of substrate nucleosides immediately downstream from P10. Both of these inhibitory components can be overcome by utilizing P9.0 and P10 elongated ribozymes. This work sets forth an initial framework for rationally designing more effective trans excision-splicing ribozymes.
Reaction of alpha,beta-unsaturated aromatic carboxylic acids with KBr and H2O2 in the presence of Na2MoO4.2H2O in aqueous medium affords beta-bromo alkenes in high yield.
A group I intron-derived ribozyme from Pneumocystis carinii has been previously shown to bind an exogenous RNA substrate, splice out an internal segment, and then ligate the two ends back together (the trans excision-splicing reaction). We demonstrate that this same ribozyme can perform a trans insertion-splicing (TIS) reaction, where the ribozyme binds two exogenous RNA substrates and inserts one directly into the other. Reactions were optimized for both yield and rate, with optimum reactions carried out in 10 mM MgCl(2) for 2 h. Reaction products are stable, with no visible loss at extended times. The ribozyme recognizes the two substrates primarily through base pairing and requires an omegaG on the ribozyme and an omegaG on the sequence being inserted. We give evidence that the reaction mechanism is not the reverse of the trans excision-splicing reaction, but is composed of three steps, with intermediates attached to the ribozyme. Surprisingly, the internal guide sequence of the ribozyme is utilized to sequentially bind both substrates, forming independent P1 helices. This is an indication that ribozymes with essentially the native intron sequence can catalyze reactions significantly more dynamic and complex than self-splicing. The implications of group I intron-derived ribozymes being able to catalyze this unique reaction, and via this mechanism, are discussed.
In the trans excision-splicing reaction, a Pneumocystis carinii group I intron-derived ribozyme binds an RNA substrate, excises a specific internal segment, and ligates the flanking regions back together. This reaction can occur both in vitro and in vivo. In this report, the first of the two reaction steps was analyzed to distinguish between two reaction mechanisms: ribozyme-mediated hydrolysis and nucleotide-dependent intramolecular transesterification. We found that the 3'-terminal nucleotide of the ribozyme is the first-reaction step nucleophile. In addition, the 3'-half of the RNA substrate becomes covalently attached to the 3'-terminal nucleotide of the ribozyme during the reaction, both in vitro and in vivo. Results also show that the identity of the 3'-terminal nucleotide influences the rate of the intramolecular transesterification reaction, with guanosine being more effective than adenosine. Finally, expected products of the hydrolysis mechanism do not form during the reaction. These results are consistent with only the intramolecular transesterification mechanism. Unexpectedly, we also found that ribozyme constructs become truncated in vivo, probably through intramolecular 3'-hydrolysis (self-activation), to create functional 3'-terminal nucleotides.
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