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Modulation of intein activity by its neighboring extein substrates.

ABSTRACT: Inteins comprise a large family of phylogenetically widespread self-splicing protein catalysts that colonize diverse host proteins. The evolutionary and functional relationship between the intein and the split-host protein, the exteins, is largely unknown. To probe an association, we developed an in vivo and in vitro intein assay based on FRET. The FRET assay reports cleavage of the intein from its N-terminal extein. Applying this assay to randomized extein libraries, we show that the nature of the extein substrate bordering the intein can profoundly influence intein activity. Residues proximal to the intein-splicing junction in both N- and C-terminal exteins can accelerate the N-terminal cleavage rate by >4-fold or attenuate cleavage by 1,000-fold, both resulting in compromised self-splicing efficiency. The existence and the magnitude of extein effects require consideration for maximizing the utility of inteins in biotechnological applications, and they predict biases in intein integration sites in nature.

SUBMITTER: Amitai G

PROVIDER: S-EPMC2708771 | biostudies-literature | 2009 Jul

REPOSITORIES: biostudies-literature

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Modulation of intein activity by its neighboring extein substrates.

Amitai Gil G Callahan Brian P BP Stanger Matt J MJ Belfort Georges G Belfort Marlene M

Proceedings of the National Academy of Sciences of the United States of America 20090617 27

Inteins comprise a large family of phylogenetically widespread self-splicing protein catalysts that colonize diverse host proteins. The evolutionary and functional relationship between the intein and the split-host protein, the exteins, is largely unknown. To probe an association, we developed an in vivo and in vitro intein assay based on FRET. The FRET assay reports cleavage of the intein from its N-terminal extein. Applying this assay to randomized extein libraries, we show that the nature of ...[more]

PMID: 19541659

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Project description:Inteins are intervening proteins that undergo an autocatalytic splicing reaction that ligates flanking host protein sequences termed exteins. Some intein-containing proteins have evolved to couple splicing to environmental signals; this represents a new form of posttranslational regulation. Of particular interest is RadA from the archaeon Pyrococcus horikoshii, for which long-range intein-extein interactions block splicing, requiring temperature and single-stranded DNA (ssDNA) substrate to splice rapidly and accurately. Here, we report that splicing of the intein-containing RadA from another archaeon, Thermococcus sibericus, is activated by significantly lower temperatures than is P. horikoshii RadA, consistent with differences in their growth environments. Investigation into variations between T. sibericus and P. horikoshii RadA inteins led to the discovery that a nonconserved region (NCR) of the intein, a flexible loop where a homing endonuclease previously resided, is critical to splicing. Deletion of the NCR leads to a substantial loss in the rate and accuracy of P. horikoshii RadA splicing only within native exteins. The influence of the NCR deletion can be largely overcome by ssDNA, demonstrating that the splicing-competent conformation can be achieved. We present a model whereby the NCR is a flexible hinge which acts as a switch by controlling distant intein-extein interactions that inhibit active site assembly. These results speak to the repurposing of the vestigial endonuclease loop to control an intein-extein partnership, which ultimately allows exquisite adaptation of protein splicing upon changes in the environment.IMPORTANCE Inteins are mobile genetic elements that interrupt coding sequences (exteins) and are removed by protein splicing. They are abundant elements in microbes, and recent work has demonstrated that protein splicing can be controlled by environmental cues, including the substrate of the intein-containing protein. Here, we describe an intein-extein collaboration that controls temperature-induced splicing of RadA from two archaea and how variation in this intein-extein partnership results in fine-tuning of splicing to closely match the environment. Specifically, we found that a small sequence difference between the two inteins, a flexible loop that likely once housed a homing endonuclease used for intein mobility, acts as a switch to control intein-extein interactions that block splicing. Our results argue strongly that some inteins have evolved away from a purely parasitic lifestyle to control the activity of host proteins, representing a new form of posttranslational regulation that is potentially widespread in the microbial world.

Dataset Information

Modulation of intein activity by its neighboring extein substrates.

Publications

Modulation of intein activity by its neighboring extein substrates.

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