Pyrococcus Abyssi Research Articles

Structural analysis of an intein from an extreme thermophile

Protein splicing is a post‐translational event by which an intervening polypeptide, the intein, facilitates its own excision from the flanking polypeptides, the exteins, and the ligation of the exteins. We have studied the structure of two inteins that interrupt the DNA PolII from the extreme thermophiles Pyrococcus abyssi (Pab) and Pyrococcus horikoshii (Pho). The two inteins have 71% sequence identity. Solutions to the NMR structure of the Pab PolII intein revealed a long flexible loop in an otherwise rigid protein; the sequence corresponding to this loop is absent in the Pho intein. We have used susceptibility to digestion by the thermostable protease thermolysin to show that the Pho intein is more rigid than the Pab intein. The Pho intein also is less active at low temperature than the Pab intein. Perhaps due to this increased rigidity, we were able to identify crystallization conditions for the Pho PolII intein. Our first crystals give diffraction data to about 2.9 Å; we hope to optimize crystallization conditions and solve the structure in collaboration with colleagues at the Wadsworth Center. We also hypothesize that the flexible loop may play a role in mechanism‐linked conformational changes that might help to coordinate the steps of splicing.This material is based upon work supported by the National Science Foundation under grant MCB‐0950245 and by the Camille and Henry Dreyfus Foundation.

A simple procedure to determine the infectivity and host range of viruses infecting anaerobic and hyperthermophilic microorganisms

Plaque assay is the method traditionally used to isolate and purify lytic viruses, to determine the viral titer and host range. Whereas most bacterioviruses are either temperate or lytic, the majority of known archeoviruses are not lytic (i.e. they are temperate or chronic). In view of the widespread occurrence of such viruses in extreme environments, we designed an original method, called the inverted spot test, to determine the host range and infectivity of viruses isolated from anaerobic hyperthermophilic and sulfur-reducing microorganisms. Here, we used this approach to prove for the first time the infectivity of Pyrococcus abyssi virus 1 (PAV1) and to confirm the host range of Thermococcus prieurii virus 1 (TPV1), the only two viruses isolated so far from any of the described marine hyperthermophilic archaea (Euryarchaeota phylum, Thermococcales order).

Insights into Dynamics of Mobile Genetic Elements in Hyperthermophilic Environments from Five New Thermococcus Plasmids

Mobilome of hyperthermophilic archaea dwelling in deep-sea hydrothermal vents is poorly characterized. To gain insight into genetic diversity and dynamics of mobile genetic elements in these environments we have sequenced five new plasmids from different Thermococcus strains that have been isolated from geographically remote hydrothermal vents. The plasmids were ascribed to two subfamilies, pTN2-like and pEXT9a-like. Gene content and phylogenetic analyses illuminated a robust connection between pTN2-like plasmids and Pyrococcus abyssi virus 1 (PAV1), with roughly half of the viral genome being composed of genes that have homologues in plasmids. Unexpectedly, pEXT9a-like plasmids were found to be closely related to the previously sequenced plasmid pMETVU01 from Methanocaldococcus vulcanius M7. Our data suggests that the latter observation is most compatible with an unprecedented horizontal transfer of a pEXT9a-like plasmid from Thermococcales to Methanococcales. Gene content analysis revealed that thermococcal plasmids encode Hfq-like proteins and toxin-antitoxin (TA) systems of two different families, VapBC and RelBE. Notably, although abundant in archaeal genomes, to our knowledge, TA and hfq-like genes have not been previously found in archaeal plasmids or viruses. Finally, the plasmids described here might prove to be useful in developing new genetic tools for hyperthermophiles.

NucS DNA Flap Interaction Mechanism Kinetics Revealed by Single Molecule Imaging

DNA reparation enzymes are essential to preserve genome integrity. The understanding of the dynamics of the regulation pathways of their activity is thus crucial. Pyrococcus abyssi NucS is a recently discovered archaeal endonuclease acting on branched DNA. The biochemical characterization of PabNucS has shown that this protein binds ssDNA at nanomolar concentrations and has a rare bidirectional activity both on 5’ and 3’ free ssDNA extremities. Although ensemble measurements indicate that free ssDNA extremity is necessary for NucS activity, the detailed mechanisms that regulate NucS activity remain unclear. In particular, it remains unknown whether NucS loads at the extremity of the flap and then diffuses to the junction, or whether protein directly binds to the ss/dsDNA junction.To probe the dynamics of PabNucS ssDNA interactions, we use single-molecule tracking of fluorescently-labeled PabNucS acting on a DNA substrate tethered to the surface. We report a bidirectional and oriented binding on 3' and 5' flaps at the single molecule level. We measured binding/unbinding kinetics with unprecedent accuracy and revealed that NucS/DNA flap unbinding follows a diffusion independent multistep mechanism that depends on the NucS/ssDNA energy landscape.Consistently with the structural information of the high affinity ssDNA binding site that displays a hydrophobic core surrounded by charged residues, we propose a two-step unbinding model that allows a stochastic unbinding of the flap and characterize two forces contributing to the interaction: (i) an electrostatic force that controls binding and (ii) a non electrostatic (hydrophobic) force that is dominant at high salt concentration and maintains NucS-DNA complex stability.Our results constitute a notable contribution to the characterization of the ssDNA-NucS interactions and more generally to the nucleotide excision repair mechanism. The method we developed furthermore constitutes a powerful way to probe DNA/protein intramolecular kinetics.

Archaeal β-CASP ribonucleases of the aCPSF1 family are orthologs of the eukaryal CPSF-73 factor

Bacterial RNase J and eukaryal cleavage and polyadenylation specificity factor (CPSF-73) are members of the β-CASP family of ribonucleases involved in mRNA processing and degradation. Here we report an in-depth phylogenomic analysis that delineates aRNase J and archaeal CPSF (aCPSF) as distinct orthologous groups and establishes their repartition in 110 archaeal genomes. The aCPSF1 subgroup, which has been inherited vertically and is strictly conserved, is characterized by an N-terminal extension with two K homology (KH) domains and a C-terminal motif involved in dimerization of the holoenzyme. Pab-aCPSF1 (Pyrococcus abyssi homolog) has an endoribonucleolytic activity that preferentially cleaves at single-stranded CA dinucleotides and a 5′–3′ exoribonucleolytic activity that acts on 5′ monophosphate substrates. These activities are the same as described for the eukaryotic cleavage and polyadenylation factor, CPSF-73, when engaged in the CPSF complex. The N-terminal KH domains are important for endoribonucleolytic cleavage at certain specific sites and the formation of stable high molecular weight ribonucleoprotein complexes. Dimerization of Pab-aCPSF is important for exoribonucleolytic activity and RNA binding. Altogether, our results suggest that aCPSF1 performs an essential function and that an enzyme with similar activities was present in the last common ancestor of Archaea and Eukarya.

4-Demethylwyosine Synthase from Pyrococcus abyssi Is a Radical-S-adenosyl-l-methionine Enzyme with an Additional [4Fe-4S]+2 Cluster That Interacts with the Pyruvate Co-substrate

Wybutosine and its derivatives are found in position 37 of tRNA encoding Phe in eukaryotes and archaea. They are believed to play a key role in the decoding function of the ribosome. The second step in the biosynthesis of wybutosine is catalyzed by TYW1 protein, which is a member of the well established class of metalloenzymes called "Radical-SAM." These enzymes use a [4Fe-4S] cluster, chelated by three cysteines in a CX(3)CX(2)C motif, and S-adenosyl-L-methionine (SAM) to generate a 5'-deoxyadenosyl radical that initiates various chemically challenging reactions. Sequence analysis of TYW1 proteins revealed, in the N-terminal half of the enzyme beside the Radical-SAM cysteine triad, an additional highly conserved cysteine motif. In this study we show by combining analytical and spectroscopic methods including UV-visible absorption, Mössbauer, EPR, and HYSCORE spectroscopies that these additional cysteines are involved in the coordination of a second [4Fe-4S] cluster displaying a free coordination site that interacts with pyruvate, the second substrate of the reaction. The presence of two distinct iron-sulfur clusters on TYW1 is reminiscent of MiaB, another tRNA-modifying metalloenzyme whose active form was shown to bind two iron-sulfur clusters. A possible role for the second [4Fe-4S] cluster in the enzyme activity is discussed.

In vitro reconstitution of RNA primer removal in Archaea reveals the existence of two pathways

Using model DNA substrates and purified recombinant proteins from Pyrococcus abyssi, I have reconstituted the enzymatic reactions involved in RNA primer elimination in vitro. In my dual-labelled system, polymerase D performed efficient strand displacement DNA synthesis, generating 5'-RNA flaps which were subsequently released by Fen1, before ligation by Lig1. In this pathway, the initial cleavage event by RNase HII facilitated RNA primer removal of Okazaki fragments. In addition, I have shown that polymerase B was able to displace downstream DNA strands with a single ribonucleotide at the 5'-end, a product resulting from a single cut in the RNA initiator by RNase HII. After RNA elimination, the combined activities of strand displacement DNA synthesis by polymerase B and flap cleavage by Fen1 provided a nicked substrate for ligation by Lig1. The unique specificities of Okazaki fragment maturation enzymes and replicative DNA polymerases strongly support the existence of two pathways in the resolution of RNA fragments.

Molecular Recognition of Canonical and Deaminated Bases by P. abyssi Family B DNA Polymerase

Euryarchaeal polymerase B can recognize deaminated bases on the template strand, effectively stalling the replication fork 4nt downstream the modified base. Using Pyrococcus abyssi DNA B family polymerase (PabPolB), we investigated the discrimination between deaminated and natural nucleotide(s) by primer extension assays, electrophoretic mobility shift assays, and X-ray crystallography. Structures of complexes between the protein and DNA duplexes with either a dU or a dH in position +4 were solved at 2.3Å and 2.9Å resolution, respectively. The PabPolB is found in the editing mode. A new metal binding site has been uncovered below the base-checking cavity where the +4 base is flipped out; it is fully hydrated in an octahedral fashion and helps guide the strongly kinked template strand. Four other crystal structures with each of the canonical bases were also solved in the editing mode, and the presence of three nucleotides in the exonuclease site caused a shift in the coordination state of its metal A from octahedral to tetrahedral. Surprisingly, we find that all canonical bases also enter the base-checking pocket with very small differences in the binding geometry and in the calculated binding free energy compared to deaminated ones. To explain how this can lead to stalling of the replication fork, the full catalytic pathway and its branches must be taken into account, during which the base is checked several times. Our results strongly suggest a switch from elongation to editing modes right after nucleotide insertion when the modified base is at position +5.

The box C/D sRNP dimeric architecture is conserved across domain Archaea

Box C/D small (nucleolar) ribonucleoproteins [s(no)RNPs] catalyze RNA-guided 2'-O-ribose methylation in two of the three domains of life. Recent structural studies have led to a controversy over whether box C/D sRNPs functionally assemble as monomeric or dimeric macromolecules. The archaeal box C/D sRNP from Methanococcus jannaschii (Mj) has been shown by glycerol gradient sedimentation, gel filtration chromatography, native gel analysis, and single-particle electron microscopy (EM) to adopt a di-sRNP architecture, containing four copies of each box C/D core protein and two copies of the Mj sR8 sRNA. Subsequently, investigators used a two-stranded artificial guide sRNA, CD45, to assemble a box C/D sRNP from Sulfolobus solfataricus with a short RNA methylation substrate, yielding a crystal structure of a mono-sRNP. To more closely examine box C/D sRNP architecture, we investigate the role of the omnipresent sRNA loop as a structural determinant of sRNP assembly. We show through sRNA mutagenesis, native gel electrophoresis, and single-particle EM that a di-sRNP is the near exclusive architecture obtained when reconstituting box C/D sRNPs with natural or artificial sRNAs containing an internal loop. Our results span three distantly related archaeal species--Sulfolobus solfataricus, Pyrococcus abyssi, and Archaeoglobus fulgidus--indicating that the di-sRNP architecture is broadly conserved across the entire archaeal domain.

Lyase activity of glycogen synthase: Is an elimination/addition mechanism a possible reaction pathway for retaining glycosyltransferases?

Despite the biological relevance of glycosyltrasferases (GTs) and the many efforts devoted to this subject, the catalytic mechanism through which a subclass of this large family of enzymes, namely those that operate with net retention of the anomeric configuration, has not been fully established. Here, we show that in the absence of an acceptor, an archetypal retaining GT such as Pyrococcus abyssi glycogen synthase (PaGS) reacts with its glucosyl donor substrate, uridine 5'-diphosphoglucose (UDP-Glc), to produce the scission of the covalent bond between the terminal phosphate oxygen of UDP and the sugar ring. X-ray diffraction analysis of the PaGS/UDP-Glc complex shows no electronic density attributable to the UDP moiety, but establishes the presence in the active site of the enzyme of a glucose-like derivative that lacks the exocyclic oxygen attached to the anomeric carbon. Chemical derivatization followed by gas chromatography/mass spectrometry of the isolated glucose-like species allowed us to identify the molecule found in the catalytic site of PaGS as 1,5-anhydro-D-arabino-hex-1-enitol (AA) or its tautomeric form, 1,5-anhydro-D-fructose. These findings are consistent with a stepwise S(N) i-like mechanism as the modus operandi of retaining GTs, a mechanism that involves the discrete existence of an oxocarbenium intermediate. Even in the absence of a glucosyl acceptor, glycogen synthase (GS) promotes the formation of the cationic intermediate, which, by eliminating the proton of the adjacent C2 carbon atom, yields AA. Alternatively, these observations could be interpreted assuming that AA is a true intermediate in the reaction pathway of GS and that this enzyme operates through an elimination/addition mechanism.

Regulation of the ATPase activity of ABCE1 from Pyrococcus abyssi by Fe–S cluster status and Mg2+: Implication for ribosomal function

Ribosomal function is dependent on multiple proteins. The ABCE1 ATPase, a unique ABC superfamily member that bears two Fe4S4 clusters, is crucial for ribosomal biogenesis and recycling. Here, the ATPase activity of the Pyrococcus abyssi ABCE1 (PabABCE1) was studied using both apo- (without reconstituted Fe–S clusters) and holo- (with full complement of Fe–S clusters reconstituted post-purification) forms, and is shown to be jointly regulated by the status of Fe–S clusters and Mg2+. Typically ATPases require Mg2+, as is true for PabABCE1, but Mg2+ also acts as a negative allosteric effector that modulates ATP affinity of PabABCE1. Physiological [Mg2+] inhibits the PabABCE1 ATPase (Ki of ∼1μM) for both apo- and holo-PabABCE1. Comparative kinetic analysis of Mg2+ inhibition shows differences in degree of allosteric regulation between the apo- and holo-PabABCE1 where the apparent ATP Km of apo-PabABCE1 increases >30-fold from ∼30μM to over 1mM with Mg2+. This effect would significantly convert the ATPase activity of PabABCE1 from being independent of cellular energy charge (φ) to being dependent on φ with cellular [Mg2+]. These findings uncover intricate overlapping effects by both [Mg2+] and the status of Fe–S clusters that regulate ABCE1’s ATPase activity with implications to ribosomal function.

Programmable sequence-specific click-labeling of RNA using archaeal box C/D RNP methyltransferases

Biophysical and mechanistic investigation of RNA function requires site-specific incorporation of spectroscopic and chemical probes, which is difficult to achieve using current technologies. We have in vitro reconstituted a functional box C/D small ribonucleoprotein RNA 2′-O-methyltransferase (C/D RNP) from the thermophilic archaeon Pyrococcus abyssi and demonstrated its ability to transfer a prop-2-ynyl group from a synthetic cofactor analog to a series of preselected target sites in model tRNA and pre-mRNA molecules. Target selection of the RNP was programmed by changing a dodecanucleotide guide sequence in a 64-nt C/D guide RNA leading to efficient derivatization of three out of four new targets in each RNA substrate. We also show that the transferred terminal alkyne can be further appended with a fluorophore using a bioorthogonal azide-alkyne 1,3-cycloaddition (click) reaction. The described approach for the first time permits synthetically tunable sequence-specific labeling of RNA with single-nucleotide precision.

Modulation of the Pyrococcus abyssi NucS Endonuclease Activity by Replication Clamp at Functional and Structural Levels

Pyrococcus abyssi NucS is the founding member of a new family of structure-specific DNA endonucleases that interact with the replication clamp proliferating cell nuclear antigen (PCNA). Using a combination of small angle x-ray scattering and surface plasmon resonance analyses, we demonstrate the formation of a stable complex in solution, in which one molecule of the PabNucS homodimer binds to the outside surface of the PabPCNA homotrimer. Using fluorescent labels, PCNA is shown to increase the binding affinity of NucS toward single-strand/double-strand junctions on 5' and 3' flaps, as well as to modulate the cleavage specificity on the branched DNA structures. Our results indicate that the presence of a single major contact between the PabNucS and PabPCNA proteins, together with the complex-induced DNA bending, facilitate conformational flexibility required for specific cleavage at the single-strand/double-strand DNA junction.

Side‐chain cyclization of Gln or Asn residues coupled to peptide bond cleavage in an intein and a model peptide

Protein splicing is a post‐translational event by which an intervening polypeptide, or intein, facilitates its own excision from the flanking polypeptides, or exteins, and the ligation of the exteins. An epimerase found in Trichodesmium erythraeum contains two inteins, Ndse1 and Ndse2. The first intein, Ndse1, can facilitate protein splicing efficiently in E. coli, even with a native C‐terminal Gln replacing the highly conserved Asn. In addition, activity of the intein may be influenced two downstream Cys residues and the presence of disulfide bonds, as we observed differential reactivity in inteins expressed under reducing and non‐reducing conditions. Substitution of Gln for Asn results in an increase in the rate of C‐terminal bond cleavage for the PolII intein from Pyrococcus abyssi. To study side chain cyclization in a model system, we obtained three pentapeptides of Abz‐His‐Xxx‐ Src‐Lys, where Xxx is Gln, Asn or Ala. Dnp, dinitrophenyl, is attached to the C‐terminal Lys and ortho‐aminobenzoic acid (Abz) serves as the N‐terminal residue. Peptide bond cleavage separates the Abz fluorophore from the Dnp quencher.This material is based upon work supported by the National Science Foundation under grant MCB‐0950245, the Camille and Henry Dreyfus Foundation (KM), and a Holy Cross Alumni/Parents Summer Research Scholarship (LU).

Protein splicing of a temperature‐dependent intein from an extreme thermophile

Inteins are intervening polypeptides that are excised during the process of protein splicing. Protein splicing is a self‐catalyzed process in which the intein is removed and the flanking polypeptides are ligated (N‐ and C‐ exteins). The Pyrococcus abyssi (Pab) PolII intein only splices at elevated temperatures. An NMR structure of the intein recently was solved in a collaborator's lab (Du, Z., et al. (2011) Structural and Mutational Studies of a Hyperthermophilic Intein from DNA Polymerase II of Pyrococcus abyssi. J. Biol. Chem. 286(44) 38638–38648.). The NMR data suggest increased rigidity of the intein in comparison to a mesophilic intein from Mycobacterium tuberculosis (Mtu), as well as a β‐hairpin found only in the structures of thermophilic inteins. We have replaced the β‐hairpin from the Pab intein with a linker inspired by the Mtu intein sequence to determine if the β‐hairpin plays a role in the structural stability/rigidity of the intein. We also have designed a genetic screening system that links protein splicing to cell growth on kanamycin in order to select for mutants that allow for splicing at lower temperatures.This material is based upon work supported by the National Science Foundation under grant MCB‐0950245, the Camille and Henry Dreyfus Foundation (KVM), and the Arnold and Mae Beckman Foundation (KMC).

Non‐canonical inteins: Protein splicing by alternate mechanisms

Protein splicing is the post‐translational excision of an intervening polypeptide (intein) from its flanking domains (the exteins), concomitant with extein ligation. The first step of splicing is an amide‐to‐thioester rearrangement of the peptide bond linking the N‐extein and intein. Class three inteins bypass this step. The Clostridium thermocellum TerA intein is class three, whereas the Thermobifida fusca Tfu2914 intein is a traditional intein despite having class three sequence motifs. The third step of splicing is Asn cyclization coupled to cleavage of the peptide bond linking the intein and C‐extein. Both the Methanoculleus marisnigri (Mma) and the Pyrococcus abyssi (Pab) PolII inteins promote efficient splicing with C‐terminal Gln in place of Asn. The activity of the Mma PolII intein may be regulated by disulfide bond formation. The Pab PolII intein splices only at elevated temperatures, allowing for isolation of a stable precursor and study each step of splicing in vitro. An NMR solution structure of the Pab PolII intein reveals a particularly rigid structure, a disordered loop absent in a highly similar P. horikoshii intein, and a β‐hairpin specific to thermophilic inteins.This material is based upon work supported by the National Science Foundation under grant MCB‐0950245 and the Camille and Henry Dreyfus Foundation.

The Box C/D sRNP dimeric architecture is conserved across Kingdom Archaea

Site‐directed 2′‐O‐methylation of RNA is catalyzed, in eukaryotes and archaea, by box C/D small (nucleolar) ribonucleoproteins (box C/D s(no) RNPs). Recent structural studies have led to a controversy over whether box C/D sRNPs are monomeric or dimeric macromolecules. Here we examine the role of the omnipresent sRNA loop as a structural determinant of multimeric sRNP assembly. Mutational analysis of the sRNA shows that the conserved loop between boxes C′ and D′ is required for dimeric sRNP formation, and explains how mutations in sRNAs can effect oligomeric architecture. In this study, we present negative‐stain electron microscopy single particle reconstructions of catalytically active dimeric box C/D sRNPs from three distantly related archaeal species, Solfolobus solfataricus, Pyrococcus abyssi, and Archaeoglobus fulgidus, Our results demonstrate that the dimeric architecture of box C/D sRNPs is broadly conserved across the Archaeal Kingdom.

Protein Splicing of inteins from Synechococcus sp. PCC 7002 and Pyrococcus abyssi

Protein splicing is a four‐step intramolecular reaction, in which an intein flanked by N‐ and C‐terminal exteins is removed, with ligation of the exteins. We are studying inteins that interrupt genes from Synechococcus sp. PCC 7002 (Ssp 7002), Trichodesmium erythraeum (Tery) or Pyrococcus abyssi DNA Polymerase II (Pab Pol II). The cyanobacterium Ssp 7002 has an intein that interrupts a putative dTDP glucose dehydratase. The Ssp 7002 intein has a highly conserved C‐terminal Asn, unlike an intein from Tery, which has 63% sequence identity with the Ssp 7002 intein but has a C‐terminal Gln. Both inteins splice with their native C‐terminal residue. We plan to study the effect of switching Gln for Asn (and vice versa), as well as the influence of conserved residues on the mechanism of splicing. The Pyrococcus abysii PolII intein has a conserved, catalytic His residue. Mutation of this residue to Ala or Gly prevents splicing. We will explore whether we can rescue activity in the His‐Gly mutant in vivo by incubation with imidazole derivatives.This material is based upon work supported by the National Science Foundation under grant MCB‐0950245 and by the Camille and Henry Dreyfus Foundation.

Intein‐mediated peptide bond cleavage adjacent to aspargine or glutamine

Protein splicing is a post‐translational, self‐catalyzed process in which an intervening polypeptide, the intein, is self‐excised from the flanking polypeptides, or exteins. The intein that interrupts the DNA polymerase of Pyrococcus abyssi can facilitate protein splicing in a temperature‐dependent fashion. This intein has a Cterminal Gln, which facilitates the third step of splicing, side chain cyclization coupled to peptide bond cleavage. We plan to induce C‐terminal cleavage of an intein (uncoupled from splicing) to facilitate purification from a solid affinity resin. We have fused the intein with an N‐terminal affinity domain and a C‐terminal target protein for purification, such that C‐terminal cleavage of the intein via cyclization of Asn or Gln liberates the C‐terminal domain with an N‐terminal Cys. This fragment will serve as a substrate for expressed protein ligation. In addition, we will compare the rate of the cyclization between glutamine and asparagine in model peptides with an N‐terminal Abz fluorophore and a C‐terminal Lys‐DNP quencher, using a fluorescence assay to measure cleavage. We will determine the rate of cleavage as a function of temperature and pH.This material is based upon work supported by the National Science Foundation under grant MCB‐0950245 and by the Camille and Henry Dreyfus Foundation.

Protein splicing facilitated by highly similar inteins from two extreme thermophiles

Protein splicing is a post‐translational event by which an intervening polypeptide, the intein, facilitates its own excision from the flanking polypeptides, the exteins, and the ligation of the exteins. Splicing of the Pyrococcus abyssi (Pab) PolII intein is initiated by an increase in temperature, which allows us to measure the rate of individual steps of the splicing reaction in vitro. We explored the rate of the first step of splicing for versions of the Pab PolII intein modified by mutation at conserved residues Asp74 and Ser166. We found no influence on the rate of the first step of splicing, but mutations at Ser166 influence the efficiency of the overall splicing reaction. The Pyrococcus horikoshii PolII intein has 71% sequence identity with the Pab PolII intein, but lacks an unstructured loop found in the Pab PolII intein. We hypothesize that this loop plays a role either in protein stability or in allowing for a mechanism‐linked conformational change to coordinate the steps of splicing. We will compare the temperature dependence of the rate and extent of splicing reactions and of the overall fold for both inteins.This material is based upon work supported by the National Science Foundation under grant MCB‐0950245 and by the Camille and Henry Dreyfus Foundation.