Chemical mapping of cytosines enzymatically flipped out of the DNA helix

Haloacetaldehydes can be employed for probing unpaired DNA structures involving cytosine and adenine residues. Using an enzyme that was structurally proven to flip its target cytosine out of the DNA helix, the HhaI DNA methyltransferase (M.HhaI), we demonstrate the suitability of the chloroacetaldehyde modification for mapping extrahelical (flipped-out) cytosine bases in protein-DNA complexes. The generality of this method was verified with two other DNA cytosine-5 methyltransferases, M.AluI and M.SssI, as well as with two restriction endonucleases, R.Ecl18kI and R.PspGI, which represent a novel class of base-flipping enzymes. Our results thus offer a simple and convenient laboratory tool for detection and mapping of flipped-out cytosines in protein-DNA complexes.     

Binding of EcoP15I DNA methyltransferase to DNA reveals a large structural distortion within the recognition sequence

EcoP15I DNA methyltransferase, a member of the type III restriction-modification system, binds to the sequence 5'-CAGCAG-3' transferring a methyl group from S-adenosyl-l-methionine to the second adenine base. We have investigated protein-DNA interactions in the methylase-DNA complex by three methods. Determination of equilibrium dissociation constants indicated that the enzyme had higher affinity for DNA containing mismatches at the target base within the recognition sequence. Potassium permanganate footprinting studies revealed that there was a hyper-reactive permanganate cleavage site coincident with adenine that is the target base for methylation. More importantly, to detect DNA conformational alterations within the enzyme-DNA complexes, we have used a fluorescence-based assay. When EcoP15I DNA methyltransferase bound to DNA containing 2-aminopurine substitutions within the cognate sequence, an eight to tenfold fluorescent enhancement resulting from enzymatic flipping of the target adenine base was observed. Furthermore, fluorescence spectroscopy analysis showed that the changes attributable to structural distortion were specific for only the bases within the recognition sequence. More importantly, we observed that both the adenine bases in the recognition site appear to be structurally distorted to the same extent. While the target adenine base is probably flipped out of the DNA duplex, our results also suggest that fluorescent enhancements could be derived from protein-DNA interactions other than base flipping. Taken together, our results support the proposed base flipping mechanism for adenine methyltransferases.     

2-Aminopurine as a fluorescent probe for DNA base flipping by methyltransferases.

DNA base flipping, which was first observed for the C5-cytosine DNA methyltransferase M. Hha I, results in a complete removal of the stacking interactions between the target base and its neighbouring bases. We have investigated whether duplex oligodeoxynucleotides containing the fluorescent base analogue 2-aminopurine can be used to sense DNA base flipping. Using M. Hha I as a paradigm for a base flipping enzyme, we find that the fluorescence intensity of duplex oligodeoxynucleotides containing 2-aminopurine at the target site is dramatically enhanced (54-fold) in the presence of M. Hha I. Duplex oligodeoxynucleotides containing 2-aminopurine adjacent to the target cytosine show little fluorescence increase upon addition of M. Hha I. These results clearly demonstrate that duplex oligodeoxynucleotides containing 2-aminopurine at the target site can serve as fluorescence probes for base flipping. Another enzyme hypothesized to use a base flipping mechanism is the N6-adenine DNA methyltransferase M. Taq I. Addition of M. Taq I to duplex oligodeoxynucleotides bearing 2-aminopurine at the target position, also results in a strongly enhanced fluorescence (13-fold), whereas addition to duplex oligodeoxynucleotides containing 2-aminopurine at the 3'- or 5'-neighbouring position leads only to small fluorescence increases. These results give the first experimental evidence that the adenine-specific DNA methyltransferase M. Taq I also flips its target base.     

High resolution footprinting of a type I methyltransferase reveals a large structural distortion within the DNA recognition site.

The type I DNA methyltransferase M.EcoR124I is a multi-subunit enzyme that binds to the sequence GAAN6RTCG, transferring a methyl group from S-adenosyl methionine to a specific adenine on each DNA strand. We have investigated the protein-DNA interactions in the complex by DNase I and hydroxyl radical footprinting. The DNase I footprint is unusually large: the protein protects the DNA on both strands for at least two complete turns of the helix, indicating that the enzyme completely encloses the DNA in the complex. The higher resolution hydroxyl radical probe shows a smaller, but still extensive, 18 bp footprint encompassing the recognition site. Within this region, however, there is a remarkably hyper-reactive site on each strand. The two sites of enhanced cleavage are co-incident with the two adenines that are the target bases for methylation, showing that the DNA is both accessible and highly distorted at these sites. The hydroxyl radical footprint is unaffected by the presence of the cofactor S-adenosyl methionine, showing that the distorted DNA structure induced by M.EcoR124I is formed during the initial DNA binding reaction and not as a transient intermediate in the reaction pathway.     

Structure of pvu II DNA-(cytosine N4) methyltransferase, an example of domain permutation and protein fold assignment.

We have determined the structure of Pvu II methyltransferase (M. Pvu II) complexed with S -adenosyl-L-methionine (AdoMet) by multiwavelength anomalous diffraction, using a crystal of the selenomethionine-substituted protein. M. Pvu II catalyzes transfer of the methyl group from AdoMet to the exocyclic amino (N4) nitrogen of the central cytosine in its recognition sequence 5'-CAGCTG-3'. The protein is dominated by an open alpha/beta-sheet structure with a prominent V-shaped cleft: AdoMet and catalytic amino acids are located at the bottom of this cleft. The size and the basic nature of the cleft are consistent with duplex DNA binding. The target (methylatable) cytosine, if flipped out of the double helical DNA as seen for DNA methyltransferases that generate 5-methylcytosine, would fit into the concave active site next to the AdoMet. This M. Pvu IIalpha/beta-sheet structure is very similar to those of M. Hha I (a cytosine C5 methyltransferase) and M. Taq I (an adenine N6 methyltransferase), consistent with a model predicting that DNA methyltransferases share a common structural fold while having the major functional regions permuted into three distinct linear orders. The main feature of the common fold is a seven-stranded beta-sheet (6 7 5 4 1 2 3) formed by five parallel beta-strands and an antiparallel beta-hairpin. The beta-sheet is flanked by six parallel alpha-helices, three on each side. The AdoMet binding site is located at the C-terminal ends of strands beta1 and beta2 and the active site is at the C-terminal ends of strands beta4 and beta5 and the N-terminal end of strand beta7. The AdoMet-protein interactions are almost identical among M. Pvu II, M. Hha I and M. Taq I, as well as in an RNA methyltransferase and at least one small molecule methyltransferase. The structural similarity among the active sites of M. Pvu II, M. Taq I and M. Hha I reveals that catalytic amino acids essential for cytosine N4 and adenine N6 methylation coincide spatially with those for cytosine C5 methylation, suggesting a mechanism for amino methylation.     

Uracil interference, a rapid and general method for defining protein-DNA interactions involving the 5-methyl group of thymines: the GCN4-DNA complex.

We describe a novel uracil interference method for examining protein contacts with the 5-methyl group of thymines. The protein of interest is incubated with target DNA containing randomly distributed deoxyuracil substitutions that is generated by carrying out the polymerase chain reaction in the presence of a mixture of TTP and dUTP. After separating DNA-protein complexes away from unbound DNA, the locations of deoxyuracil residues that either do or do not interfere with DNA-binding are determined by cleavage with uracil-N-glycosylase followed by piperidine. Using this uracil interference assay, we show that the methyl groups of the four core thymines, but not the two peripheral thymines, of the optimal binding site (ATG-ACTCAT) are important for high affinity binding of GCN4. Similar, but not identical, results are obtained using KMnO4 interference, another method used for studying protein-DNA interactions involving thymine residues. These observations strongly suggest that GCN4 directly contacts the 5-methyl groups of the four core thymines that lie in the major groove of the target DNA. Besides providing specific structural information about protein-DNA complexes, uracil interference should also be useful for identifying DNA-binding proteins and their target sites in eukaryotic promoter regions.     

Footprint analysis of the bsp RI DNA methyltransferase-DNA interaction.

The interaction between the GGCC-specific Bsp RI DNA methyltransferase (M. Bsp RI) and substrate DNA was studied with footprinting techniques using a DNA fragment that was unmodified on both strands. Footprinting with DNase I revealed an approximately 14 bp protected region. Footprinting with dimethylsulfate detected major groove interactions with the guanine bases of the recognition sequence. Reaction with 1,10-phenanthroline-copper did not show protection, suggesting that minor groove interactions play little role in sequence-specific recognition by M. Bsp RI. Hydroxyl radical footprinting revealed a protected stretch of 6 nt. The hydroxyl radical footprint of M. Bsp RI differs markedly from the the footprint reported for the Hha I and Sss I methyltransferases. The pattern of protection from dimethylsulfate and hydroxyl radicals suggests that the interactions of M. Bsp RI with DNA are similar to those detected in the co-crystal structure of the Hae III methyltransferase.     

M.HhaI binds tightly to substrates containing mismatches at the target base.

The (cytosine-5) DNA methyltransferase M.HhaI causes its target cytosine base to be flipped completely out of the DNA helix upon binding. We have investigated the effects of replacing the target cytosine by other, mismatched bases, including adenine, guanine, thymine and uracil. We find that M.HhaI binds more tightly to such mismatched substrates and can even transfer a methyl group to uracil if a G:U mismatch is present. Other mismatched substrates in which the orphan guanine is changed exhibit similar behavior. Overall, the affinity of DNA binding correlates inversely with the stability of the target base pair, while the nature of the target base appears irrelevant for complex formation. The presence of a cofactor analog. S-adenosyl-L-homocysteine, greatly enhances the selectivity of the methyltransferase for cytosine at the target site. We propose that the DNA methyltransferases have evolved from mismatch binding proteins and that base flipping was, and still is, a key element in many DNA-enzyme interactions.     

Clustered Adenine/Thymine Stretches Are Essential for Function of a Fission Yeast Replication Origin

We have determined functional elements required for autonomous replication of the Schizosaccharomyces pombe ars2004 that acts as an intrinsic chromosomal replication origin. Internal deletion analysis of a 940-bp fragment (ars2004M) showed three regions, I to III, to be required for autonomously replicating sequence (ARS) activity. Eight-base-pair substitutions in the 40-bp region I, composed of arrays of adenines on a DNA strand, resulted in a great reduction of ARS activity. Substitutions of region I with synthetic sequences showed that no specific sequence but rather repeats of three or more consecutive adenines or thymines, without interruption by guanine or cytosine, are required for the ARS activity. The 65-bp region III contains 11 repeats of the AAAAT sequence, while the 165-bp region II has short adenine or thymine stretches and a guanine- and cytosine-rich region which enhances ARS activity. All three regions in ars2004M can be replaced with 40-bp poly(dA/dT) fragments without reduction of ARS activity. Although spacer regions in the ars2004M enhance ARS activity, all could be deleted when an 40-bp poly(dA/dT) fragment was added in place of region I. Our results suggest that the origin activity of fission yeast replicators depends on the number of adenine/thymine stretches, the extent of their clustering, and presence of certain replication-enhancing elements.     

Sequence dependence for bypass of thymine glycols in DNA by DNA polymerase I.

Single-stranded phage DNAs containing thymine glycols were prepared by oxidation with osmium tetroxide (OsO4) and were used as templates for DNA synthesis by E. coli DNA polymerase I. The induction of thymine glycol lesions in DNA, as measured by immunoassay, quantitatively accounted for an inhibition of in vitro DNA synthesis on modified templates. Analysis of termination sites for synthesis by DNA polymerase I (Klenow fragment) showed that DNA synthesis terminated at most template thymine sites in OsO4-treated DNA, indicating that incorporation occurred opposite putative thymine glycols in DNA. Nucleotides 5' and 3' to putative thymine glycol sites affect the reaction, however, since termination was not observed at thymines in the sequence 5'-CTPur-3'. Conversion of thymine glycols to urea residues in DNA by alkali treatment caused termination of DNA synthesis one nucleotide 3' to template thymine sites, including thymines in the 5'-CTPur-3' sequence, showing that the effect of surrounding sequence is on the elongation reaction by DNA polymerase rather than differential damage induction by OsO4.     

2-Pyrimidinone as a probe for studying the EcoRII DNA methyltransferase-substrate interaction.

EcoRII DNA methyltransferase (M.EcoRII) recognizes the 5' em leader CC*T/AGG em leader 3' DNA sequence and catalyzes the transfer of the methyl group from S-adenosyl-l-methionine to the C5 position of the inner cytosine residue (C*). Here, we study the mechanism of inhibition of M.EcoRII by DNA containing 2-pyrimidinone, a cytosine analogue lacking an NH(2) group at the C4 position of the pyrimidine ring. Also, DNA containing 2-pyrimidinone was used for probing contacts of M.EcoRII with functional groups of pyrimidine bases of the recognition sequence. 2-Pyrimidinone was incorporated into the 5' em leader CCT/AGG em leader 3' sequence replacing the target and nontarget cytosine and central thymine residues. Study of the DNA stability using thermal denaturation of 2-pyrimidinone containing duplexes pointed to the influence of the bases adjacent to 2-pyrimidinone and to a greater destabilizing influence of 2-pyrimidinone substitution for thymine than that for cytosine. Binding of M.EcoRII to 2-pyrimidinone containing DNA and methylation of these DNA demonstrate that the amino group of the outer cytosine in the EcoRII recognition sequence is not involved in the DNA-M.EcoRII interaction. It is probable that there are contacts between the functional groups of the central thymine exposed in the major groove and M.EcoRII. 2-Pyrimidinone replacing the target cytosine in the EcoRII recognition sequence forms covalent adducts with M.EcoRII. In the absence of the cofactor S-adenosyl-l-methionine, proton transfer to the C5 position of 2-pyrimidinone occurs and in the presence of S-adenosyl-l-methionine, methyl transfer to the C5 position of 2-pyrimidinone occurs.     

In vitro selection of oligonucleotides that bind double-stranded DNA in the presence of triplex-stabilizing agents

A SELEX approach has been developed in order to select oligonucleotides that bind double-stranded DNA in the presence of a triplex-stabilizing agent, and was applied to a target sequence containing an oligopurine–oligopyrimidine stretch. After only seven rounds of selection, the process led to the identification of oligonucleotides that were able to form triple helices within the antiparallel motif. Inspection of the selected sequences revealed that, contrary to GC base pair which were always recognized by guanines, recognition of AT base pair could be achieved by either adenine or thymine, depending on the sequence context. While thymines are strongly preferred for several positions, some others can accommodate the presence of adenines. These results contribute to set the rules for designing oligonucleotides that form stable triple helices in the presence of triplex-stabilizing agents at physiological pH. They set the basis for further experiments regarding extension of potential target sequences for triple-helix formation or recognition of ligand–DNA complexes.     

Structure of the N6-adenine DNA methyltransferase M.TaqI in complex with DNA and a cofactor analog

The 2.0 A crystal structure of the N6-adenine DNA methyltransferase M.TaqI in complex with specific DNA and a nonreactive cofactor analog reveals a previously unrecognized stabilization of the extrahelical target base. To catalyze the transfer of the methyl group from the cofactor S-adenosyl-l-methionine to the 6-amino group of adenine within the double-stranded DNA sequence 5'-TCGA-3', the target nucleoside is rotated out of the DNA helix. Stabilization of the extrahelical conformation is achieved by DNA compression perpendicular to the DNA helix axis at the target base pair position and relocation of the partner base thymine in an interstrand pi-stacked position, where it would sterically overlap with an innerhelical target adenine. The extrahelical target adenine is specifically recognized in the active site, and the 6-amino group of adenine donates two hydrogen bonds to Asn 105 and Pro 106, which both belong to the conserved catalytic motif IV of N6-adenine DNA methyltransferases. These hydrogen bonds appear to increase the partial negative charge of the N6 atom of adenine and activate it for direct nucleophilic attack on the methyl group of the cofactor.     

Chemical reactivity of potassium permanganate and diethyl pyrocarbonate with B DNA: specific reactivity with short A-tracts

We have examined the reactivity of B DNA with two chemical probes of DNA structure, potassium permanganate (KMnO4; thymine specific) and diethyl pyrocarbonate (DEPC; purine specific, A greater than G). The DNA probed is from the beta-lactamase promoter region of the vector pBR322, and from the 3' noncoding region of a chicken embryonic myosin heavy chain gene. The chemical probes display variable reactivity with the susceptible bases in these fragments, suggesting that modification of these bases by KMnO4 and DEPC is quite sequence dependent. In contrast, these probes react with the short A-tracts present in these DNA fragments in a reproducible fashion, generating two related patterns of reactivity. In the majority of the A-tracts, all but the 3'-terminal thymine are protected from KMnO4 attack, while DEPC reacts significantly with all but the 3'-terminal adenine of the A-tracts. Some A-tracts also display a very high DEPC reactivity at the adenine adjacent to the 3'-terminal unreactive adenine. Little qualitative difference in the KMnO4 reactivity of the A-tracts was found between 12 and 43 degrees C. However, at lower temperatures the elevated KMnO4 reactivity at the 3'-terminal A-tract thymine is sometimes lost. Raising the temperature of the KMnO4 reaction can cause relatively large increases in the reactivity of some single thymines, suggesting that significant local changes in stacking occur at these thymines at elevated temperatures. The data presented suggest that many short A-tracts embedded in long fragments of DNA can assume a number of related structures in solution, each of which possess distinct junctions with the flanking DNA. This result is consistent with high-resolution structural studies on oligonucleotides containing short A-tracts. The relevance of these results to current models of A-tract structure and DNA bending is discussed. Our data also indicate that KMnO4 and DEPC are potentially useful reagents for the study of sequence-dependent variations in B DNA structure.     

Human thymine DNA glycosylase (TDG) and methyl-CpG-binding protein 4 (MBD4) excise thymine glycol (Tg) from a Tg:G mispair

The repair enzymes thymine DNA glycosylase (TDG) and methyl-CpG-binding protein 4 (MBD4) remove thymines from T:G mismatches resulting from deamination of 5-methylcytosine. Thymine glycol, a common DNA lesion produced by oxidative stress, can arise from oxidation of thymine or from oxidative deamination of 5-methylcytosine, and is then present opposite adenine or opposite guanine, respectively. Here we have used oligonucleotides with thymine glycol incorporated into different sequence contexts and paired with adenine or guanine. We show that TDG and MBD4 can remove thymine glycol when present opposite guanine but not when paired with adenine. The efficiency of these enzymes for removal of thymine glycol is about half of that for removal of thymine in the same sequence context. The two proteins may have evolved to act specifically on DNA mismatches produced by deamination and by oxidation-coupled deamination of 5-methylcytosine. This repair pathway contributes to mutation avoidance at methylated CpG dinucleotides.     

Identification of the binding site for the extrahelical target base in N6-adenine DNA methyltransferases by photo-cross-linking with duplex oligodeoxyribonucleotides containing 5-iodouracil at the target position.

DNA methyltransferases flip their target bases out of the DNA double helix for catalysis. Base flipping of C5-cytosine DNA methyltransferases was directly observed in the protein-DNA cocrystal structures of M.HhaI and M.HaeIII. Indirect structural evidence for base flipping of N6-adenine and N4-cytosine DNA methyltransferases was obtained by modeling DNA into the three-dimensional structures of M.TaqI and M.PvuII in complex with the cofactor. In addition, biochemical evidence of base flipping was reported for different N6-adenine DNA methyltransferases. As no protein-DNA cocrystal structure for the related N6-adenine and N4-cytosine DNA methyltransferases is available, we used light-induced photochemical cross-linking to identify the binding site of the extrahelical target bases. The N6-adenine DNA methyltransferases M.TaqI and M.CviBIII, which both methylate adenine within the double-stranded 5'-TCGA-3' DNA sequence, were photo-cross-linked to duplex oligodeoxyribonucleotides containing 5-iodouracil at the target position in 50-60% and almost quantitative yield, respectively. Proteolytic fragmentation of the M. CviBIII-DNA complex followed by Edman degradation and electrospray ionization mass spectrometry indicates photo-cross-linking to tyrosine 122. In addition, the mutant methyltransferases M. TaqI/Y108A and M.TaqI/F196A were photo-cross-linked with 6-fold and 2-fold reduced efficiency, respectively, which suggests that tyrosine 108 is the primary site of modification in M.TaqI. Our results indicate a close proximity between the extrahelical target base and tyrosine 122 in M.CviBIII or tyrosine 108 in M.TaqI. As both residues belong to the conserved motif IV ((N/D/S)(P/I)P(Y/F/W)) found in all N6-adenine and N4-cytosine DNA as well as in N6-adenine RNA methyltransferases, a similar spatial relationship between the target bases and the aromatic amino acid residue within motif IV is expected for all these methyltransferases.     

Transition from nonspecific to specific DNA interactions along the substrate-recognition pathway of dam methyltransferase

DNA methyltransferases methylate target bases within specific nucleotide sequences. Three structures are described for bacteriophage T4 DNA-adenine methyltransferase (T4Dam) in ternary complexes with partially and fully specific DNA and a methyl-donor analog. We also report the effects of substitutions in the related Escherichia coli DNA methyltransferase (EcoDam), altering residues corresponding to those involved in specific interaction with the canonical GATC target sequence in T4Dam. We have identified two types of protein-DNA interactions: discriminatory contacts, which stabilize the transition state and accelerate methylation of the cognate site, and antidiscriminatory contacts, which do not significantly affect methylation of the cognate site but disfavor activity at noncognate sites. These structures illustrate the transition in enzyme-DNA interaction from nonspecific to specific interaction, suggesting that there is a temporal order for formation of specific contacts.     

M.TaqI facilitates the base flipping via an unusual DNA backbone conformation

MD simulations have been carried out to understand the dynamical behavior of the DNA substrate of the Thermus aquaticus DNA methyltransferase (M.TaqI) in the methylation process at N6 of adenine. As starting structures, an x-ray structure of M.TaqI in complex with DNA and cofactor analogue (PDB code: 1G 38) and free decamer d(GTTCGATGTC)(2) were taken. The x-ray structure shows two consecutive BII substates that are not observed in the free decamer. These consecutive BII substates are also observed during our simulation. Additionally, their facing backbones adopt the same conformations. These double facing BII substates are stable during the last 9 ns of the trajectories and result in a stretched DNA structure. On the other hand, protein-DNA contacts on 5' and 3' phosphodiester groups of the partner thymine of flipped adenine have changed. The sugar and phosphate parts of thymine have moved further into the empty space left by the flipping base without the influence of protein. Furthermore, readily high populated BII substates at the GpA step of palindromic tetrad TCGA rather than CpG step are observed in the free decamer. On the contrary, the BI substate at the GpA step is observed on the flipped adenine strand. A restrained MD simulation, reproducing the BI/BII pattern in the complex, demonstrated the influence of the unusual backbone conformation on the dynamical behavior of the target base. This finding along with the increased nearby interstrand phosphate distance is supportive to the N6-methylation mechanism.     

Structure of RsrI methyltransferase, a member of the N6-adenine beta class of DNA methyltransferases

DNA methylation is important in cellular, developmental and disease processes, as well as in bacterial restriction-modification systems. Methylation of DNA at the amino groups of cytosine and adenine is a common mode of protection against restriction endonucleases afforded by the bacterial methyltransferases. The first structure of an N:6-adenine methyltransferase belonging to the beta class of bacterial methyltransferases is described here. The structure of M. RSR:I from Rhodobacter sphaeroides, which methylates the second adenine of the GAATTC sequence, was determined to 1.75 A resolution using X-ray crystallography. Like other methyltransferases, the enzyme contains the methylase fold and has well-defined substrate binding pockets. The catalytic core most closely resembles the PVU:II methyltransferase, a cytosine amino methyltransferase of the same beta group. The larger nucleotide binding pocket observed in M. RSR:I is expected because it methylates adenine. However, the most striking difference between the RSR:I methyltransferase and the other bacterial enzymes is the structure of the putative DNA target recognition domain, which is formed in part by two helices on an extended arm of the protein on the face of the enzyme opposite the active site. This observation suggests that a dramatic conformational change or oligomerization may take place during DNA binding and methylation.     

A prediction of the amino acids and structures involved in DNA recognition by type I DNA restriction and modification enzymes.

The S subunits of type I DNA restriction/modification enzymes are responsible for recognising the DNA target sequence for the enzyme. They contain two domains of approximately 150 amino acids, each of which is responsible for recognising one half of the bipartite asymmetric target. In the absence of any known tertiary structure for type I enzymes or recognisable DNA recognition motifs in the highly variable amino acid sequences of the S subunits, it has previously not been possible to predict which amino acids are responsible for sequence recognition. Using a combination of sequence alignment and secondary structure prediction methods to analyse the sequences of S subunits, we predict that all of the 51 known target recognition domains (TRDs) have the same tertiary structure. Furthermore, this structure is similar to the structure of the TRD of the C5-cytosine methyltransferase, Hha I, which recognises its DNA target via interactions with two short polypeptide loops and a beta strand. Our results predict the location of these sequence recognition structures within the TRDs of all type I S subunits.