Structural requirements for N-trimethylation of lysine 115 of calmodulin

Calmodulin is trimethylated at lysine 115 by a highly specific methyltransferase that utilizes S-adenosylmethionine as a co-substrate. Lysine 115 is found within a highly conserved six-amino acid loop (LGEKLT) that forms a 90 degrees turn between EF-hand III and EF-hand IV in the carboxyl-terminal lobe. In the present work a mutagenesis approach was used to investigate the structural features of the carboxyl-terminal lobe that lead to the specificity of calmodulin methylation. Three structural regions within the carboxyl-terminal lobe appear to be involved in methyltransferase recognition: the highly conserved six-amino acid loop-turn region that contains lysine 115 as well as the adjacent alpha-helices (helix 6 and helix 7) from EF-hands III and IV. Site-directed mutagenesis of residues in the loop show that three residues, glycine 113, glutamate 114, and leucine 116 are essential for methylation. In addition, subdomain (individual helix or Ca(2+) binding loop) exchange mutants show that the substitutions of either helix 6 (EF-hand III) with helix 2 (EF-hand I) or helix 7 (EF-hand IV) with helix 3 (EF-hand II) compromises methylation. Charge-to-alanine mutations in helix 7 show that substitution of conserved charged residues at positions 118, 120, 122, 126, and 127 reduced lysine 115 methylation rates, suggesting possible electrostatic interactions between this helix and the methyltransferase. Single substitutions in helix 6 did not affect calmodulin methylation, suggesting this region may play a more indirect role in stabilizing the conformation of the methyltransferase recognition sequence.     

Conserved Structural Regions Involved in the Catalytic Mechanism of Escherichia coli K-12 WaaO (RfaI)

Escherichia coli K-12 WaaO (formerly known as RfaI) is a nonprocessive α-1,3 glucosyltransferase, involved in the synthesis of the R core of lipopolysaccharide. By comparing the amino acid sequence of WaaO with those of 11 homologous α-glycosyltransferases, four strictly conserved regions, I, II, III, and IV, were identified. Since functionally related transferases are predicted to have a similar architecture in the catalytic sites, it is assumed that these four regions are directly involved in the formation of α-glycosidic linkage from α-linked nucleotide diphospho-sugar donor. Hydrophobic cluster analysis revealed a conserved domain at the N termini of these α-glycosyltransferases. This domain was similar to that previously reported for β-glycosyltransferases. Thus, this domain is likely to be involved in the formation of β-glycosidic linkage between the donor sugar and the enzyme at the first step of the reaction. Site-directed mutagenesis analysis of E. coli K-12 WaaO revealed four critical amino acid residues.     

Structural fingerprints of the Ras-GTPase activating proteins neurofibromin and p120GAP

Ras specific GTPase activating proteins (GAPs), neurofibromin and p120GAP, bind GTP bound Ras and efficiently complement its active site. Here we present comparative data from mutations and fluorescence-based assays of the catalytic domains of both RasGAPs and interpret them using the crystal structures. Three prominent regions in RasGAPs, the arginine-finger loop, the phenylalanine-leucine-arginine (FLR) region and alpha7/variable loop contain structural fingerprints governing the GAP function. The finger loop is crucial for the stabilization of the transition state of the GTPase reaction. This function is controlled by residues proximal to the catalytic arginine, which are strikingly different between the two RasGAPs. These residues specifically determine the orientation and therefore the positioning of the arginine finger in the Ras active site. The invariant FLR region, a hallmark for RasGAPs, indirectly contributes to GTPase stimulation by forming a scaffold, which stabilizes Ras switch regions. We show that a long hydrophobic side-chain in the FLR region is crucial for this function. The alpha7/variable loop uses several conserved residues including two lysine residues, which are involved in numerous interactions with the switch I region of Ras. This region determines the specificity of the Ras-RasGAP interaction.     

Mutational analysis of TraM correlates oligomerization and DNA binding with autoregulation and conjugative DNA transfer

F plasmid TraM, an autoregulatory homotetramer, is essential for F plasmid bacterial conjugative transfer, one of the major mechanisms for horizontal gene dissemination. TraM cooperatively binds to three sites (sbmA, -B, and -C) near the origin of transfer in the F plasmid. To examine whether or not tetramerization of TraM is required for autoregulation and F conjugation, we used a two-plasmid system to screen for autoregulation-defective traM mutants generated by random PCR mutagenesis. A total of 72 missense mutations in TraM affecting autoregulation were selected, all of which also resulted in a loss of TraM function during F conjugation. Mutational analysis of TraM defined three regions important for F conjugation, including residues 3-10 (region I), 31-53 (region II), and 80-121 (region III); in addition, residues 3-47 were also important for the immunoreactivity of TraM. Biochemical analysis of mutant proteins indicated that region I defined a DNA binding domain that was not involved in tetramerization, whereas regions II and III were important for both tetramerization and efficient DNA binding. Mutations in region III affected the cooperativity of binding of TraM to sbmA, -B, and -C. Our results suggest that tetramerization is important for specific DNA binding, which, in turn, is essential for traM autoregulation and F conjugation. These findings support the hypothesis that TraM functions as a "signaling" factor that triggers DNA transport during F conjugation.     

Acidic residues critical for the activity and biological function of yeast DNA polymerase eta

Rad30 is a member of the newly discovered UmuC/DinB/Rad30 family of DNA polymerases. The N-terminal regions of these proteins are highly homologous, and they contain five conserved motifs, I to V, while their C-terminal regions are quite divergent. We examined the contributions of the C-terminal and N-terminal regions of Rad30 to its activity and biological function. Although deletion of the last 54 amino acids has no effect on DNA polymerase or thymine-thymine (T-T) dimer bypass activity, this C-terminal deletion-containing protein is unable to perform its biological function in vivo. The presence of a bipartite nuclear targeting sequence within this region suggests that at least one function of this portion of Rad30 is nuclear targeting. To identify the active-site residues of Rad30 important for catalysis, we generated mutations of nine acidic residues that are invariant or highly conserved among Rad30 proteins from different eukaryotic species. Mutations of the Asp30 and Glu39 residues present in motif I and of the Asp155 residue present in motif III to alanine completely inactivated the DNA polymerase and T-T dimer bypass activities, and these mutations did not complement the UV sensitivity of the rad30Delta mutation. Mutation of Glu156 in motif III to alanine confers a large reduction in the efficiency of nucleotide incorporation, whereas the remaining five Rad30 mutant proteins retain wild-type levels of DNA polymerase and T-T dimer bypass activities. From these observations, we suggest a role for the Asp30, Glu39, and Asp155 residues in the binding of two metal ions required for the reaction of the incoming deoxynucleoside 5'-triphosphate with the 3'-hydroxyl in the primer terminus, while Glu156 may participate in nucleotide binding.     

Functional analysis of exotoxin A-related protein of Pseudomonas aeruginosa lacking residues 225-412

The crystal structure of the exotoxin A (ETA) of Pseudomonas aeruginosa showed that this protein is folded into three distinct domains. Domain I (Ia and Ib), the amino-terminal domain, is the receptor-binding domain of ETA and domain III, the carboxy-terminal domain, is responsible for the ADP-ribosyl transferase activity of the toxin. To elucidate the function(s) of domains 1b and II in the intoxication process and to define the region of the domain III necessary for ADP-ribosylating activity, a defined deletion in the structural gene of P. aeruginosa ETA encompassing residues 225-412 was constructed and an ETA-related product DeID, (from which all of domains II and Ib were deleted) was expressed. The ETA-related protein did not penetrate sensitive cells, but retained the same specific activity to ADP-ribosylate elongation factor-2 as wild-type toxin. This suggests that domain II is necessary to allow toxin internalization by sensitive cells and that the absence of domain Ib does not interfere with enzymic activity. The domain strictly involved in ADP-ribosylation activity encompasses residues 412-613.     

Exploring the ubiquinone binding cavity of respiratory complex I

Proton pumping respiratory complex I is a major player in mitochondrial energy conversion. Yet little is known about the molecular mechanism of this large membrane protein complex. Understanding the details of ubiquinone reduction will be prerequisite for elucidating this mechanism. Based on a recently published partial structure of the bacterial enzyme, we scanned the proposed ubiquinone binding cavity of complex I by site-directed mutagenesis in the strictly aerobic yeast Yarrowia lipolytica. The observed changes in catalytic activity and inhibitor sensitivity followed a consistent pattern and allowed us to define three functionally important regions near the ubiquinone-reducing iron-sulfur cluster N2. We identified a likely entry path for the substrate ubiquinone and defined a region involved in inhibitor binding within the cavity. Finally, we were able to highlight a functionally critical structural motif in the active site that consisted of Tyr-144 in the 49-kDa subunit, surrounded by three conserved hydrophobic residues.     

Alterations in the adenine-plus-thymine-rich region of CEN3 affect centromere function in Saccharomyces cerevisiae.

Centromere DNA from 11 of the 16 chromosomes of the yeast Saccharomyces cerevisiae have been analyzed and reveal three sequence elements common to each centromere, referred to as conserved centromere DNA elements (CDE). The adenine-plus-thymine (A + T)-rich central core element, CDE II, is flanked by two short conserved sequences, CDE I (8 base pairs [bp]) and CDE III (25 bp). Although no consensus sequence exists among the different CDE II regions, they do have three common features of sequence organization. First, the CDE II regions are similar in length, ranging from 78 to 86 bp measured from CDE I to the left boundary of CDE III. Second, the base composition is always greater than 90% A + T. Finally, the A and T residues in these segments are often arranged in runs of A and runs of T residues, sometimes with six or seven bases in a stretch. We constructed insertion, deletion, and replacement mutations in the CDE II region of the centromere from chromosome III, CEN3, designed to investigate the length and sequence requirements for function of the CDE II region of the centromere. We analyzed the effect of these altered centromeres on plasmid and chromosome segregation in S. cerevisiae. Our results show that increasing the length of CDE II from 84 to 154 bp causes a 100-fold increase in chromosome nondisjunction. Deletion mutations removing segments of the A + T-rich CDE II DNA also cause aberrant segregation. In some cases partial function could be restored by replacing the deleted DNA with fragments whose primary sequence or base composition is very different from that of the wild-type CDE II DNA. In addition, we found that identical mutations introduced into different positions in CDE II have very similar effects.     

Evolutionary conservation of the chlorophyll a/b-binding proteins: cDNAs encoding type I, II and III LHC I polypeptides from the gymnosperm Scots pine

Summary cDNAs encoding three different LHC I polypeptides (Type I, Type II and Type III) from the gymnosperm Scots pine (Pinus sylvestris L.) were isolated and sequenced. Comparisons of the deduced amino acid sequences with the corresponding tomato sequences showed that all three proteins were highly conserved although less so than the LHC II proteins. The similarities between mature Scots pine and tomato Types I, II and III LHC I proteins were 80%, 87% and 85%, respectively. Two of the five His residues that are found in AXXXH sequences, which have been identified as putative chlorophyll ligands in the Type I and Type II proteins, were not conserved. The same two regions of high homology between the different LHC proteins, which have been identified in tomato, were also found in the Scots pine proteins. Within the conserved regions, the Type I and Type II proteins had the highest similarity; however, the Type II and Type III proteins also showed a similarity in the central region. The results suggest that all flowering plants (gymnosperms and angiosperms) probably have the same set of LHC polypeptides. A new nomenclature for the genes encoding LHC polypeptides (formerly cab genes) is proposed. The names lha and lhb are suggested for genes encoding LHC I and LHC II proteins, respectively, analogous to the nomenclature for the genes encoding other photosynthetic proteins.     

Isolation and characterization of the gene from Pseudomonas syringae pv. phaseolicola encoding the phaseolotoxin-insensitive ornithine carbamoyltransferase

Summary The gene coding for the phaseolotoxin-insensitive ornithine carbamoyltransferase (OCTase) fromPseudomonas syringae pv.phaseolicola has been cloned and sequenced. The gene has a deduced coding capacity for a polypeptide with a calculated M, of 36520 daltons. Comparison of the amino acid sequence of the OCTase enzymes encoded by theP. aeruginosa argF and theEscherichia coli argI andargF genes with the deduced sequence of the newly identified gene shows that 79 amino acid residues are strictly conserved in all four polypeptides; among these 7 out of 9 residues are involved in enzyme function. Of three amino acid regions that have been implicated in substrate binding or catalysis, two are strictly conserved, and the third involved in carbamoylphosphate binding differs. This correlates well with published data showing that phaseolotoxin competes for the carbamoylphosphate binding site in the phaseolotoxin-sensitive OCTases. We propose that the gene be namedargK.     

Mutational analysis of the Tetrahymena telomerase RNA: identification of residues affecting telomerase activity in vitro.

Telomere-specific repeat sequences are essential for chromosome end stability. Telomerase maintains telomere length by adding sequences de novo onto chromosome ends. The template domain of the telomerase RNA component dictates synthesis of species-specific telomeric repeats and other regions of the RNA have been suggested to be important for enzyme structure and/or catalysis. Using enzyme reconstituted in vitro with RNAs containing deletions or substitutions we identified nucleotides in the RNA component that are important for telomerase activity. Although many changes to conserved features in the RNA secondary structure did not abolish enzyme activity, levels of activity were often greatly reduced, suggesting that regions other than the template play a role in telomerase function. The template boundary was only altered by changes in stem II that affected the conserved region upstream of the template, not by changes in other regions, such as stems I, III and IV, consistent with a role of the conserved region in defining the 5' boundary of the template. Surprisingly, telomerase RNAs with substitutions or deletion of residues potentially abolishing the conserved pseudoknot structure had wild-type levels of telomerase activity. This suggests that this base pairing interaction may not be required for telomerase activity per se but may be conserved as a regulatory site for the enzyme in vivo.     

Mutational Analysis of Meiotic and Mitotic Centromere Function in Saccharomyces cerevisiae

A centromere (CEN) in Saccharomyces cerevisiae consists of approximately 150 bp of DNA and contains 3 conserved sequence elements: a high A + T region 78-86 bp in length (element II), flanked on the left by a conserved 8-bp element I sequence (PuTCACPuTG), and on the right by a conserved 25-bp element III sequence. We have carried out a structure-function analysis of the element I and II regions of CEN3 by constructing mutations in these sequences and subsequently determining their effect on mitotic and meiotic chromosome segregation. We have also examined the mitotic and meiotic segregation behavior of ARS plasmids containing the structurally altered CEN3 sequences. Replacing the periodic tracts of A residues within element II with random A + T sequences of equal length increases the frequency of mitotic chromosome nondisjunction only 4-fold; whereas, reducing the A + T content of element II while preserving the length results in a 40-fold increase in the frequence of chromosome nondisjunction. Structural alterations in the element II region that do not decrease the overall length have little effect on the meiotic segregation behavior of the altered chromosomes. Centromeres containing a deletion of element I or a portion of element II retain considerable mitotic activity, yet plasmids carrying these same mutations segregate randomly during meiosis I, indicating these sequences to be essential for maintaining attachment of the replicated sister chromatids during the first meiotic division. The presence of an intact element I sequence properly spaced from the element III region is absolutely essential for proper meiotic function of the centromere.     

Primary structure of belladonna mottle virus coat protein

The coat protein of belladonna mottle virus (a tymovirus) was cleaved by trypsin and chymotrypsin, and the peptides were separated by high performance liquid chromatography using a combination of gel permeation, reverse phase, and ion pair chromatography. The peptides were sequenced manually using the 4-N, N-dimethylaminoazobenzene-4'-isothiocyanate/phenyl isothiocyanate double-coupling method. The chymotryptic peptides were aligned by overlapping sequences of tryptic peptides and by homology with another tymovirus, eggplant mosaic virus. The belladonna mottle virus is more closely related to eggplant mosaic virus than to turnip yellow mosaic virus, the type member of this group, as evident from the sequence homologies of 57 and 32%, respectively. The accumulation of basic residues at the amino terminus implicated in RNA-protein interactions in many spherical plant viruses was absent in all the three sequences. Interestingly, the amino-terminal region is the least conserved among the tymoviruses. The longest stretch of conserved sequence between belladonna mottle virus and eggplant mosaic virus was residues 34-44, whereas it was residues 96-102 in the case of belladonna mottle virus and turnip yellow mosaic virus. A tetrapeptide in the region (residues 154-157) was found to be common for all the three sequences. It is possible that these conserved regions (residues 34-44, 96-102, 154-157) are involved in either intersubunit or RNA-protein interactions.     

Insertion analysis of putative functional elements in the promoter region of the Aspergillus oryzae Taka-amylase A gene (amyB) using a heterologous Aspergillus nidulans amdS-lacZ fusion gene system

Expression of the Taka-amylase A gene (amyB) of Aspergillus oryzae is induced by starch or maltose. The A. oryzae amyB gene promoter contains three highly conserved sequences, designated Regions I, II, and III, compared with promoter regions of the A. oryzae glaA encoding glucoamylase and the agdA encoding alpha-glucosidase. To identify the function of these sequences within the amyB promoter, various fragments containing conserved sequences in the amyB promoter were introduced into the upstream region of the heterologous A. nidulans amdS gene (encoding acetamidase) fused to the Escherichia coli lacZ gene as a reporter. Introduction of the sequence between -290 to -233 (the number indicates the distance in base pairs from the translation initiation point (+1)) containing Region III significantly increased the expression of the lacZ reporter gene in the presence of maltose. The sequence between -377 to -290 containing Region I also increased the lacZ activity, but its maltose inducibility was less than that of Region III. The sequence between -233 to -181 containing Region II had no effect on the expression. These results indicated that Region III is most likely involved in the maltose induction of the amyB gene expression.     

COOH-terminal propeptides of the major human procollagens. Structural, functional and genetic comparisons

The sequences of the carboxy-terminal extensions (COOH-propeptides) of at least one chain of all of the major human procollagens have only recently been deduced, and include those of the interstitial (alpha 1(I), alpha 2(I), alpha 1(II), alpha 1(III)), basement membrane (alpha 1(IV)) and pericellular (alpha 2(V)) procollagens. Comparisons of DNA and protein sequences, corresponding to these COOH-propeptides domains, established the early divergence of the basement membrane alpha 1(IV) COOH-propeptide from the corresponding sequences of the interstitial and pericellular procollagens. The latter are relatively highly conserved and share 58% primary peptide sequence similarities, whereas sequence similarities relative to alpha 1(IV) are limited. Hydropathy profiles and secondary structure potentials further emphasize the clustering of conserved and variable regions among the interstitial and pericellular COOH-propeptides, and provided further evidence for significant structural differences between these sequences and the alpha 1(IV) COOH-propeptide. The most highly conserved sequences of the alpha 1(I), alpha 2(I), alpha 1(II), alpha 1(III) and alpha 2(V) COOH-propeptides include regions surrounding the carbohydrate attachment site, cysteine-containing regions and the COOH-terminal sequences. Cysteinyl, tyrosyl and tryptophanyl residues were found to be highly conserved as were most charged residues. Localization of variable regions, in general, occurs within hydrophilic sequences with high beta-turn potentials that are proximal to intron/exon splice junctions. The most variable sequences are associated with the telopeptides and adjoining NH2-terminal portions of the COOH-propeptides as demonstrated by predictive secondary structure analyses. These results, combined with similar analyses of abnormal alpha 2(I) COOH-propeptide (osteogenesis imperfecta) permitted the identification of subsequences that are likely to be a prerequisite for COOH-propeptide functions, namely procollagen chain recognition and nucleation sites for triple helix formation. These functions are also common to the alpha 1(IV) COOH-propeptide; however, the lack of cleavage of this region and its additional postulated structural role in extracellular matrix interactions likely account for its divergent primary and secondary structure.     

Essential histidine and tryptophan residues in CcsA,a system II polytopic cytochrome c biogenesis protein

Three distinct systems (I, II, and III) for catalysis of heme attachment to c-type apocytochromes are known. The CcsA and Ccs1 proteins are required in system II for the assembly of bacterial and plastid cytochromes c. A tryptophan-rich signature motif (WWD), also occurring in CcmC and CcmF found in system I, and three histidinyl residues, all strictly conserved in CcsA suggest a function in heme handling. Topological analysis of plastid CcsA in bacteria using the PhoA and LacZalpha reporters placed the WWD motif, the conserved residues His(212) and His(347) on the lumen side of the membrane, whereas His(309) was assigned a location on the stromal side. Functional analysis of CcsA through site-directed mutagenesis enabled the designation of the initiation codon of the ccsA gene and established the functional importance of the WWD signature motif and the absolute requirement of all three histidines for the assembly of plastid c-type cytochromes. In a ccsA mutant, a 200-kDa Ccs1-containing complex is absent from solubilized thylakoid membranes, suggesting that CcsA operates together with Ccs1. We propose a model where the WWD motif and histidine residues function in relaying heme from stroma to lumen and we postulate the existence of a cytochrome c assembly machinery containing CcsA, Ccs1 and additional components.     

Site-directed mutagenesis of the conserved residues in component I of Bacillus subtilis heptaprenyl diphosphate synthase.

Heptaprenyl diphosphate synthase of Bacillus subtilis is composed of two dissociable heteromeric subunits, component I and component II. Component II has highly conserved regions typical of (E)-prenyl diphosphate synthases, but it shows no prenyltransferase activity alone unless it is combined with component I. Alignment of amino acid sequences for component I and the corresponding subunits of Bacillus stearothermophilus heptaprenyl diphosphate synthase and Micrococcus luteus B-P 26 hexaprenyl diphosphate synthase shows three regions of high similarity. To elucidate the role of these regions of component I during catalysis, 13 of the conserved amino acid residues in these regions were selected for substitution by site-directed mutagenesis. Kinetic studies indicated that substitutions of Val-93 with Gly, Leu-94 with Ser, and Tyr-104 with Ser resulted in 3-10-fold increases of K(m) values for the allylic substrate and 5-15-fold decreases of V(max) values compared to those of the wild-type enzyme. The three mutated enzymes, V93G, L94S, and Y104S, showed little binding affinity to the allylic substrate in the membrane filter assay. Furthermore, product analyses showed that D97A yielded shorter chain prenyl diphosphates as the main product, while Y103S gave the final product with a C(40) prenyl chain length. These results suggest that some of the conserved residues in region B of component I are involved in the binding of allylic substrate as well as determining the chain length of the enzymatic reaction product.     

Identification of C3b-binding regions on herpes simplex virus type 2 glycoprotein C.

Glycoprotein C from herpes simplex viruses types 1 and 2 (gC-1 and gC-2) acts as a receptor for the C3b fragment of the third component of complement. Our goal is to identify domains on gC involved in C3b receptor activity. Here, we used in-frame linker-insertion mutagenesis of the cloned gene for gC-2 to identify regions of the protein involved in C3b binding. We constructed 41 mutants of gC-2, each having a single, double, or triple insertion of four amino acids at sites spread across the protein. A transient transfection assay was used to characterize the expressed mutant proteins. All of the proteins were expressed on the transfected cell surface, exhibited processing of N-linked oligosaccharides, and bound one or more monoclonal antibodies recognizing distinct antigenic sites on native gC-2. This suggested that each of the mutant proteins was folded into a native structure and that a loss of C3b binding by any of the mutants could be attributed to the disruption of a specific functional domain. When the panel of insertion mutants was assayed for C3b receptor activity, we identified three distinct regions that are important for C3b binding, since an insertion within those regions abolished C3b receptor activity. Region I was located between amino acids 102 and 107, region II was located between residues 222 and 279, and region III was located between residues 307 and 379. In addition, region III has some structural features similar to a conserved motif found in complement receptor 1, the human C3b receptor. Finally, blocking experiments indicated that gC-1 and gC-2 bind to similar locations on the C3b molecule.     

Integron integrases possess a unique additional domain necessary for activity.

Integrons are genetic elements capable of integrating genes by a site-specific recombination system catalyzed by an integrase. Integron integrases are members of the tyrosine recombinase family and possess the four invariant residues (RHRY) and conserved motifs (boxes I and II and patches I, II, and III). An alignment of integron integrases compared to other tyrosine recombinases shows an additional group of residues around the patch III motif. We have analyzed the DNA binding and recombination properties of class I integron integrase (IntI1) variants carrying mutations at residues that are well conserved among all tyrosine recombinases and at some residues from the additional motif that are conserved among the integron integrases. The well-conserved residues studied were H277 from the conserved tetrad RHRY (about 90% conserved), E121 found in the patch I motif (about 80% conserved in prokaryotic recombinases), K171 from the patch II motif (near 100% conserved), W229 and F233 from the patch III motif, and G302 of box II (about 80% conserved in prokaryotic recombinases). Additional IntI1 mutated residues were K219 and a deletion of the sequence ALER215. We observed that E121, K171, and G302 play a role in the recombination activity but can be mutated without disturbing binding to DNA. W229, F233, and the conserved histidine (H277) may be implicated in protein folding or DNA binding. Some of the extra residues of IntI1 seem to play a role in DNA binding (K219) while others are implicated in the recombination activity (ALER215 deletion).