Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “structural variants”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,567 records · Page 87Linked to original sources
PubMed1998

Differential regulation of human T cell cytokine patterns and IgE and IgG4 responses by conformational antigen variants.

Bee venom phospholipase A2 (PLA) represents the major allergen and antigen in allergic and non-allergic individuals sensitized to bee sting. We have studied specific activation of peripheral T cells by different structural and conformational variants of PLA and secretion of cytokines regulating IgE and IgG4 antibody (Ab) formation. PLA molecules expressing the correctly folded tertiary structure, which show high affinity to membrane phospholipids and were recognized by Ab from bee sting allergic patients, induced high IL-4, IL-5 and IL-13 production in peripheral blood mononuclear cell cultures. In contrast, non-refolded recombinant PLA (rPLA) and reduced and alkylated native PLA (nPLA) induced more IFN-gamma and IL-2 and higher proliferative responses. Differences in proliferation and cytokine patterns among correctly folded and non-refolded PLA resulted from conformation-dependent involvement of different antigen-presenting cell (APC) types. Antigen (Ag)-presenting B cells recognized PLA only in its natural conformation, stimulated Th2 type cytokines and induced IgE Ab. Non-refolded PLA was recognized, processed and presented exclusively by monocytes and induced a Th1 dominant cytokine profile leading to IgG4 production by B cells. The possibility that production of particular cytokine patterns and Ig isotype was influenced by the enzymatic activity of PLA was excluded by using enzymatically inactive H34Q point-mutated, refolded rPLA. These findings demonstrate the decisive role of specific Ag recognition by different APC, depending on structural features, membrane phospholipid binding property and the existence of conformational B cell epitopes, in the differential regulation of memory IgE and IgG4 Ab. Furthermore, they show that a change from IgE-mediated allergy to normal immunity against a major allergen can be induced by rPLA variants that are not recognized by specific Ab and B cells but still carry the T cell epitopes. These features may enable new applications for safer immunotherapy.

Adult↗
PubMed1994

Immunologic and molecular characterization of Amb p V allergens from Ambrosia psilostachya (western Ragweed) pollen.

We have purified and characterized the Amb p V allergen (A1 variant) from western ragweed (Ambrosia psilostachya) pollen. This allergen was found to be highly cross-reactive with the Amb a VA1 allergen from short ragweed (A. artemisiifolia) pollen in a competitive double-Ab radioimmunoassay (DARIA) and the two allergens showed concordant allergenic potency in histamine-release experiments. We cloned and sequenced several Amb p V genes from western ragweed pollen and flowers by direct PCR of genomic DNA. The amino acid sequences deduced from the nucleotide sequences indicated the presence of multiple forms of Amb p V that could be broadly classified into two groups: Amb p VA and Amb p VB variants. The sequences of the Amb p VA variants are highly homologous to Amb a V (about 90% identity) and very similar to the protein sequence that we obtained. The Amb p VB variants share approximately 65% amino acid homology with Amb a V and have five to seven cysteine residues as compared with the eight found in Amb a V and Amb t V. Two cysteine residues that form disulfide bonds in other Amb Vs (positions 19 and 43 in Amb a V) are replaced by serine and alanine in the Amb p VB1 and Amb p VB2 variants. We have generated model structures of Amb p VA1, VA2, VA3, and VB1 variants from the nuclear magnetic resonance-derived structure of Amb a VA1 by homology modeling. Comparison of antigenic epitopes predicted for the structures of Amb p V variants and Amb a VA1 explains the observed cross-reactivity of the two ragweed proteins and suggests the epitopes likely to be involved in Ab recognition.

Allergens↗
PubMed1999

Anomalous cadherin expression in osteosarcoma. Possible relationships to metastasis and morphogenesis.

Two isoforms of the human cadherin-11/OB-cadherin gene, the intact and the variant forms, had been isolated from an osteosarcoma cDNA library. The intact form has a typical cadherin structure, whereas the variant form, generated by alternative splicing, encodes a cytoplasmic domain that is completely different from that of the intact form and lacks a homophilic cell-cell adhesion ability. At the protein level, the secreted form generated from the intact cadherin-11 is present. We examined the expression of the intact and the variant forms of cadherin-11 in 23 primary and metastatic osteosarcomas from 22 patients by reverse transcriptase-polymerase chain reaction (RT-PCR) analyses, revealing that all 23 tumors in the patients expressed the variant form and three of them expressed it prominently. On the other hand, Western blot analyses of six tumors showed that the secreted form was strongly expressed, and furthermore, expression of N-cadherin was extremely low. Overexpression of the intact cadherin-11 cDNA in osteosarcoma cell lines demonstrated that the secreted form is derived from the intact form of cadherin-11 in osteosarcoma. Immunohistochemically, cadherin-11, N-cadherin, and beta-catenin were expressed at the cell surface of fetal osteoblasts, whereas in osteosarcoma cells, they were expressed only focally or weakly in the cytoplasm. Considering the function of cadherin in carcinomas, it is suggested that the anomalous expression of human cadherin-11 in osteosarcoma and the reduced expression of N-cadherin play a role in metastasis and the irregular morphology in the highly malignant mesenchymal tumor.

Bone Development↗
PubMed1981

BS I-B4 isolectin as a probe for an investigation of membrane alterations and transformation phenotypes of mouse L cells.

BS I-B4, an alpha-D-galactopyranosyl-binding isolectin from Bandeiraea simplicifolia seeds, was found to interact differently with transformed mouse L cells and non-transformed mouse 3T3 cells. The lectin induces detachment of 3T3 cells but increases adhesiveness and clustering of L cells. However, the induced cell aggregation does not lead to cell fusion. A variant clone of L cells, resistant to BS I-B4, which had lost the capacity for agglutination in the presence of the lectin, was isolated. Fluorescence binding studies of this variant suggest a lesion involving alpha and beta-D-galactopyranosyl units on its cell-surface structures. Although the variant cells form colonies in a methylcellulose medium, they do not produce tumours, as do the parental cells, when transplanted in athymic nude mice. The results demonstrate that alterations in cell membrane glyco-conjugates play an important role in tumourigenesis of animal cells, but anchorage-independent growth in vitro, as one of the transformation phenotypes, cannot be correlated absolutely with tumourigenicity in vivo.

Animals↗
PubMed2025

Integrating AlphaFold2 models and clinical data to improve the assessment of Short Linear Motifs (SLiMs) and their variants' pathogenicity.

Short Linear Motifs (SLiMs) are protein functionally relevant regions that mediate reversible protein-protein interactions. Variants that disrupt SLiMs can lead to numerous Mendelian diseases. Although various bioinformatic tools have been developed to identify SLiMs, most suffer from low specificity. In our previous work, we demonstrated that integrating sequence variant information with structural analysis can enhance the prediction of true functional SLiMs while simultaneously generating tolerance matrices that indicate whether each of the 19 possible single amino acid substitutions (SASs) is tolerated. However, the scarcity of representative crystallographic structures of SLiM-receptor complexes posed a significant limitation. In this study, we demonstrate that these interactions can be modeled using AlphaFold2 (AF2) to generate high-quality structures that serve as input for our MotSASi method. These AF2-derived structures show robust performance, both in reproducing known structures deposited in the Protein Data Bank (PDB) and in reflecting the deleterious effects of known sequence variants. This updated version of MotSASi expands the repertoire of high-confidence predicted SLiMs and provides a comprehensive catalog of variants located within SLiMs, along with their respective deleteriousness assessments. When compared to AlphaMissense, MotSASi demonstrates superior performance in predicting variant deleteriousness. By contributing to the accurate identification and interpretation of variants, this work aligns with ACMG/AMP standards and aims to improve diagnostic rates in clinical genomics.

Humans↗
PubMed2006

Structural characterization of human 8-oxoguanine DNA glycosylase variants bearing active site mutations.

The human 8-oxoguanine DNA glycosylase (hOGG1) protein is responsible for initiating base excision DNA repair of the endogenous mutagen 8-oxoguanine. Like nearly all DNA glycosylases, hOGG1 extrudes its substrate from the DNA helix and inserts it into an extrahelical enzyme active site pocket lined with residues that participate in lesion recognition and catalysis. Structural analysis has been performed on mutant versions of hOGG1 having changes in catalytic residues but not on variants having altered 7,8-dihydro-8-oxoguanine (oxoG) contact residues. Here we report high resolution structural analysis of such recognition variants. We found that Ala substitution at residues that contact the phosphate 5' to the lesion (H270A mutation) and its Watson-Crick face (Q315A mutation) simply removed key functionality from the contact interface but otherwise had no effect on structure. Ala substitution at the only residue making an oxoG-specific contact (G42A mutation) introduced torsional stress into the DNA contact surface of hOGG1, but this was overcome by local interactions within the folded protein, indicating that this oxoG recognition motif is "hardwired." Introduction of a side chain intended to sterically obstruct the active site pocket (Q315F mutation) led to two different structures, one of which (Q315F(*149)) has the oxoG lesion in an exosite flanking the active site and the other of which (Q315F(*292)) has the oxoG inserted nearly completely into the lesion recognition pocket. The latter structure offers a view of the latest stage in the base extrusion pathway yet observed, and its lack of catalytic activity demonstrates that the transition state for displacement of the lesion base is geometrically demanding.

Alanine↗
PubMed1998

Analysis of human serum albumin variants by mass spectrometric procedures.

A new strategy for the structural characterisation of human albumin variants has been developed which makes extensive use of mass spectrometric methodologies. The rationale behind the method is to provide a rapid and effective screening of the entire albumin structure. The first step in this strategy consists in the attempt to determine the accurate molecular mass of the intact variant by electrospray mass spectrometry often providing a first indication on the presence of the variant. An HPLC procedure has been developed io isolate all the seven fragments generated by CNBr hydrolysis of HSA in a single chromatographic step. A rapid screening of the entire albumin structure is achieved by the ESMS analysis of the peptide fragments and the protein region(s) carrying the structural abnormality is identified by its anomalous mass value(s). Mass mapping of the corresponding CNBr peptide, either by Fast Atom Bombardment Mass Spectrometry (FABMS) or by Matrix Assisted Laser Desorption Ionisation Mass Spectrometry (MALDIMS), leads to the definition of the site and the nature of the variation. This combined strategy was applied to the structural characterisation of three HSA genetic variants and provided to be an effective procedure for the rapid assessment of their structural modifications showing considerable advantages over the classical approach.

Humans↗
PubMed1995

Primary structure of the long and short splice variants of mouse collagen XII and their tissue-specific expression during embryonic development.

Type XII collagen, a member of the FACIT group of extracellular matrix proteins, consists of molecules that are trimers of alpha 1(XII) chains. The three chains in each molecule form a cross-shaped structure with a central globule from which a triple-helical tail and three finger-like regions (containing von Willebrand factor A-like regions (containing von Willebrand factor A-like domains and fibronectin type III repeats) extend. cDNA cloning/sequencing of chicken alpha 1(XII) collagen and protein studies with mouse, bovine, and human material suggest that the alpha 1(XII) collagen gene gives rise to two molecular variants, differing in the length of the finger-like regions, by alternative splicing of the primary transcript. To provide a basis for studies of the function of the two variants in an organism that can be genetically manipulated, we have isolated and sequenced mouse cDNAs encoding both splice variants. The sequence provides the first complete nucleotide and amino acid sequence of mammalian type XII collagen. From these cDNAs we have generated digoxigenin-labeled RNA probes for in situ hybridization of developing mouse embryos to find out whether the splicing mechanism responsible for generation of the two forms is developmentally regulated. The results, combined with Northern blot and RT-PCR analysis of RNA from embryos at various developmental stages, demonstrate that the long form of collagen XII, XIIA, is the predominant form at early stages (ED7 and 11); at later stages of development (ED15 and 17) the short form, XIIB, becomes the major form. As the short form becomes the major product, the long splice variant continues to be expressed in several tissues, even after birth. An exception is dermis, which is positive for the long form up to embryonic day 15, but negative at day 18, when only the short form RNA can be detected.

Alternative Splicing↗
PubMed1988

The same substitution, glutamic acid----lysine at position 501, occurs in three alloalbumins of Asiatic origin: albumins Vancouver, Birmingham, and Adana.

A strategy is described for identifying structural changes in genetic variants of human serum albumin (alloalbumins). By use of this strategy we have determined an amino acid substitution in three alloalbumins of Asiatic origin. The same amino acid exchange, glutamic acid----lysine at position 501, occurs in albumins Vancouver and Birmingham, both from families that migrated from northern India, and also in albumin Adana from Turkey. This exchange corresponds to a single base mutation in the codon GAG to AAG and accords with the slow mobility of the three albumins at pH 8.6. Each of the three alloalbumins had been reported to be a new variant, yet they have the same substitution. These results emphasize the need for structural study of genetic variants that have been differentiated only by nonspecific physical criteria such as dye binding and electrophoretic mobility. We know of no other description of the substitution involved in an alloalbumin originating from the Indian subcontinent. However, the same change of glutamic acid----lysine at position 501 may be present in several other named variants reported for populations in north India and the surrounding regions.

Amino Acid Sequence↗
PubMed2004

Equine catechol estrogen 4-hydroxyequilenin is a more potent inhibitor of the variant form of catechol-O-methyltransferase.

Catechol-O-methyltransferase (COMT) plays an important role in the inactivation of biologically active and toxic catechols. It has been shown that COMT is genetically polymorphic with a wild-type and variant form where a valine has been substituted with a methionine. Several, but not all, epidemiological studies have shown that women, homozygous with the variant form, have an increased risk of developing breast cancer. Previously, we showed that 4-hydroxyequilenin (4-OHEN), a cytotoxic/genotoxic equine catechol estrogen metabolite, is both a substrate of COMT and an irreversible inhibitor of the methylation activity of COMT in vitro. To further understand the mechanism(s) of the association between the breast cancer risk and the COMT polymorphism, it was of interest to study the effect of the Val/Met polymorphism on COMT-catalyzed catechol estrogen methylation and 4-OHEN-mediated inhibition. In the present study, Michaelis-Menten analysis showed no difference between the relative ability of each form to methylate 4-OHEN. However, we found that the COMT variant form was more susceptible to 4-OHEN-mediated irreversible inactivation. Electrospray ionization mass spectrometry and SDS-gel analysis of COMT modified by 4-OHEN revealed that inhibition mechanisms include alkylation and/or oxidation of certain amino acids. In addition, site-directed mutagenesis experiments showed that Cys33 played a more important role in the variant form of COMT demonstrated by the fact that the C33A mutant of the variant form of COMT decreased its catalytic capability more dramatically as compared with that of wild type. Furthermore, thermotropic studies indicated that the variant form was more thermolabile, which suggested that the valine to methionine substitution may have changed the secondary/tertiary structure of the variant form of COMT, making it more susceptible to 4-OHEN and heat inactivation. These data suggest that 4-OHEN-mediated inhibition of the variant form of COMT in vivo might affect the detoxification efficiency of endogenous and/or exogenous catechol estrogens and play a role in the association between breast cancer risk and COMT polymorphism.

Breast Neoplasms↗
PubMed2003

Medical students' application of published evidence: randomised trial.

OBJECTIVES: To determine how medical students apply research evidence that varies in validity of methods and importance of results to a clinical decision. DESIGN: Students examined a standardised patient with a whiplash injury, decided whether to order a cervical spine radiograph, and rated their confidence in their decision. They then read one of four randomly assigned variants of a structured abstract from a study of a decision rule that argued against such a procedure in this patient. Variants factorially combined two levels of validity of methods (prospective cohort or chart review) with two levels of importance of results (high sensitivity or high specificity rule). After reading the abstract, students repeated their choice and rated their confidence. SETTING: Academic medical centre in the United States. PARTICIPANTS: 164 graduating medical students. MAIN OUTCOME MEASURES: Proportion of students in each group whose beliefs shifted or stayed the same. RESULTS: When abstracts were of low importance students were more likely to shift their beliefs in favour of radiography, which was not supported by the evidence (odds ratio 3.42, 95% confidence interval 1.10 to 10.66). Neither methodological validity nor the interaction between validity and importance influenced decision shift. Few students acquired all necessary clinical data from the patient. CONCLUSIONS: Although the students could apply concepts of diagnostic testing, greater focus is needed on appraisal of validity and application of evidence to a particular patient.

Clinical Competence↗
PubMed1999

A Watson-Crick base-pair-disrupting methyl group (m1A9) is sufficient for cloverleaf folding of human mitochondrial tRNALys.

We have previously shown by chemical and enzymatic structure probing that, opposite to the native human mitochondrial tRNA(Lys), the corresponding in vitro transcript does not fold into the expected tRNA-specific cloverleaf structure. This RNA folds into a bulged hairpin, including an extended amino acid acceptor stem, an extra large loop instead of the T-stem and loop, and an anticodon-like domain. Hence, one or several of the six modified nucleotides present in the native tRNA are required and responsible for its cloverleaf structure. Phylogenetic comparisons as well as structural analysis of variant transcripts had pointed to m(1)A9 as the most likely important modified nucleotide in the folding process. Here we describe the synthesis of a chimeric tRNA(Lys) with m(1)A9 as the sole modified base and its structural analysis by chemical and enzymatic probing. Comparison of this structure to that of the unmodified RNA, the fully modified native tRNA, and a variant designed to mimic the effect of m(1)A9 demonstrates that the chimeric RNA folds indeed into a cloverleaf structure that resembles that of the native tRNA. Thus, due to Watson-Crick base-pair disruption, a single methyl group is sufficient to induce the cloverleaf folding of this unusual tRNA. This is the first direct evidence of the role of a modified nucleotide in RNA folding.

Adenine↗
PubMed1997

Scanning electron microscopy of the development of structured aerial mycelia and satellite colonies of phenotypically switched Candida albicans.

Candida albicans is an asexual diploid fungus that can express high frequency phenotypic switching. Switched variants can develop structured aerial mycelia (SAM) and cultures that have been grown for a protracted period exhibit satellite colonies. This study investigated the development of both SAM and satellite colonies by means of a freeze-drying technique and scanning electron microscopy. The results show that SAM may develop due to hyphae developing in the colony at the base of the structures. The development of satellite colonies is a result of cells that have grown deep into the agar re-emerging on to the agar/air surface and producing a new colony. Although both SAM and satellite colonies are often seen together on mature colonies, their cause and development are different.

Candida albicans↗
PubMed2000

Gene structure and promoter variation of expressed and nonexpressed variants of the KIR2DL5 gene.

Two variants of the novel KIR2DL5 gene (KIR2DL5.1 and.2) were identified in genomic DNA of a single donor. However, only the KIR2DL5.1 variant was transcribed in PBMC. In this study, analysis of seven additional donors reveals two new variants of the KIR2DL5 gene and indicates that transcription, or its lack, are consistently associated with particular variants of this gene. Comparison of the complete nucleotide sequences of the exons and introns of KIR2DL5.1 and KIR2DL5.2 reveals no structural abnormalities, but similar open reading frames for both variants. In contrast, the promoter region of KIR2DL5 shows a high degree of sequence polymorphism that is likely relevant for expression. Substitution within a putative binding site for the transcription factor acute myeloid leukemia gene 1 could determine the lack of expression for some KIR2DL5 variants.

Base Sequence↗
PubMed2006

The G473D mutation impairs dimerization and catalysis in human sulfite oxidase.

Among the mutations identified in patients with isolated sulfite oxidase deficiency, the G473D variant is of particular interest since sedimentation analysis reveals that this variant is a monomer, and the importance of the wild-type dimeric state of mammalian sulfite oxidase is not yet well understood. Analysis of recombinant G473D sulfite oxidase indicated that it is severely impaired both in the ability to bind sulfite and in catalysis, with a second-order rate constant 5 orders of magnitude lower than that of the wild type. To elucidate the specific reasons for the severe effects seen in the G473D variant, several other variants were created, including G473A, G473W, and the double mutant R212A/G473D. Despite the inability to form a stable dimer, the G473W variant had 5-fold higher activity than G473D and nearly wild-type activity at pH 7.0 when ferricyanide was the electron acceptor. In contrast, the R212A/G473D variant demonstrated some ability to oligomerize but had undetectable activity. The G473A variant retained the ability to dimerize and had steady-state activity that was comparable to that of the wild type. Furthermore, stopped-flow analysis of the reductive half-reaction of this variant yielded a rate constant nearly 3 times higher than that of the wild type. Examination of the secondary structures of the variants by CD spectroscopy indicated significant random-coil formation in G473D, G473W, and R212A/G473D. These results demonstrate that both the charge and the large size of an Asp residue in this position contribute to the severe effects seen in a patient with the G473D mutation, by causing partial misfolding and monomerization of sulfite oxidase and attenuating both substrate binding and catalytic efficiency during the reaction cycle.

Amino Acid Sequence↗
PubMed2005

Identification of variant molecules of Bacillus thermoproteolyticus ferredoxin: crystal structure reveals bound coenzyme A and an unexpected [3Fe-4S] cluster associated with a canonical [4Fe-4S] ligand motif.

During the purification of recombinant Bacillus thermoproteolyticus ferredoxin (BtFd) from Escherichia coli, we have noted that some Fe-S proteins were produced in relatively small amounts compared to the originally identified BtFd carrying a [4Fe-4S] cluster. These variants could be purified into three Fe-S protein components (designated as V-I, V-II, and V-III) by standard chromatography procedures. UV-vis and EPR spectroscopic analyses indicated that each of these variants accommodates a [3Fe-4S] cluster. From mass spectrometric and protein sequence analyses together with native and SDS gel electrophoresis, we established that V-I and V-II contain the polypeptide of BtFd associated with acyl carrier protein (ACP) and with coenzyme A (CoA), respectively, and that V-III is a BtFd dimer linked by a disulfide bond. The crystal structure of the BtFd-CoA complex (V-II) determined at 1.6 A resolution revealed that each of the four complexes in the crystallographic asymmetric unit possesses a [3Fe-4S] cluster that is coordinated by Cys(11), Cys(17), and Cys(61). The polypeptide chain of each complex is superimposable onto that of the original [4Fe-4S] BtFd except for the segment containing Cys(14), the fourth ligand to the [4Fe-4S] cluster of BtFd. In the variant molecules, the side chain of Cys(14) is rotated away to the molecular surface, forming a disulfide bond with the terminal sulfhydryl group of CoA. This covalent modification may have occurred in vivo, thereby preventing the assembly of the [4Fe-4S] cluster as observed previously for Desulfovibrio gigas ferredoxin. Possibilities concerning how the variant molecules are formed in the cell are discussed.

Bacillus↗
PubMed1990

2.9 A resolution structure of the N-terminal domain of a variant surface glycoprotein from Trypanosoma brucei.

The variant surface glycoprotein (VSG) of Trypanosoma brucei forms a coat on the surface of the parasite; by the expression of a series of antigenically distinct VSGs in the surface coat the parasite escapes the host immune response. The 2.9 A resolution crystal structure of the N-terminal domain of one variant, MITat 1.2, has been determined. The structure was solved using data collected from two crystal forms. Initially a partial model was built into an electron density map based on multiple isomorphous replacement phases and improved by phase combination methods. Subsequently this model was used to obtain the molecular replacement solution for a second crystal form, providing starting phases which were refined using 2-fold non-crystallographic symmetry averaging. The current model includes 362 residues and has been refined using X-PLOR to an R value of 0.22 for data between 7 and 2.9 A. The molecule is a dimer, approximately 100 A long, having an asymmetrical cross section with maximum dimensions of approximately 40 A x 60 A. Two long, approximately 70 A, alpha-helices from each monomer pack together to form, with several other helices, a core helix bundle that extends nearly the full length of the molecule. The "top" of the protein, which in the surface coat may be exposed to the external environment, is formed from the ends of the two long helices, a short three-stranded beta-sheet, and a strand having irregular conformation that packs above these secondary structure elements. Two conserved disulfide bridges are in this part of the molecule. Several elements of the MITat 1.2 sequence, which contribute to the formation of the helix bundle structure, have been identified. These elements can be found in the sequences of several different VSGs, suggesting that to some extent the VSG structure is conserved in those variants.

Amino Acid Sequence↗
PubMed2004

The effect of mutations surrounding and within the active site on the catalytic activity of ricin A chain.

Models for the binding of the sarcin-ricin loop (SRL) of 28S ribosomal RNA to ricin A chain (RTA) suggest that several surface exposed arginine residues surrounding the active site cleft make important interactions with the RNA substrate. The data presented in this study suggest differing roles for these arginyl residues. Substitution of Arg48 or Arg213 with Ala lowered the activity of RTA 10-fold. Furthermore, substitution of Arg213 with Asp lowered the activity of RTA 100-fold. The crystal structure of this RTA variant showed it to have an unaltered tertiary structure, suggesting that the positively charged state of Arg213 is crucial for activity. Substitution of Arg258 with Ala had no effect on activity, although substitution with Asp lowered activity 10-fold. Substitution of Arg134 prevented expression of folded protein, suggesting a structural role for this residue. Several models have been proposed for the binding of the SRL to the active site of RTA in which the principal difference lies in the conformation of the second 'G' in the target GAGA motif in the 28S rRNA substrate. In one model, the sidechain of Asn122 is proposed to make interactions with this G, whereas another model proposes interactions with Asp75 and Asn78. Site-directed mutagenesis of these residues of RTA favours the first of these models, as substitution of Asn78 with Ser yielded an RTA variant whose activity was essentially wild-type, whereas substitution of Asn122 reduced activity 37.5-fold. Substitution of Asp75 failed to yield significant folded protein, suggesting a structural role for this residue.

Amino Acid Sequence↗