Interactions between the tetratricopeptide repeat-containing transcription factor TFIIIC131 and its ligand, TFIIIB70. Evidence for a conformational change in the complex.

In the transcription of tRNA and 5 S genes by RNA polymerase III, recruitment of the transcription factor (TF)IIIB is mediated by the promoter-bound assembly factor TFIIIC. A critical limiting step in this process is the interaction between the tetratricopeptide repeat (TPR)-containing subunit of TFIIIC (TFIIIC131) and the TFIIB-related factor Brf1p/TFIIIB70. To facilitate biochemical studies of this interaction, we expressed a fragment of TFIIIC131, TFIIIC131-(1-580), that includes the minimal TFIIIB70 interaction domain defined by two-hybrid studies together with adjacent sequences, up to the end of TPR9, implicated in the assembly reaction. TFIIIC131-(1-580) interacts with TFIIIB70 in solution and inhibits the formation of TFIIIB70.TFIIIC.DNA complexes. In a coupled equilibrium binding assay, the formation of TFIIIC131-(1-580).TFIIIB70 complexes was adequately described by a single-site binding model and yielded an apparent equilibrium dissociation constant of 334 +/- 23 nm. CD spectroscopy and limited proteolysis experiments defined a well structured and largely protease-resistant core in TFIIIC131-(1-580) comprising part of the hydrophilic amino terminus, TPR1-5, the intervening non-TPR region, and TPR6-8. CD spectra showed that trifluoroethanol induced significant alpha-helical structure in TFIIIC131-(1-580). A more modest monovalent ion-dependent CD difference was observed in mixtures of TFIIIC131-(1-580) and TFIIIB70, suggesting that formation of the binary complex may proceed with the acquisition of alpha-helicity.

A family study of multiple mutations of alpha and delta glycophorins (glycophorins A and B).

Glycophorins alpha and delta are the carriers of the antigens of the MNSs blood system; this report documents the presence of three glycophorin mutations in two individuals of a 16 member family. Erythrocytes were examined by serology, sodium dodecyl sulfate electrophoresis, and immunoblotting. The inheritance pattern and immunoblot profile revealed: (1) A variant Dantu glycophorin showed properties consistent with a delta-alpha glycophorin hybrid structure, previously noted in other individuals. The gene responsible for the Dantu glycophorin in this family is linked to a gene coding for an M-specific alpha glycophorin. (2) Another variant glycophorin, Mi-III glycophorin, was first revealed by immunoblotting and subsequently confirmed by erythrocyte antigen typing. This autosomal dominant trait is associated with N blood group activity and the inheritance pattern indicates that it could be a variant of delta glycophorin. (3) In the individuals with both Dantu and Mi-III glycophorins a delta glycophorin deficiency was observed suggesting that a deletion or alteration of delta gene may exist cis to the Dantu gene. Our findings that document clustering of multiple mutations in MNSs gene loci in the propositus family are very unusual as such variants are relatively rare.

The chimpanzee M blood-group antigen is a variant of the human M-N glycoproteins.

Chimpanzee erythrocytes express strong M but weak, occasional N blood-group activity, as detected by anti-M and anti-N reagents. We have found that the M activity is carried by a major membrane glycoprotein that is similar but not identical to the human MM glycoprotein (glycophorin A). We have isolated and characterized this glycoprotein from erythrocyte membranes of four individual chimpanzees. The purified glycoproteins strongly inhibited agglutination of M cells by rabbit anti-human M sera and only weakly inhibited the agglutination of N cells by rabbit anti-human N sera. They also displayed medium-to-strong inhibitory activity against chimpanzee iso- and crossimmune antisera tested with chimpanzee erythrocytes of various V-A-B-D and Wc specificities, which are known as chimpanzee extensions of the human type M-N system and the Miltenberger counterpart, respectively. Each glycoprotein was cleaved with CNBr into three fragments, whose size, solubility, and composition were analogous to those obtained by similar treatment of the human M-N antigens. The amino-terminal fragment was found to be a glycooctapeptide whose amino acid composition and partial sequence indicated that it is an intermediate form of the human M and N glycooctapeptides. Its carbohydrate content comprised two threonine-linked saccharide units that, although similar in composition to the human threonine-linked units, were fewer in number than the three units found in the corresponding human glycooctapeptides. Structural similarities to the human antigens strongly suggest that the amino terminus bears the major antigenic determinants of the molecule, and the occurrence in this region of numerous, albeit rare, variants among humans and in chimpanzees indicates that the corresponding coding sequence of the structural gene is particularly susceptible to mutational events. We conclude that the chimpanzee M gene product is a variant of the human type and that the chimpanzee gene is an allele of the human polymorphic M-N locus.

Amino acid and carbohydrate structural variants of glycoprotein products (M-N glycoproteins) of the M-N allelic locus.

Major glycoprotein of MgM, MM Miltenberger III (MiIII), and M-N erythrocyte membranes from individual donors were cleaved with CNBr and their amino-terminal octapeptides were examined with respect to amino acid and carbohydrate composition. The amino-terminal octapeptides from the heterozygous MgM donor were resolved into two types, A and A'. MgM A was identical to octapeptide A from MM glycoproteins in carbohydrate and amino acid compositions. MgM A' exhibited amino acid composition similar to NN peptide A except for a single substitution of an Asx for a Thr and, as a result, was not glycosylated. MM(MiIII) octapeptide A was identical to M peptide A in amino acid composition, but differed in carbohydrate content. This glycopeptide contained three O-glycosidically linked carbohydrate units, one of which contained GlcNAc bound to a core of NeuAc, Gal, and GalNAc. About two such units were also present in the CNBr glycopeptide B of the glycoprotein, and on the basis of studies with alkaline borohydride and alkaline sulfite degradations, these units are believed to have the following structure: (formula see text) The Mg is an allelomorph of the M-N locus, likely evolved from a single base substitution in the N gene. The resulting single amino acid substitution effects the posttranslational carbohydration of neighboring Ser and Thr residues. The MM(MiIII) appears to be a product of the M gene that undergoes sequences of posttranslational glycosylations different from those of the M-N glycoproteins.

Preparation of cyanogen bromide fragments of MM, NN, and MN glycoproteins (glycophorins) from human erythrocyte membranes of single donors.

A procedure is described for the preparation of three cyanogen bromide fragments of the MM, NN, or MN glycoprotein (glycophorin) of the human erythrocyte membranes, from erythrocytes of single donors. The fragments are obtained in pure form and excellent yields by employing procedures which include proteolytic inhibitors during membrane processing, thorough delipidation of the glycoprotein, and CNBr cleavage conditions which lead to quantitative fragmentation without loss of carbohydrates. A phenol-urea extraction resolves the two glycopeptide fragments from the carbohydrate-free fragment. The two glycopeptides are further purified by Bio-Gel P-6 and P-100 chromatography. The three fragments include the amino terminal 8 residue glycopeptide, a large glycopeptide form the middle of the molecule which bears the Asn-linked oligosaccharide and 8--9 O-glycosidically linked units, and a carboxyl terminal, carbohydrate-free, approx. 50 residue fragment. Their amino acid and carbohydrate composition, and size, are in close agreement with the sequence data of Tomita, M., Furthmayr, H. and Marchesi, V.T. (Biochemistry (1978), 17, 4756--4770). The fragments represent three well delineated portions of the glycoprotein molecule.