Vitamin K-dependent carboxylase: Synthesis of an inhibitor of the glutamyl binding site

Liver microsomes contain a vitamin K and O₂-dependent carboxylase that converts peptide-bound glutamyl residues to γ-carboxyglutamyl residues. The peptide Boc-O-phospho—Ser-O-phospho—Ser—Leu-OMe has now been synthesized. This peptide inhibits the carboxylation of endogenous protein precursors by a detergent-solubilized preparation of the carboxylase and is an apparent competitive inhibitor of the carboxylation of Phe—Leu—Glu—Glu—Leu.     

The effect of tRNA and tryptophanyl adenylate on limited proteolysis of beef pancreas tryptophanyl-tRNA synthetase.

Limited proteolysis of tryptophanyl-tRNA synthetase was used to detect changes in the enzyme molecule in the presence of substrates. Trypsinolysis of each of the two identical subunits occurs in succession from the N-terminus as follows: 60 leads to 51 leads to 40 leads to 24 kilodaltons. The transition 51 leads to 40 is hindered in tryptophanyl adenylate.enzyme complex. Yeast tRNATrp accelerates the first steps of hydrolysis and decelerates the transition 40 leads to 24. Once tRNATrp is added to the synthetase.adenylate complex, the protective effect of the adenylate disappears. The same effects are found also in the presence of tRNATrp oxidized with NaI04 and tRNATrp lacking the 3'-terminal adenosine. Oxidized tRNATrp (but not tRNATrp without the 3'-A) accelerates tryptophan-dependent hydrolysis of ATP catalyzed by the enzyme. A scheme is proposed for the interaction of yeast tRNATrp with beef pancreas tryptophanyl-tRNA synthetase involving the association of tRNA with a positively charged site(s) of the enzyme and the changes in the conformation of enzyme manifesting itself in unfolding of the acidic N-terminal fragment of the polypeptide chain and in the exposure of the adenylate.     

Directed cleavage of ribopolynucleotides with nucleases restricted by multiple modification of substrate.

Simultaneous exhaustive modification of cytidine and uridine residues of rRNA with methoxyamine and sodium metabisulfite renders adjacent phosphodiester bonds resistant to pancreatic and T2 ribonucleases. Another method of T2 RNAase restriction is modification of cytidine with methoxyaminebisulfite followed by modification of guanosine residues with beta-ethoxy-alpha-ketobutyraldehyde. Mild alkaline treatment leads to demodification of uridine and guanosine residues leaving intact modified cytidine residues, thus providing a means of stepwise, directed cleavage of the polynucleotide. The series of combined cleavage procedures and methods of isolation of oligo(C), oligo(G) and oligopyrimidine tracts, as well as the procedure of selective cleavage at uridine residues elaborated in the course of the present studies may serve as a basis for more rational procedures of RNA sequencing.     

Human nucleotide sequences related to the transforming gene of a murine sarcoma virus: studies with cloned viral and cellular DNAs

A recombinant plasmid, pI26, has been constructed by cloning into pBR322 a transforming gene of murine sarcoma virus (a Moloney strain, clone 124, MSV) synthesized by detergent-treated virions. From this plasmid a XbaI-HindIII fragment has been isolated which contains only mos-specific sequences. This mos-specific probe has been used for screening a human gene library cloned in bacteriophage λ Charon 4A. Of these, 19 clones have been isolated containing mos-related sequences. By physical mapping and molecular hybridization it has been shown that these sequences are neighboured by DNA regions related to Moloney murine leukemia virus. Recombinant phages have also been found containing human inserts related to MLV, not to the mos gene. The possible existence of murine-like endogenous retroviruses in the normal human genome, including that of a sarcoma type, is discussed. By Northern blotting, expression of the cellular c-mos gene has been detected in mouse liver treated with a hepatocarcinogen. The general significance of the suggested model for evaluating the relationship between chemical carcinogenesis and oncogene expression is discussed.     

Ap4A induces apoptosis in human cultured cells.

Diadenosine oligophosphates (Ap(n)A) have been proposed as intracellular and extracellular signaling molecules in animal cells. The ratio of diadenosine 5',5'-P1,P3-triphosphate to diadenosine 5',5'-P1,P4-tetraphosphate (Ap3A/Ap4A) is sensitive to the cellular status and alters when cultured cells undergo differentiation or are treated with interferons. In cells undergoing apoptosis induced by DNA topoisomerase II inhibitor VP16, the concentration of Ap3A decreases significantly while that of Ap4A increases. Here, we have examined the effects of exogenously added Ap3A and Ap4A on apoptosis and morphological differentiation. Penetration of Ap(n)A into cells was achieved by cold shock. Ap4A at 10 microM induced programmed cell death in human HL60, U937 and Jurkat cells and mouse VMRO cells and this effect appeared to require Ap4A breakdown as hydrolysis-resistant analogues of Ap4A were inactive. On its own, Ap3A induced neither apoptosis nor cell differentiation but did display strong synergism with the protein kinase C activators 12-deoxyphorbol-13-O-phenylacetate and 12-deoxyphorbol-13-O-phenylacetate-20-acetate in inducing differentiation of HL60 cells. We propose that Ap4A and Ap3A are physiological antagonists in determination of the cellular status: Ap4A induces apoptosis whereas Ap3A is a co-inductor of differentiation. In both cases, the mechanism of signal transduction remains unknown.     

Structural Organization of the Human Genome: Distribution of Nucleotides,AluRepeats, and Exons in Chromosomes 21 and 22

Analysis of DNA sequences of the human chromosomes 21 and 22 performed using a specially designed MegaGene software allowed us to obtain the following results. Purine and pyrimidine nucleotide residues are unevenly distributed along both chromosomes, displaying maxima and minima (waves) with a period of about 3 Mbp. Distribution of G+C along both chromosomes has no distinct maxima and minima, however, chromosome 21 contains considerably less G+C than chromosome 22. Both exons and Alurepeats are unevenly distributed along chromosome 21: they are scarce in its left part and abundant in the right part, while MIR elements are quite monotonously spread along this chromosome. The Alurepeats show a wave-like distribution pattern similar for both repeat orientations. The number of the Alurepeats of opposite orientations was equal for both studied chromosomes, and this may be considered a new property of the human genome. The positive correlation between the exon and Aludistribution patterns along the chromosome, the concurrent distribution of Alurepeats in both orientations along the chromosome, and the equal copy numbers for Aluin direct and inverted orientations within an individual chromosome point to their important role in the human genome, and do not fit the notion that Alurepeats belong to parasitic (junk) DNA.     

Class-1 translation termination factors: invariant GGQ minidomain is essential for release activity and ribosome binding but not for stop codon recognition

Previously, we have shown that all class-1 polypeptide release factors (RFs) share a common glycine-glycine-glutamine (GGQ) motif, which is critical for RF activity. Here, we subjected to site-directed mutagenesis two invariant amino acids, Gln185 and Arg189, situated in the GGQ minidomain of human eRF1, followed by determination of RF activity and the ribosome binding capacity for mutant eRF1. We show that replacement of Gln185 with polar amino acid residues causes partial inactivation of RF activity; Gln185Ile, Arg189Ala and Arg189Gln mutants are completely inactive; all mutants that retain partial RF activity respond similarly to three stop codons. We suggest that loss of RF activity for Gln185 and Arg189 mutants is caused by distortion of the conformation of the GGQ minidomain but not by damage of the stop codon recognition site of eRF1. Our data are inconsistent with the model postulating direct involvement of Gln185 side chain in orientation of water molecule toward peptidyl-tRNA ester bond at the ribosomal peptidyl transferase centre. Most of the Gln185 mutants exhibit reduced ability to bind to the ribosome, probably, to rRNA and/or (peptidyl)-tRNA(s). The data suggest that the GGQ motif is implicated both in promoting peptidyl-tRNA hydrolysis and binding to the ribosome.     

Mg2+-induced tRNA folding

Mg(2+)-induced folding of yeast tRNA(Phe) was examined at low ionic strength in steady-state and kinetic experiments. By using fluorescent labels attached to tRNA, four conformational transitions were revealed when the Mg(2+) concentration was gradually increased. The last two transitions were not accompanied by changes in the number of base pairs. The observed transitions were attributed to Mg(2+) binding to four distinct types of sites. The first two types are strong sites with K(diss) of 4 and 16 microM. The sites of the third and fourth types are weak with a K(diss) of 2 and 20 mM. Accordingly, the Mg(2+)-binding sites previously classified as "strong" and "weak" can be further subdivided into two subtypes each. Fluorescent transition I is likely to correspond to Mg(2+) binding to a unique strong site selective for Mg(2+); binding to this site causes only minor A(260) change. The transition at 2 mM Mg(2+) is accompanied by substantial conformational changes revealed by probing with ribonucleases T1 and V1 and likely enhances stacking of the tRNA bases. Fast and slow kinetic phases of tRNA refolding were observed. Time-resolved monitoring of Mg(2+) binding to tRNA suggested that the slow kinetic phase was caused by a misfolded tRNA structure formed in the absence of Mg(2+). Our results suggest that, similarly to large RNAs, Mg(2+)-induced tRNA folding exhibits parallel folding pathways and the existence of kinetically trapped intermediates stabilized by Mg(2+). A multistep scheme for Mg(2+)-induced tRNA folding is discussed.