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Biomedical subjects

I A Krasheninnikov

Publications and source records attributed to I A Krasheninnikov.

At least 19 recordsLinked to original sources
PubMed1997

Cotranslational folding of globin.

Globin synthesis in a wheat germ cell-free translation system was performed in the presence of [3H]hemin and [35S]methionine to determine the minimal length of the nascent ribosome-bound globin chain capable of heme binding. Nascent polypeptides of predetermined size were synthesized on ribosomes by translation of truncated mRNA molecules. Analysis with the use of sucrose gradient centrifugation and puromycin reaction revealed that the ribosome-bound N-terminal alpha-globin fragments of 140, 100, and 86 amino acid residues are capable of an efficient heme binding, whereas those of 75, 65, and 34 amino acid residues display a significantly weaker, or just nonspecific, affinity to heme. This indicates that the ribosome-bound nascent chain of 86 amino acid residues has already acquired a spatial structure that allows its interaction with the heme group or that heme attachment promotes the formation of the proper tertiary structure in the ribosome-bound nascent peptide. In any case the cotranslational folding of globin is suggested.

Animals↗
PubMed1996

Non-random usage of 'degenerate' codons is related to protein three-dimensional structure.

We report an analysis of a novel sequence-structure database of mammalian proteins incorporating nucleotide sequences of the exon regions of their genes together with protein sequence and structural information. We find that synonymous codon families (i.e. coding the same residue) have non-random codon distribution frequencies between protein secondary structure types. Their structural preferences are related to the third, 'silent' nucleotide position in a codon. We also find that some synonymous codons show very different or even opposite structural preferences at the N- or C-termini of structure fragments, relative to those observed for their amino acid residues.

Animals↗
PubMed1996

Mitochondrial import of a yeast cytoplasmic tRNA (Lys): possible roles of aminoacylation and modified nucleosides in subcellular partitioning.

The yeast tRNA(CUU)LYS is transcribed from a nuclear gene and then unequally redistributed between the cytosol (97-98%) and mitochondria (2-3%). We have optimized the conditions for its specific import into isolated mitochondria. However, only a minor fraction (about 0.5%) of the added tRNA was translocated into the organelles. An in vitro transcript, once aminoacylated, appeared to be a better import substrate than the natural tRNA which carries modified nucleosides. The tRNA is translocated across mitochondrial membranes in its aminoacylated form and remains relatively stable inside the organelle. Possible roles of aminoacylation, tRNA-protein interactions and nucleoside modification in subcellular partitioning of the tRNA are discussed.

Cell Fractionation↗
PubMed1993

Cotranslational heme binding to nascent globin chains.

Globin synthesis in cell-free extracts of rabbit reticulocytes was carried out in the presence of 3H-labeled hemin. Sucrose gradient centrifugation analysis revealed [3H]hemin in the polyribosome fraction. The addition of puromycin resulted in the release of both [3H]hemin- and [14C]leucine-labeled polypeptide from the polyribosomes. The data suggest cotranslational folding of the globin molecule on the ribosome and cotranslational heme binding to the nascent globin chain.

Animals↗
PubMed1993

[Import of cytoplasmic lysyl tRNA in mitochondria of baker's yeasts: prospects for study in vivo and in vitro].

To study the mechanisms of targeting tRNA(CUULys) from the cytoplasm into the mitochondrial compartment of the yeast cells, two test systems have been developed. The in vivo system based on the electroporation of intact yeast cells was used to introduce labelled tRNAs into the cytoplasm; however, only tRNA(CUULys) was found in the mitochondrial compartment. The in vitro import of this tRNA into isolated mitochondria required the presence of ATP and soluble cellular proteins in the reaction mixture. Two protein fractions were found to be necessary to direct the import in vitro. The first one had a high heparin-binding affinity, while the other one was not retained on heparin-Sepharose. Aminoacylation of the tRNA(Lys) before the transport and/or addition of the correspondent lysyl-tRNA synthetase to the in vitro system increased the efficiency of the import, but the protein fraction with heparin-binding properties is still required. The unmodified transcripts of the tRNA(CUULys) gene were shown to be able to be transported into isolated yeast mitochondria.

Amino Acyl-tRNA Synthetases↗
PubMed1992

A novel nonhistone protein (MENT) promotes nuclear collapse at the terminal stage of avian erythropoiesis.

The terminal stage of differentiation of nucleated chicken erythrocytes is associated with an overall gene repression and a condensation of the repressed chromatin portion. Two-dimensional DNP electrophoresis has been used to separate transcriptionally active and repressed chromatin of mature chicken erythrocytes. The repressed chromatin fraction is shown to be enriched with histone H5 as well as with a 42-kDa nonhistone chromosomal protein. The 42-kDa protein designated here as MENT (mature erythrocyte nuclear termination stage-specific protein) is hyperexpressed at the terminal stage of chicken erythropoiesis and is accumulated in adult chicken erythrocyte nuclei. This protein was purified by ion-exchange chromatography from 0.4 M NaCl extracts of the erythrocyte nuclei. It appeared to be a basic polypeptide (pI 9.2) which, however, precipitated at low pH. When reconstituted in vitro with immature erythrocyte nuclei, MENT promoted condensation of intact nuclear chromatin and enhanced the solubilization of nuclease-digested polynucleosomes, thus mimicking the processes occurring in vivo at the final stage of erythrocyte maturation. The extent of dissociation of specific gene sequences from the nuclear matrix in MENT-treated nuclei is in striking correlation with their transcriptional activity. No other basic proteins (H5, cytochrome c, RNase A) added to the nuclear preparation at the same level as MENT (protein/DNA = 0.005) caused any effect on nuclear organization. No alterations were observed when MENT was mixed with erythroblasts and nonerythroid nuclei having little or no histone H5. We propose that MENT cooperates with histone H5 to complete the nuclear collapse in mature nucleated erythrocytes.

Animals↗
PubMed1991

Nonuniform size distribution of nascent globin peptides, evidence for pause localization sites, and a contranslational protein-folding model.

Examination of nascent globin peptides accumulating in vitro during globin synthesis in rabbit reticulocyte lysates was carried out. A view was supported that nonrandom distribution of codons with different usage frequencies in mRNA may determine the messenger's translation kinetics. Regions of reduced translation of alpha- and beta-globin polypeptide chains were localized, and the cotranslational protein-folding model suggested previously was substantiated. An active conjunction of synthesis and folding of proteins was proposed as one of the main destinations of a translation nonuniformity.

Animals↗
PubMed1990

Loosened nucleosome linker folding in transcriptionally active chromatin of chicken embryo erythrocyte nuclei.

We have investigated the mechanism of the electrophoresis-driven chromatin aggregation which had been described by Weintraub (1984, Cell 38, 17-27) as a putative mean for propagation of genetic repression in eukaryotes. We show that the oligonucleosome aggregates are assembled de novo at the starting zone of DNP electrophoresis. A new system of native two-dimensional DNP electrophoresis has been worked out to separate the oligonucleosome aggregates ('A' particles) and the freely-migrating oligonucleosomes ('B' particles). The 'B' particle fraction which is derived from transcriptionally-active chromatin regions undergoes an extensive nuclease degradation of its DNA termini during the nuclease digestion. This fraction is partially depleted of histones H1 and H5 and is enriched in HMG nonhistone proteins. 'A' particles comprise the repressed chromatin DNA fragments which are about 60 b.p. longer than the corresponding DNA oligomers of 'B' particles. An oligonucleosome preparation containing the elongated DNA oligomers has been also isolated by means of sucrose gradient ultracentrifugation. Exonuclease III mapping reveals that the two chromatin fractions differ by an extent of terminal linker DNA trimming during the Micrococcal nuclease digestion rather than by the nucleosome repeat length. The complex character of nuclease digestion is not observed when the chromatin is digested in solution after the nuclear lysis. We argue that the protection of terminal oligonucleosome linkers is due to selective condensation of inactive chromatin in chicken erythrocyte nuclei and that the terminal DNA tails together with linker histones bound to them mediate the aggregation of repressed chromatin fragments.

Animals↗
PubMed1989

[Role of the code redundancy in determining cotranslational protein folding].

It has been demonstrated earlier in our laboratory that rare codon clusters can determine the boundaries of the polypeptide chain fragments of the same secondary structure type during the co-translational protein folding. According to this data, co-translational protein folding can occur under condition of a correlation between the frequency of codon choice in mRNAs and the relative abundance of their isoaccepting tRNAs. The alterations in the spectrum and concentrations of the isoaccepting tRNAs in different cells were demonstrated by many authors. The existence of a mechanism of the coordinate regulation of the levels (activities) of the isoaccepting tRNAs, corresponding aminoacyl-tRNA synthetases and mRNAs predominantly translated at a given moment of time can be suggested. Such a mechanism can ensure the needed accuracy of the protein folding process. Analysis of gene sequences of various pro- and eukaryotic organisms carried out in the present work revealed that the codon usage frequency spectra of simultaneously synthesized proteins are similar. The relative appearance of the most rare and frequent codons in investigated gene sequences displays a high degree of conservatism. It has also been found that structural-homologous proteins from different organisms (cytochromes c, myoglobins) have very similar codon frequency distribution profiles. This property retains despite the significant variations in the codon usage spectra in the investigated gene sequences. The data obtained indicate that the codon distribution in mRNAs whose diversity is mainly conditioned by the genetic code redundance is a program that determines translational rates of different mRNA parts thus controlling the spatial folding of the synthesized peptide chain.

Animals↗
PubMed1983

A topological model for chromatin transcription and a role for nucleosome linkers.

There is a good deal of evidence that transcribing RNA polymerase may translocate across nucleosomes without their displacement and (or) rearrangement. A topological model for RNA chain elongation on a nucleosome is considered here. A new mechanism of RNA polymerase translocation is suggested in order to avoid the steric hindrances inherent in the model. It is shown that a transcribed nucleoprotein fiber should be interrupted by protein-free DNA stretches (nucleosome linkers) to allow release of nascent RNA. Possible verifications and consequences of the model are discussed.

Animals↗
PubMed1982

The central tryptic fragment of histones H1 and H5 is a fully compacted domain and is the only folded region in the polypeptide chain. A thermodynamic study.

Scanning microcalorimetry has been used to show that the globular structure in histones H1 and H5 melts reversibly over a relatively narrow temperature range as a single domain with enthalpy of melting very close to the van't Hoff enthalpy. These proteins therefore exhibit macroscopic cooperativity like that of many well-studied small proteins. For histone H5 the observed melting temperature and melting enthalpy coincide with those previously obtained [Eur. J. Biochem. 67, 379-388 (1976)] using intrinsic Cotton effects in the circular dichroism spectrum, but not with those from extrinsic Cotton effects or NMR chemical shifts. Calorimetric measurements on a central tryptic fragment from histones H1 and H5 show these fragments to have the same molar melting enthalpy as the parent histones. The folded structures of H1 and H5 are therefore located only within these fragments. For histone H1 this conclusion is supported by the finding that the C-terminal fragment (residues 122-213) shows no peak in the specific heat curve.

Calorimetry, Differential Scanning↗
PubMed1982

Transient unfolding of trypsin-digested chromatin core particles.

Limited digestion of nucleosome core particles with trypsin caused cleavage and removal of N-terminal histone sequences of 10-30 amino acids. The proteolyzed core particles exhibited salt-dependent structural transitions revealed by sedimentation, circular dichroism and nuclease-cutting assays, while the intact nucleosome cores were not affected under the experimental conditions. The results obtained indicate that the observed transitions correspond to the transient unfolding of terminal segments of core particle nucleoprotein caused by the increase of its net negative charge. The excision of the N-terminal histone domains therefore leads to partial destabilization but not to irreversible disruption of the compact nucleosome structure.

Amino Acid Sequence↗